For Peer Review http://mc.manuscriptcentral.com/fems Adhesive Organelles of Gram-Negative Pathogens Assembled with the Сlassical Сhaperone/Usher Machinery: Structure and Function with a Clinical Bend Journal: FEMS Microbiology Reviews Manuscript ID: FEMSRE-09-06-0029.R2 Manuscript Type: Review - Invited Date Submitted by the Author: Complete List of Authors: Zaviyalov, Vladimir; University of Turku, Joint Biotechnology Laboratory Zavialov, Anton; Swedish University of Agricultural Sciences, Department of Molecular Biology Zaviyalova, Galina; University of Turku, Joint Biotechnology Laboratory Korpela, Timo; University of Turku, Joint Biotechnology Laboratory Keywords: Adhesins, Gram-Negativ Pathogens, Chaperone/Usher Machinery ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews For Peer Review INTRODUCTION A necessary step for the development of an infectious disease in the host organism is the formation of a firm link between the pathogen and target cells of the host. The link is mediated by surface-exposed adhesive organelles and is required for internalization of bacteria or extracellular colonization of host tissues. The adhesive organelles mediate bacterial adhesion via specific interaction with surface structures presented on host cells. The adhesin-binding to the target cells triggers subversive signals that allow pathogens to evade immune defense and facilitate bacterial colonization or invasion (reviewed by Zavialov et al., 2007). There are two major classes of protein adhesins of Gram-negative pathogens: (1) The fimbrial adhesins, represented by the linear homopolymers or heteropolymers (up to 7 distinct subunits) of hundreds to thousands of subunits; (2) The non-fimbrial adhesins consisted of a single protein or homotrimers. The fimbrial adhesins in Gram-negative bacteria are typically formed by non-covalent homo- or hetero- polymerization of subunit proteins (reviewed by Fronzes et al., 2008; Kline et al., 2009; Waksman & Hultgren, 2009; Zavialov et al., 2007). In contrast, the more recently discovered fimbrial adhesins in Gram-positive bacteria are formed by covalent polymerization of protein subunits in a process that requires a dedicated sortase enzyme (reviewed by Proft & Baker, 2009). The assembly of fimbrial and non-fimbrial adhesins of Gram-negative pathogens involves the function of different secretion systems. Protein transport across the outer membrane of Gram-negative bacteria can be subdivided into Sec-independent and Sec-dependent pathways (reviewed by Gerlach & Hensel, 2007). Depending on the system of secretion, adhesive proteins presented on the surface of Gram-negative bacteria may be divided in a few major families: (1) The fimbrial adhesins, assembled on outer membrane by the classical chaperone/usher pathway (Choudhury et al., 1999; Hung et al., 1996; Fronzes et al., 2008; Knight et al., 2000; Page 1 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Remaut et al., 2006, 2008; Sauer et al., 1999, 2000, 2002, 2004; Thanassi et al., 1998; Verger et al., 2007; Waksman & Hultgren, 2009; Yu et al., 2009; Zavialov et al., 2001, 2002, 2003, 2005, 2007; Zavialov & Knight, 2007). (2) CS pili, assembled on outer membrane by the “alternate chaperone/usher pathway” (Soto & Hultgren, 1999). Assembly of the surface antigen 1 (CS1) pili of enterotoxigenic Escherihia coli shows high functional similarities to the classical chaperone/usher pathway but the proteins involved share no detectable sequence similarities. Poole et al. (2007) demonstrated that like the classical chaperone/usher pathway the donor strand complementation mechanism governs intersubunit interaction of fimbriae of the alternate chaperone/usher pathway. (3) Type IV pili, formed in distinction to (1) and (2), by polymerization of pilin subunits at the cytoplasmic membrane. The assembled pilus structure is extruded across the outer membrane and forms long and flexible surface appendages (Craig et al., 2004; Fronzes et al., 2008, 2009a,b). Components of the type IV pilus assembly machinery are structurally related to Type 2 secretion system, where homologous proteins are called “pseudopilins”. (4) Curli or thin aggregative fimbrial adhesins, assembled at the bacterial surface through extracellular nucleation precipitation: the major fiber subunit CsgA polymerizes on the surface- exposed nucleator CsgB (Hammar et al., 1996; Fronzes et al., 2008). (5) The non-fimbrial “trimeric autotransported adhesins”, secreted by the Type 5 secretion system (Linke et al., 2006). Adhesin YadA is the prototypical member of non-fimbrial adhesins. YadA is expressed by several Yersinia species and is a member of the family of very stable trimeric autotransporter adhesins anchored in the outer membrane with a β-barrel. (6) Integral outer-membrane proteins (e.g. OmpA, invasin and intimin), anchored to the outer membrane with a unique mechanism, in which the bacteria provide the cognate receptor for the adhesin intimin by translocating it into the host cell in a Type III secretion system-dependent manner (Niemann et al., 2004). Page 2 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (7) Non-fimbrial adhesins, secreted by the Type 1 secretion system (Delepelaire, 2004). E.g. two-partner-secreted filamentous hemagglutinins from Bordetella pertussis exist in a membrane- bound and secreted form, each having distinct functions. Dominant class (1) of the fibrillar adhesive organelles of Gram-negative pathogens is assembled by the conserved classical chaperone/usher protein secretion system (Choudhury et al., 1999; Hung et al., 1996; Fronzes et al., 2008; Knight et al., 2000; Remaut et al., 2006, 2008; Sauer et al., 1999, 2000, 2002, 2004; Thanassi et al., 1998; Verger et al., 2007; Waksman & Hultgren, 2009; Yu et al., 2009; Zavialov et al., 2001, 2002, 2003, 2005, 2007; Zavialov & Knight, 2007). This system can assemble fimbrial organelles of diverse subunit composition, architecture and function. The assembled organelles consist of two main structurally and functionally distinct families. One family consists of only one or two types of subunits and at low resolution typically shows non-pilus, amorphous or capsule-like morphology (Hung et al., 1996; Remaut et al., 2006; Soto & Hultgren, 1999; Zavialov et al., 2003; 2005; 2007). The assembly of this family is assisted with the FGL (having a long F1-G1 loop) family of periplasmic chaperones (Hung et al., 1996; Remaut et al., 2006; Zavialov et al., 2003; 2005; 2007; Zavialov & Knight, 2007; Zav’yalov et al., 1995b). The notable property of the organelles is that all subunits possess two independent binding sites specific to different host cell receptors (Anderson et al., 2004a, b; Korotkova et al., 2006a,b; 2008a; Pettigrew et al., 2004). Because of this function-structural property they were named as “FGL chaperone-assembled polyadhesins” (Zavialov et al., 2007). The other family consists of thick rigid and thin flexible adhesive pili (also known as adhesive fimbriae) of a complex subunit composition (up to 7 different subunits). The majority of the pili displays only one adhesive domain on the tip of the pilus (mono- adhesive fimbriae/pili) (Choudhury et al., 1999; Hung et al., 1996; Fronzes et al., 2008; Knight et al., 2000; Remaut et al., 2008; Sauer et al., 1999, 2000, 2002, 2004; Thanassi et al., 1998; Verger et al., 2007; Waksman & Hultgren, 2009). The assembly of mono-adhesive pili/fimbriae Page 3 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review is assisted with the FGS (having a short F1-G1 loop) family of periplasmic chaperones (Hung et al., 1996). The discovery of two families of organelles and chaperones was based on the different morphologies of organelles and the sequence comparison of chaperones (Hung et al., 1996; Zav’yalov et al., 1995b). The relevance of the division was confirmed later by high-resolution 3D analysis of the typical representatives of the two families of chaperones and the organelle subunits folded by them (Choudhury et al., 1999; Remaut et al., 2006; Sauer et al., 1999, 2002; Salih et al., 2008; Verger et al., 2007; Waksman & Hultgren, 2009; Zavialov et al., 2003, 2005, 2007; Zavialov & Knight, 2007). Both types of organelles are made of fibers of linearly polymerized subunits. The subunits are connected with the donor strand exchange mechanism (Salih et al., 2008; Sauer et al., 1999; Verger et al., 2007; Waksman & Hultgren, 2009; Zavialov et al., 2003, 2005, 2007). Comparison of the subunit 3D structures in fiber and bound to chaperone (Sauer et al., 2002; Zavialov et al., 2003) together with calorimetric studies (Zavialov et al., 2005) revealed that the fiber formation in both of the families is driven by the chaperone- preserved folding energy. The last comprehensive review on the superfamily of Gram-negative bacterial adhesins assembled via the classical chaperone/usher pathway was published a decade ago (Soto & Hultgren, 1999). A remarkable progress in understanding the structure and function of the classical chaperone/usher assembly-translocation machinery (Remaut et al., 2008; Yu et al., 2009), the structure and function of the surface-exposed adhesive organelles (Anderson et al., 2004a,b; Bouckaert et al., 2005, 2006; De Greve et al., 2007; Korotkova et al., 2006a,b, 2008a; Li et al., 2007; Pettigrew et al., 2004; Salih et al., 2008; Verger et al., 2007; Westerlund- Wikström & Korhonen, 2005; Zavialov et al., 2003, 2005, 2007), and the phylogenesis of ushers/chaperones (Nuccio & Bäumler, 2007) has been achieved since that time. It has become now evident that adhesins trigger subversive signals directed to mislead the immune system (Bergsten et al., 2005; Betis et al., 2003a, b; Cane et al., 2007; Diard et al., 2006; Sharma et al., Page 4 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2005a, b; Sodhi et al., 2004). Numerous examples of application of the organelles for development of vaccines have been described (Alvarez et al., 2006; Chichester et al., 2009; Del Prete et al., 2009; Elvin et al., 2006; Eyles et al., 2000; Honko et al., 2006; Glynn et al., 2005; Goluszko et al., 2005; Hu et al., 2009; Jones et al., 2006; Langermann et al., 2000; Lopes et al., 2006; Powell et al., 2005; Remer et al., 2009; Santi et al., 2006; Strindelius et al., 2004; Verdonck et al., 2009; Williamson et al., 2005). In addition, it has been demonstrated that adhesive domains of monoadhesins and chaperone/usher assembly-translocation machinery are promising targets for new generations of antimicrobials that specifically inhibit adhesion (Wellens et al., 2008) or interfere the fimbrial adhesion assembly (Aberg & Almqvist, 2007; Pinkner et al., 2006). Although several excellent reviews have been published recently, they either focused on specialized aspects of the chaperone-usher assembly (Fronzes et al., 2008; Sauer et al., 2004) or covered the results of studies of only particular chaperone-usher system (Waksman & Hultgren, 2009). Our previous review was also devoted only to FGL chaperone-assembled polyadhesins (Zavialov et al., 2007). We believe that recently accumulated significant new knowledge on different aspects of biogenesis of the superfamily of Gram-negative bacterial adhesins assembled via the classical chaperone/usher pathway and their medical applications require new analysis and generalizations. During this work we found that number of different types of subunits, composing the organelles, strongly correlates with the length of F1-G1 loop of chaperone proteins. Based on this, we suggest here a novel function-structural classification of the superfamily of adhesive organelles assembled with the classical chaperone/usher machinery. General Properties of Adhesive Organelles Adhesive organelles assembled with the classical chaperone/usher machinery are found in Gram-negative bacteria, primarily in genuses Escherichia, Klebsiella, Photorhabdus, Proteus, Page 5 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Salmonella, and Yersinia of Enterobacteriaceae. The organelles are also found in species from genuses Bordetella of Alcaligenaceae, Haemophilus of Pasteurellaceae family, Pseudomonas of Pseudomonadaceae family, and Acinetobacter of Moraxellaceae. All these bacteria cause various diseases including fatal systemic diseases, like bubonic and pneumonic plague (Yersinia pestis), enteric typhoid fever (Salmonella typhi and Salmonella paratyphi A), sepsis or extra- intestinal focal infections (Salmonella choleraesuis), gastroenteritis (Yersinia pseudotuberculosis, Salmonella typhimurium and Salmonella enteritidis), pyelonephritis, cystitis, diarrhea (different pathogenic E. coli strains and Proteus mirabilis), whooping cough (Bordetella pertussis), Brazilian purpuric fever, meningitis, otitis media (Haemophilus influenzae), pneumonia (Klebsiella pneumoniae), insect infections (Photorhabdus temperata) and plant infections (Pseudomonas syringae). Table 1 describes assembly assisting proteins, species distribution and associated diseases for fifty currently known adhesive organelles, the assembly of which on bacterial surface has been confirmed experimentally and is assisted with the classical chaperone/usher machinery. The information is placed in Table 1 in alphabetical order of the names of the chaperone/usher proteins. Gene Clusters Encoding for Adhesive Organelles Genes of proteins involved in expression and assembly of adhesive fibres via the classical chaperone/usher pathway are arranged into compact gene clusters, which are located either on the chromosome or plasmids of Gram-negative bacteria. Depending on the structural properties of periplasmic chaperones they can be divided into two families: (1) FGL chaperone-comprising gene clusters; (2) FGS chaperone-comprising gene clusters. Page 6 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review FGL chaperone-comprising gene clusters. Encoded by the caf gene cluster fraction 1 (F1), capsular antigen from Y. pestis is comprised of aggregated high-molecular-weight linear polymers of a single subunit Caf1 (Zavialov et al., 2002, 2003, 2005, 2007). The genes of caf gene cluster, caf1, caf1M, caf1A and caf1R, encode, respectively, for Caf1 subunit, periplasmic chaperone Caf1M, an outer membrane assembler, the molecular usher Caf1A, and the protein Caf1R regulating of the gene cluster transcription (Galyov et al., 1990, 1991; Karlyshev et al., 1992a, b; 1994). The psa gene cluster from Y. pestis encodes proteins for expression and assembly of the fimbrial pH6 antigen comprised of high-molecular-weight polymer of PsaA subunit (Lindler & Tall, 1993). PsaB functions as the periplasmic chaperone, PsaC is the molecular usher. Two additional proteins, PsaE and PsaF, have been shown to regulate transcription of psaA gene (Yang & Isberg, 1997). Another transcriptional regulator, RovA, interacts with the psaE and psaA promoter regions, suggesting that RovA is an upstream regulator of psa gene cluster (Cathelyn et al., 2006). Identical psa gene clusters are present in Y. pestis and Y. pseudotuberculosis (Lindler & Tall, 1993). Closely related to psa gene cluster of Y. pestis, Y. enterocolitica contains myf encoding the Myf fimbriae, which are built up of MyfA subunits. The psa and myf clusters have similar general organisation. Moreover, proteins encoded by these gene clusters display a significant sequence similarity, suggesting that pH6 antigen and Myf fibriae have a common function in the different species of Yersinia. As PsaE and PsaF encoded by psa, the MyfE and MyfF proteins encoded by myf have a role in regulation of the cluster transcription (Iriarte & Cornelis, 1995). The cs-3 gene cluster from E. coli encodes for proteins for expression and assembly of the colonization factor-3 that forms CS-3 fimbriae comprised of high-molecular-weight polymer of CS-3 subunit (Jalajakumar et al., 1989). CS3-E functions as the periplasmic chaperone, and CS3- D is the molecular usher. Page 7 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review The nfa gene cluster from E. coli encodes proteins for expression and assembly of the nonfimbrial adhesin, NFA-I, comprised of high-molecular-weight polymer of NfaA subunit (Ahrens et al., 1993). NfaE functions as the periplasmic chaperone, and NfaE is the molecular usher. A group of E. coli gene clusters, afa-3, afa-8, agg, aaf, agg-3, dafa, dra, daa, that encode proteins for expression and assembly of the afimbrial adhesins Afa-III and AfaE-VIII, the aggregative adherence fimbria type I, II and III (AAF/I, AAF/II and AAF-III), the diffuse adherence fibrillar adhesin (Dafa), the Dr hemagglutinin flexible fimbriae and the F1845 (DaaE) fimbrial adhesin, respectively, have a peculiar feature: each gene cluster encodes additional subunit D for which an invasive function was suggested (putative invasin subunit; Jouve et al., 1997; Servin, 2005). DraE and AfaE-III adhesins may assemble into a flexible fibre that provides the link between the usher at the outer membrane and the putative invasin subunit located at the tip of the fibre (Anderson et al., 2004a, b; Pettigrew et al., 2004). However, expression of DraD invasin subunit is independent of the DraC usher and DraE fimbrial subunit (Zalewska et al., 2005). In addition, polymerization of DraE fimbrial subunits into fimbrial structures does not require expression of DraD. Recently, Zalewska-Piątek et al. (2008) showed that type II secretion in E. coli strain Dr+ leads to DraD translocation to the bacterial cell surfaces. Very recently, Korotkova et al. (2008b) and Guignot et al. (2009) demonstrated that the DraD subunit is not required for β1 integrin recruitment or bacterial internalization. Therefore, the function of D subunits is still under the question. A group of Salmonella spp. gene clusters saf, sef, cs6-1, cs6-2 that encodes proteins for expression and assembly of the atypical fimbriae Saf, the filamentous fimbriae-like structures SEF14/18, and the colonization factors CS6-1 and -2 has another common peculiar feature: all of these gene clusters encode two adhesin subunits. The SefB chaperone of S. enteritidis assists in the assembly of two distinct cell-surface structures, SEF14 and SEF18, which are homopolymers of SefA and SefD subunits, respectively (Clouthier et al., 1994). The CssC chaperone assists in Page 8 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review assembling thin CS6 fibrillae that are composed of two heterologous CssA and CssB subunits (Wolf et al., 1997). FGS chaperone-comprising gene clusters. A group of two gene clusters atf and pef that encodes proteins for expression and assembly of the ambient-temperature fimbriae ATF of P. mirabilis (Massad et al., 1996) and plasmid-encoded (PE) fimbriae of S. typhimurium have common peculiar feature: they encode only one structural subunit which is probably functioning as an adhesin subunit. Recently a cosmid carrying the pef operon was introduced into E. coli and expression of fimbrial filaments composed of PefA was confirmed by flow cytometry and immune-electron microscopy (Chessa et al., 2008). Plasmid-encoded fimbriae were purified from the surface of E. coli and the resulting preparation was shown to contain PefA as the sole major protein component. Binding of purified plasmid-encoded fimbriae to a glycan array suggested that this adhesin specifically binds the tri-saccharide Galss1-4 (Fuca1-3) GlcNAc, also known as the Lewis X (Lex) blood group antigen. The aciad gene cluster of Acinetobacter sp., strain ADP1 (Barbe et al., 2004; Gohl et al., 2006) encodes only one structural subunit which may function as an adhesin subunit. This cluster contains genes for two periplasmic chaperones. A group of gene clusters f17a, acu and fim/fha that encodes proteins for expression and assembly of the F17 pili of E. coli (Lintermans et al., 1988), thin pili of Acinetobacter sp., strain BD413 (Barbe et al., 2004; Gohl et al., 2006) and type 2 and 3 pili of B. pertussis (Willems et al., 1992). They encode one structural and one adhesin subunit that are exposed on the tip of pili. A group of gene clusters hif, haf, mrk, lpf and pmf that encodes proteins for expression and assembly of the H. influenzae fimbriae (van Ham et al., 1994), H. influenzae biogroup aegyptius fimbriae (Read et al., 1996), K. pneumoniae type 3 fimbriae (Allen et al., 1991), S. typhimurium long polar fimbriae (Bäumler & Heffron, 1995) and P. mirabilis PMF pili (Massad & Mobley, 1994) encode two structural subunits and one adhesive subunit that is exposed on the tip of fimbriae. Page 9 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review A group of gene clusters fas, csw and fot that encodes proteins for expression and assembly of the 987P (Edwards et al., 1996), CS12 (EMBL accession number Q9ALL0) and CS18 (Honarvar et al., 2003) fimbriae of E. coli have very unusual feature since they encode three distinct chaperones which assist in assembling of fibers composed of two structural subunits and one adhesive subunit exposed on the tip of fimbriae. E.g., in the case of 987P fimbriae the FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA (Edwards et al., 1996). The periplasmic chaperone FasC specifically interacts and stabilizes the adhesin FasG (Edwards et al., 1996). FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly for other subunits (Edwards et al., 1996). Two gene clusters fos and stf that encode proteins for expression and assembly of the F1C pili of E. coli (Riegman et al., 1990) and Stf fimbriae of S. typhimurium (Emmerth et al., 1999) and they encode three structural and one adhesin subunit exposed on the tip of fimbriae. A group of gene clusters fim, sfp, sfa and mrp that encodes proteins for expression and assembly of the type 1 pili of E. coli (Jones et al., 1993), Sfp fimbriae of E. coli (Brunder et al., 2001), S pili of E. coli (Dobrindt et al., 2001) and mannose-resistant/Proteus-like MR/P pili of P. mirabilis (Bahrani & Mobley, 1994). They encode four structural subunits and one adhesin subunit which is exposed on the tip of fimbriae. The gene clusters fan, lda, fae and ral encode proteins for expression and assembly of the F4 (K88), Lda and F5 (K99) thin flexible pili and REPEC fimbriae of E. coli, respectively (Adams et al., 1997; Bakker et al., 1991; Scaletsky et al., 2005). These pili/fimbriae consist of four or five subunits. However F4 (K88), F5 (K99) and Lda pili do not display specialized adhesive domains on the tip of the pilus, but carry binding sites on their main structural subunit (FanH, FaeH and LdaH) (Bakker et al., 1991; Scaletsky et al., 2005). The overall arrangement of the ral gene cluster closely resembles that of the fae cluster with homologous genes occupying the same relative position in each cluster. The ral cluster also has some of the more specific features of the fae cluster such as the overlapping reading frames of the genes encoded chaperone and usher and Page 10 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review the apparent absence of promoters within the region carrying the structural genes (Adams et al., 1997). This general similarity together with the significant levels of homology exhibited by individual genes makes it reasonable to propose functions for the ral gene products based on the known roles of their Fae counterparts. Thus, Adams et al. (1997) proposed that RalC, RalF, and RalH are minor fimbrial subunits of the fimbrial structure which is primarily composed of RalG, the major fimbrial subunit. The gene cluster afr encodes proteins for expression and assembly of the E. coli AF/R1 pili (Cantey et al., 1999). The subunits encoded by afr gene cluster have the highest percentage amino acid identity with the subunits encoded by ral cluster (Adams et al., 1997). The mrf and pap gene clusters that encode proteins for expression and assembly of mannose- resistant fimbriae of Ph. temperata (Meslet-Cladiere et al., 2004) and P pili of E. coli (Marklund et al., 1992) have the most complex composition: they encode six structural and one adhesin subunit that is exposed on the tip of fimbriae. Fig. 1 shows the general organization of gene clusters for which the expression has been experimentally confirmed. STRUCTURE AND MECHANISM OF FUNCTION OF CHAPERONE/USHER MACHINERY Molecular Functions Periplasmic chaperones and outer membrane molecular usher proteins function in co- operation as the chaperone/usher machinery that drives the assembly of surface-exposed fimbrial adhesins (Fig. 2). Periplasmic chaperones possess the following main functions (Hung et al., 1996; Knight et al., 2000; Remaut et al., 2006; Sauer et al., 2000, 2004; Thanassi et al., 1998; Verger et al., Page 11 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2007; Zavialov et al., 2001, 2003, 2005, 2007; Zavialov & Knight, 2007; Zav’yalov et al., 1995b): (1) Binding to nascent subunits as they enter the periplasm via the Sec pathway; (2) Protection of subunits from non-productive aggregation and proteolytic degradation by capping their assembly surfaces; (3) Transport of subunits to an outer membrane molecular usher. Periplasmic chaperones either form stable complexes with subunits emerging to the periplasm or form dimers (PapD) (Hung et al., 1999) or tetramers (Caf1M) (Zavialov & Knight, 2007), where the subunit-binding sequences are protected against proteolysis and unspecific binding (see the chapter “3D structures of chaperones”). Outer membrane molecular ushers possess the following main functions (Fronzes et al., 2008; Nishiyama et al., 2005; Remaut et al., 2006, 2008; Yu et al., 2009): (1) Release of subunit from the chaperone; (2) Formation of assembly platform for polymerization of subunits in linear fibers; (3) Formation of twinned-pore translocation machinery for secretion of linear fibers on the cell surface. The recently decoded structure and proposed mechanism of the function of molecular usher proteins are described in the chapters "Structure of outer membrane molecular usher proteins" and "Mechanism of function of chaperone/usher machinery". Structures of Chaperones 3D structure of chaperones. For a long time the PapD chaperone has been serving as a prototype protein for the superfamily of periplasmic chaperones (Holmgren & Branden, 1989) and was the first structure of a molecular chaperones in general. The PapD chaperone structure consists of two domains joined at approximately right angle with a large cleft between the Page 12 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review domains (Fig. 3). Both domains are 7-stranded β-sandwiches with immunoglobulin-like topology (Holmgren & Branden, 1989). The F1 and G1 β-strands in the N-terminal domain of PapD are connected by a long and flexible loop that protrudes like a handle from the body of the domain. Two families of the periplasmic chaperones were suggested with sequence analysis, FGS (having a short F1-G1 loop) and FGL (having a long F1-G1 loop) (Hung et al., 1996; Zav'yalov et al., 1995a) (Fig. 4). PapD chaperone represents the FGS class of chaperones. High resolution crystal structures for two FGL chaperones, Caf1M from Y. pestis and SafB from S. typhimurium, in complex with corresponding subunits, Caf1 of the F1 capsular antigen and SafA of atypical fimbriae Saf, have been determined (Remaut et al., 2006; Zavialov et al., 2003, 2005). As FGS chaperones (Choudhury et al., 1999; Holmgren & Branden, 1989; Knight et al., 2000; Sauer et al., 1999, 2002; Verger et al., 2007), FGL chaperones consist of two domains joined at approximately right angle with a large cleft between the domains (Fig. 3). Both domains are 7- stranded β-sandwiches with immunoglobulin-like topology (Remaut et al., 2006; Zavialov et al., 2003, 2005). The F1 and G1 β-strands in the N-terminal domain of Caf1M and SafB are connected by a flexible loop that is much longer than in PapD chaperone (Fig. 4). When PapD is not engaged in binding to subunits, it is capable of interacting transiently with itself to form a weakly but specifically bound dimer (Hung et al., 1999). The crystal structures of two dimeric forms of PapD were solved to gain insight into the molecular basis of PapD dimer formation (Hung et al., 1999). The structure–function analysis revealed that PapD interacts with itself by means of the same interactive surfaces that it uses to bind subunits, possibly representing a self-capping mechanism that protects the subunit-binding sequences against proteolysis and unspecific binding. Zavialov & Knight (2007) found that typical representative of FGL chaperones, subunit-free Caf1M, exists predominantly as a tetramer. A 2.9 Å resolution crystal structure of the Caf1M tetramer revealed that each of the four molecules contribute to its subunit binding sequences (the Page 13 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review A1 and G1 strands) to form an eight-stranded hetero-sandwich with a well-packed phenylalanine-rich hydrophobic core (Zavialov & Knight, 2007). Tetramerization protects chaperone molecules against enzymatic proteolysis. Deletions in the subunit binding motifs completely abolish tetramer assembly, suggesting that the hetero-sandwich is the main structural feature holding the tetramer together. Deletions in the VGVFVQFAI motif abolish both tetramer assembly and aggregation, consistent with the predicted high β-aggregation propensity for this motif. Such a packing of the aggregation-prone subunit binding sequences into the hetero- domain is a novel molecular mechanism preventing unspecific aggregation of free chaperones. Assembly of F4 fimbriae of enterotoxigenic E. coli, indicate that self-capping of the pilin- interactive interfaces is not the mechanism that is conservedly applied by all periplasmic chaperones, but is rather a case-specific solution to cap aggregation-prone surfaces (Van Molle et al., 2009). FaeE crystal structure shows a dimer formed by interaction between pilin-binding interfaces of two monomers. Thermodynamic and biochemical data show that FaeE occurs as a stable monomer in solution (Van Molle et al., 2009). Characteristic features of FGL and FGS chaperones. Comparison of structures of FGS and FGL chaperones reveals differences, which appear to locate in the functionally important segments. These class-specific differences were correctly predicted earlier based on sequence comparison and modelling using known structure of the FGS chaperone PapD (Chapman et al., 1999; Hung et al., 1996; Zav'yalov et al., 1995b), and functional studies (Chapman et al., 1999; MacIntyre et al., 2001; Zav'yalov et al., 1997): (1) FGL chaperones contain a significantly longer binding motif in the F1-G1 loop and G1 β- strand than FGS chaperones, due to the extension of this motif into the F1-G1 loop region (typically by two hydrophobic alternating residues) (Fig. 4) (Hung et al., 1996; MacIntyre et al., 2001; Remaut et al., 2006; Zavialov et al., 2003, 2005; Zav'yalov et al., 1995b); Page 14 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (2) FGL chaperones also contain a longer binding motif at the N-terminus with three alternating bulky hydrophobic residues than in FGS chaperones (two), which extends to A1 strand in FGL chaperones (at least 3 residues) (Chapman et al., 1999; Remaut et al., 2006; Zavialov et al., 2003, 2005; 2007). (3) In contrast to FGS chaperones, the massive subunit binding hairpin, F1 strand-loop-G1 strand of FGL chaperones is stabilized by a disulphide bridge between two conserved Cys-residues, one of which is localized in F1 β-strand, and the other in G1 β-strand (Figs 3 and 4; C98 and C137 in Caf1M and C98 and C125 in SafB) (Remaut et al., 2006; Zavialov et al., 2003, 2005). Biochemical and mutagenesis studies showed that the unique structural features of FGL chaperones are crucial for function (see below). Importance of the disulphide bond in provision of FGL chaperone functions in vitro and in vivo. Reduction of the disulphide bond and alkylation of the cysteine residues in a FGL chaperone considerably increase dissociation constant for Caf1M–Caf1 complex (Zav'yalov et al., 1997). Later on, it was also showed that cysteine residues of FGL chaperone DraB form a disulfide bond and are crucial for the formation of the DraB–DraE binary complex (Piątek et al., 2005). Probing the conformation and stability of Caf1M at different temperatures, pH, and concentration of urea by measurements of circular dichroism and fluorescence suggested that disulphide bond does not affect the general conformation, but induces changes in the local structure around the bond (Zav'yalov et al., 1997). However, the level of expression of Caf1M in E. coli was clearly affected by disulphide isomerase DsbA. Caf1M accumulated in considerably larger quantities in DsbA+ rather than in DsbA- strain, suggesting an important role of the disulphide bond in provision of Caf1M functions in vivo (MacIntyre et al., 2001). G1 and A1 β-strands are crucial for chaperone function. The studies of MacIntyre et al. (2001) had highlighted the importance of G1 β-strand hydrophobic residues in protecting newly secreted Caf1 from proteolytic degradation. This could be explained in part by the observed importance of some residues (F132) in stabilizing chaperone–subunit complex. The mutation, Page 15 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Caf1MV128A, which also resulted in subunit degradation, however, enhanced chaperone– subunit stability (communicated by A. Zavialov). Contrary to FGS chaperones, the A1 β-strand of Caf1M also significantly adds to the binding contact in this region (Figs 3 and 6). Deletion of the sequence 10-15 of Caf1M in A1 β-strand (communicated by A. Zavialov) led to complete loss of the chaperone function. Structure of the Caf1M–Caf1 complex suggests that the chaperone A1 and G1 strands are likely to form a binding platform, which is rigid enough to prevent collapse of the open subunit conformation in this very unstable region (Fig. 3). Correlation between the length of FG loop and the number of different types of subunits operated by a chaperone. Fig. 5 shows the plot of correlation between the number of deleted residues in a F1-G1 loop of chaperones (in comparison with the longest F1-G1 loop of the Caf1M chaperone) and the number of different types of subunits operating by the chaperones. These parameters show a strong correlation, but the slopes of the plots of correlation for FGL and FGS chaperones are different. That may be explained by an influence of the disulfide bond connecting F1 and G1 strands in FGLs. The coefficient of correlation for FGL chaperones is equal to 0.80 and for FGS chaperones it is equal to 0.72. The longer F1-G1 sequence creates a longer subunit recognition motif for a more specific binding of one or two subunits forming FGL chaperone-assembled fimbrial polyadhesins (Zavialov et al., 2003, 2007). Probably, the shorter F1-G1 sequence in FGS chaperones was evolved as a consequence of a need in less specific binding of subunits because monoadhesive fimriae/pili are composed of up to 7 different subunits. Structure of Subunits and Molecular Architecture of Adhesive Organelles 3D structure of chaperone-complemented subunits. Chaperone-free subunits of fimbrial polyadhesins (Zavialov et al., 2005; Zav'yalov et al., 1997) and mono-adhesive fimbriae/pili (Bann et al., 2004; Nishiyama et al., 2003) are highly unstable and prone to form aggregates. Page 16 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Hence, structural information on many subunits of these organelles was obtained by studying chaperone-subunit complexes (Choudhury et al., 1999; Remaut et al., 2006; Sauer et al., 1999, 2002; Verger et al., 2007; Zavialov et al., 2003, 2005). The crystal structures of the type-1 pilus FimC–FimH (Choudhury et al., 1999) and of the P pilus PapD–PapK, PapD–PapE and PapD–PapA (Sauer et al., 1999, 2002; Verger et al., 2007) chaperone–subunit complexes show that pilus subunits (pilins), like chaperones, have immunoglobulin-like folds (Figs 3 and 6). However, the C-terminal (G) β-strand of the fold is missing, creating a deep hydrophobic groove on the surface of the subunit (Figs 3 and 6). Chaperones bind pilins by inserting their G1 β-strand into this groove in a process called donor strand complementation (Choudhury et al., 1999; Sauer et al., 1999, 2002; Verger et al., 2007) (Figs 3 and 6). Three hydrophobic side chains in the conserved G1 motif are inserted into the hydrophobic acceptor groove and become an integral part of the subunit hydrophobic core. The crystal structures of the Caf1M-Caf1 and SafB-SafA chaperone-subunit complexes reveal the chaperone bound conformation of FGL chaperone-assembled polyadhesin subunits (Remaut et al., 2006; Zavialov et al., 2003, 2005). As pilins, the polyadhesin subunits Caf1 and SafA have an incomplete immunoglobulin-like fold (Figs 3 and 6). Despite the lack of significant sequence similarity, polyadhesin subunits and pilins display similar organization of the B, C, E, and F β-strands (Fig. 6a), which are known to form a common structural core of the immunoglobulin-like fold (Bork et al., 1994). However the β-strand A has different structures. In pilins, the β-strands A starts hydrogen bonding to the β-strand B, participating in formation of the ABED β-sheet, then it makes a switch in the middle and continues as part of the A’G1FC β- sheet (Fig. 6a). In polyadhesin subunits the A β-strand either switches very late (in Caf1) or becomes disordered (in SafA). A region between C and E β-strands shows a large structural variability for both pilus and polyadhesin subunits (Fig. 6a). Pilins tend to have larger loop between the β-strands D’’ and E. Polyadhesin subunits have considerably longer sequences which are involved in the region between the β-strands C and D’’. This region is clearly more Page 17 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review structurally variable in polyadhesin subunits than in pilins and potentially it might participate in formation of binding sites and organelle-specific epitopes (see the chapter “Binding of FGL chaperone-assembled polyadhesins to host cell receptors”). The major differences between the two classes of subunits and corresponding chaperones are found in the chaperone-subunit interactive area. Fig. 3 shows ribbon diagrams of the PapD–PapA (Verger et al., 2007), SafB–SafA (Remaut et al., 2006) and Caf1M–Caf1 (Zavialov et al., 2003, 2005) complexes. The end of the F1-G1 loop and the beginning of G1 β-strand in PapD harbour a four-residue subunit binding motif of one small hydrophilic (N101) and three alternating bulky hydrophobic residues (L107, I105 and L103) (Verger et al., 2007). The same region in SafB molecule harbours a similar five-residue motif of one small hydrophobic (A114) and four bulky hydrophobic residues (L116, L118, L120, and I122) (Remaut et al., 2006). The end of F1-G1 loop and the beginning of G1 β-strand in Caf1M harbour a subunit binding motif of five alternating bulky hydrophobic residues (V126, V128, V130, F132, and I134) (Zavialov et al., 2003, 2005). The rest of F1-G1 loop (residues 96-102 in PapD, 104-113 in SafB and 104-123 in Caf1M) is disordered in the crystal structures. Another subunit binding motif in FGL chaperones of three alternating hydrophobic residues (Y12 in Caf1M/F12 in SafB, V14, and I16) is localized in a long N-terminal sequence, which forms A1 strand. A1 and G1 β-strands are the edge strands of the β-sandwich fold of the N-terminal domain. In the complex A1 and G1 β-strands are extended due to the partial ordering of the N-terminal sequence and F1-G1 loop, respectively, to form a binding platform, exposing the hydrophobic residues of the binding motifs. In addition to this binding structure, PapD, Caf1M and SafB chaperones apply a pair of conserved positively charged residues (R8 and K112 in PapD, R20 and K127 in SafB, R20 and K139 in Caf1M) to bind subunits by anchoring their C-terminal carboxyl groups. Fig. 6a illustrates how Caf1 subunit is complemented by Caf1M chaperone (Zavialov et al., 2003, 2005). The absence of the 7th (G) strand results in a 6-stranded β-sandwich where the hydrophobic core of Caf1 is partially exposed in a long and deep hydrophobic groove. Caf1 Page 18 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review interacts mainly with N-terminal domain in Caf1M (Fig. 3). These two proteins bind via edge strands in Caf1 and in the N-terminal domain of Caf1M to form closed barrel with a common core (Zavialov et al., 2003). Strand G1 in Caf1M is hydrogen bonded to strand F in Caf1. Chaperone A1 strand is hydrogen bonded to subunit strand A. As in FGS chaperone–pilin complexes (Choudhury et al., 1999; Sauer et al., 1999, 2002; Verger et al., 2007), hydrophobic residues from Caf1M chaperone G1 strand are donated to Caf1 subunit to compensate for the missing G strand (Figs 3 and 6). The longer G1 donor strand of the Caf1M chaperone inserts motif of five bulky hydrophobic residues (P1 to P5 residues; see Figs 3 and 4) into five binding pockets in the hydrophobic groove of the Caf1 subunit (P1 to P5 binding pockets). As a result, the acceptor groove of Caf1 subunit is significantly longer than in the pilus subunits (Fig. 6a). The longer A1 strand in Caf1M also interacts more extensively with the subunit than the A1 strand in FGS chaperone–pilin complexes. The crystal structure of the SafB‒SafA complex also shows a considerably larger interactive area between the chaperone and subunit than that in the FGS chaperone-pilin complexes (Fig. 3). As in the Caf1M-Caf1 complex, this is a result from the presence of a more extended hydrophobic groove in the SafA subunit than in pilus subunits, which is complemented by subunit binding motifs of SafB containing the additional FGL specific sequences. However, the major F1-G1-loop-G1 β-strand binding motif of SafB contains four rather than five bulky hydrophobic residues (L116, L118, L120, and I122), interacting with the hydrophobic P4–P1 pockets of the subunit’s groove. The fifth donor residue inserting to the pocket P5 is a small A114. It was observed two crystal forms of the SafB–SafA complex that differ in the extent of ordering around A114 (Fig. 3) (Remaut et al., 2006). In type I crystals, A114 is ordered and is inserted into the P5 pocket of the SafA subunit (Fig. 3). In type II crystals this residue is disordered and does not insert into the P5 pocket (Fig. 3). As a result, the loops and secondary structure elements in the SafA subunit that form this P5 pocket are also disordered and are not observed in the electron density map. These two structures suggest equilibrium between the two Page 19 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review states of the SafB–SafA complex as a result of a weak binding of chaperone G1 donor strand at P5 site of the SafA binding groove (Remaut et al., 2006). 3D structure of fiber subunits. Elucidation of the crystal structure of Y. pestis F1 minimal fiber Caf1M–Caf1′–Caf1″ (ternary complex) made an important step in understanding general principles of subunit assembly via the chaperone/usher pathway, revealing the fiber conformation of the organelle subunit (Caf1″) and subunit-subunit interactions in fibers (Zavialov et al., 2003, 2005). The structure of Caf1M and the chaperone-bound Caf1′ subunit is virtually the same as in the Caf1M–Caf1 binary pre-assembling complex. However, in contrast to the disordered N-terminal region of Caf1 in binary complex, the N-terminal region of Caf1′ is ordered and forms an antiparallel donor β-strand interaction with the last (F) β-strand of the chaperone free Caf1″ subunit (Fig. 6b). The donated strand produces a bona fide immunoglobulin-like topology in the fibre subunit. The N-terminal donor strand was denoted as “Gd” (d for donor) because it plays in the fibre the same structural role as the (C-terminal) G strand of the canonical immunoglobulin fold (Zavialov et al., 2003). Thus, the release of the subunit from the chaperone-subunit complex and its incorporation into a growing fiber involves an exchange of G1 and A1 donor strands of the chaperone to Gd strand of the neighboring subunit in the fiber. The replacement of G1 strand by Gd strand also involves the change of direction of the donor strand from parallel to anti-parallel to F β-strand of the subunit. This process was predicted earlier for FGS chaperone-assembled adhesive pili (Choudhury et al., 1999; Sauer et al., 1999) and for FGL chaperone-assembled polyadhesins (Zavialov et al., 2002) and was termed “donor strand exchange”. A similar “topological transition” (Sauer et al., 2002) was also observed for the P pilus subunit PapE bound to a peptide designed to have sequence of the proposed donor strand of the PapK subunit, suggesting that the donor strand exchange takes place during assembly of both types of organelles. Recently, the structure of a ternary complex of PapD bound to PapA (through donor–strand complementation) was solved (Verger et al., 2007). The structure of this complex is shown in Page 20 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Fig. 6b. The structure provides a snapshot of PapA before and after donor strand exchange. PapD–PapA′–PapA″ complex is similar to the one obtained for the Caf system (Zavialov et al., 2003, 2005). The core sheet structure of donor strand-exchanged PapA is in a closer conformation than that of donor strand complemented PapA, as the β-strands on each side of the groove of donor strand exchanged PapA are nearer to each other. Also, the ‘‘63–74’’ loop is ordered in donor strand exchanged PapA and not in donor strand complemented PapA, as this molecule is missing residues 70 to 73 in this region. High resolution structures of several other subunits of fimbrial polyadhesins AfaE/DraE, DaaE and SafA have been determined (Anderson et al., 2004a, b; Korotkova et al., 2006b; Pettigrew et al., 2004; Remaut et al., 2006). Artificially engineered constructs were made to facilitate the structure determination in each of these studies. Structural information on the DaaE and AfaE/DraE subunits was obtained by structure determination of cytoplasm-assembled trimers of these subunits (Anderson et al., 2004a; Korotkova et al., 2006b; Pettigrew et al., 2004; Remaut et al., 2006). The crystal structures revealed that the trimers are connected together by β- strand swapping mechanism. Although non-native, the β-strand swapping is similar to the donor strand complementation. A different approach was chosen to determine an NMR structure of a circularly permuted self-complemented AfaE subunit (Anderson et al., 2004b). This construct contains the donor sequence fused at the C-terminus, rather than at N-terminus, which allows insertion of this self-complementing Gd strand in the acceptor groove, restoring classical immunoglobulin-like fold. To determine structure of the fiber form of the SafA subunit, the group of Gabriel Waksman (Remaut et al., 2006) employed the same technique, which they used earlier for structure determination of PapE (Sauer et al., 2002). SafA was co-crystallized with a peptide corresponding to the N-terminal sequence, which was predicted to form to the donor strand. Biological relevance of the constructs used by (Anderson et al., 2004b; Remaut et al., 2006) in their structural studies rely on the correctness of the prediction of the donor sequence, which is not easy to prove. Hence, these structures potentially may contain errors. The self- Page 21 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review complemented AfaE (Anderson et al., 2004b) has slightly distorted structure at the beginning of the donor strand, which makes it dissimilar to Caf1 and SafA subunits (authors' observation). Comparison with the crystal structure of the cytoplasm assembled trimers of the same subunit suggests that this could be a result of changes introduced by the artificially engineered linker connecting the donor strand to the C-terminus of the subunit. Nevertheless, all these structures show a similar incomplete immunoglobulin-like fold for the subunits and suggest that subunit- subunit interactions in polyadhesins fibers involve N-terminal Gd donor-strand complementation. The Caf1 polyadhesin subunit (Zavialov et al., 2003, 2005) has a longer acceptor groove, which accommodates longer Gd donor strand than the P pilus subunit PapA (Verger et al., 2007) (Fig. 6b). This is in agreement with the observation of a more extended contact area between FGL chaperone and polyadhesin subunit in structures of Caf1M–Caf1 and SafB-SafA complexes than that between FGS chaperone and pilus subunit in structures of FimC–FimH, PapD–PapK, PapD–PapE and PapD–PapA complexes (Choudhury et al., 1999; Sauer et al., 1999, 2002; Verger et al., 2007; Zavialov et al., 2003, 2005). Chaperone preserves folding energy of subunit for driving fiber assembly. No energy input from external sources is required to convert periplasmic chaperone–subunit pre-assembly complexes to free chaperone and secreted fibers (Jacob-Dubuisson et al., 1994), in spite of a much more extensive interface between chaperone and subunit than between fiber subunits (Zavialov et al., 2003). Some clues as to how the process can be energetically driven have been provided by structural studies (Sauer et al., 2002; Verger et al., 2007; Zavialov et al., 2003, 2005). Comparison of chaperone complemented (Caf1′) with fiber subunit (Caf1″) revealed a large conformational difference (Zavialov et al., 2003, 2005). The fiber conformation was referred to as the “closed” or “condensed” conformation (Zavialov et al., 2005). The observed difference between open and closed conformations, involving a rearrangement and condensation of the subunit hydrophobic core, suggested that periplasmic chaperones might trap subunits in a Page 22 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review high-energy molten globule-like folding-intermediate state (Zavialov et al., 2003). A model was proposed in which release of the subunit followed by Gd donor strand complementation allows folding to be completed, driving fiber formation (Zavialov et al., 2003). In contrast to the bulky hydrophobic donor residues in the chaperone G1 donor strand, much smaller donor residues in the subunit N-terminal Gd donor segment do not intercalate between the two sheets of the subunit β-sandwich, allowing close contact between the two sheets (Zavialov et al., 2003, 2005). A significant stabilizing contribution from the final fine packing of the subunit hydrophobic core is suggested by the melting of the native ternary complex. The structurally observed complete collapse of the Gd complemented fiber Caf1'' subunit results in a dramatic increase in the enthalpy and transition temperature for the melting of the fiber module. The thermodynamic studies provide strong evidence for the hypothesis that collapse of the subunit hydrophobic core shifts the equilibrium towards fiber formation (Zavialov et al., 2005). Zip-in-zip-out mechanism of the donor strand exchange. Zavialov et al. (2003) proposed a model for the usher-catalyzed fiber assembly involving a sequential concerted donor strand exchange in which G1 is gradually replaced by Gd with a zip-in-zip-out mechanism. Remaut et al. (2006) using real-time electrospray ionization mass spectrometry detected a transient ternary complex between the chaperone-subunit complex and a peptide mimicking the donor strand of the subunit, providing an experimental support to the hypothesis. Exchange of the donor strand of the chaperone to the peptide was highly dependent on the interactions at the P5 pocket of the subunit (Fig. 7). This site may recruit the incoming subunit donor strand. Indeed, the observation of two crystal forms with SafB−SafA complex (Fig. 3) (Remaut et al., 2006) suggests that the acceptor cleft of SafA subunit could be easily uncapped at this site, providing a starting point for the donor strand exchange. Recently, Verger et al. (2008) solved the structure of the PapD: PapF complex in order to understand why PapF undergoes slow donor strand exchange. The structure reveals that the PapF P5 pocket is partially obstructed. Molecular dynamics simulations show that this region of PapF Page 23 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review is flexible compared with its equivalent in PapH, a subunit that also has an obstructed P5 pocket and is unable to undergo donor strand exchange. Using electro spray-ionization mass spectrometry, Verger et al. (2008) showed that mutations in the P5 region result in increased donor strand exchange rates. Thus, the partial obstruction of the P5 pocket serves as a modulating mechanism of donor strand exchange. Rose et al. (2008) used molecular dynamics simulations to probe the donor strand exchange mechanism during formation of the Saf pilus from S. enterica at the atomic level, allowing the direct investigation of the zip-in–zip-out hypothesis. The simulations provide an explanation of how the incoming Gd is able to dock and initiate donor strand exchange due to inherent dynamic fluctuations within the chaperone–subunit complex. In the simulations, the chaperone donor strand was seen to unbind from the pilus subunit, residue by residue, in direct support of the zip- in–zip-out hypothesis. In addition, an interaction of a residue towards the N-terminus of the Gd with a specific binding pocket on the adjacent pilus subunit was seen to stabilise the donor strand exchange product against unbinding, which also proceeded in the simulations by a zippering mechanism. The study provides an in-depth picture of donor strand exchange, including the first atomistic insights into the molecular events occurring during the zip-in–zip-out mechanism. Fig. 7 schematically presents the donor strand exchange mechanism in vivo based on the experimental data obtained in vitro by Remaut et al. (2006). Zavialov et al. (2005) and Vitagliano et al. (2008) reported molecular dynamics characterizations of Y. pestis Caf1 subunit in its monomeric-unbound and dimeric states. Data on properties of the monomeric form show that it is highly reactive and tends to evolve toward compact states, which likely hamper subunit–subunit association. The chaperone release and subunit–subunit association evidently take place concerted. Molecular architecture of adhesive organelles. The final architecture and morphology of linear fibres depend on subunit composition and mode of subunit–subunit interactions. These factors determine the coiling of secreted linear fibres into different structures such as FGS Page 24 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review chaperone-assembled thick rigid mono-adhesive pili with a diameter of 7-8 nm (Fig. 8a), reviewed in Knight et al. (2000); Sauer et al. (2000, 2004); Soto & Hultgren (1999) and Thanassi et al. (1998), FGS chaperone-assembled thin flexible hetero-polyadhesins with a diameter 2-4 nm (Fig. 8b), reviewed in van den Broeck et al. (2000), FGS chaperone-assembled homo- polyadhesins with a diameter about 2 nm (Fig. 8c) (Chessa et al., 2008) and FGL chaperone- assembled polyadhesins with a diameter about 2 nm (Fig. 8d) (Zavialov et al., 2007). The latter polyadhesins can aggregate to form amorphous masses or capsules, e.g. the F1 capsular antigen (Chen & Elberg, 1977), nonfimbrial adhesin I (NFA-I) (Ahrens et al., 1993), NFA-I-like, Dr-II (Pham et al., 1997), or afimbrial adhesins, III, VII, and VIII (Jouve et al., 1997; Lalioui et al., 1999). In the case of FGS chaperone-assembled mono-adhesive fimbriae/pili, the specialized adhesive subunit always occurs at the tip of fimbriae, either as the distal end of thin (~2.5 nm) and flexible fimbriae (e.g. F17G from F17 fimbriae), or at the edge of a thin (~2.5 nm) tip fibrillum that is stuck onto a relatively rigid, 1–2 mm long and ~7.5 nm wide right-handed helical pilus rod (e.g. PapG of P pili and FimH of type 1 pili) (Fig. 8a) (de Greve et al., 2007). This specialized subunit is called as adhesin. All adhesive subunits of mono-adhesive fimbriae/pili are two-domain adhesins (Choudhury et al., 1999; Bouckaert et al., 2005, 2006; de Greve et al., 2007; Li et al., 2007; Westerlund- Wikström & Korhonen, 2005). A two-domain adhesin consists of N-terminal receptor-binding domain that can be stably expressed on its own, and a rather conserved C-terminal pilin domain. Both domains have an immunoglobulin-like fold and are joined via a short interdomain linker. The few known crystal structures of tip-located receptor-binding N-terminal adhesin domains of mono-adhesive fimbriae/pili, PapGII, FimH, F17G/GafD, show that, despite little or no sequence identity, common to them all is an elongated beta-barrel jelly roll fold that contains the receptor- binding groove (Fig. 9) (Choudhury et al., 1999; Bouckaert et al., 2005, 2006; de Greve et al., 2007; Li et al., 2007; Westerlund-Wikström & Korhonen, 2005). The adhesin domains differ in Page 25 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review disulfide patterns, in size and location of the ligand-binding groove, as well as in mechanism of receptor binding. In particular, their glycan-binding sites have evolved in different locations onto this similar scaffold, and with distinct, highly specific binding properties. Subunits of mono-adhesive fimbriae are called as pilins. In particular, P fimbriae are composed of ~1000 copies of the major subunit protein, PapA, which polymerize to form a rigid stalk connected to a flexible tip consisting of limited copies of the minor subunit proteins, PapE and PapF, and receptor-binding adhesin, PapG, at the distal end (Kuehn et al., 1992; Lindberg et al., 1987). Type 1 pili is composed of up to 3000 copies of the subunit FimA, which form a stiff, helical pilus rod, and subunits FimF, FimG, and FimH, which form the linear tip fibrillum. All subunits in the pilus interact via the donor strand complementation, in which the incomplete immunoglobulin-like fold of each subunit is complemented by insertion of an N-terminal extension from the following subunit. Gossert et al. (2007) determined the NMR structure of a monomeric, self-complemented variant of FimF, FimFF, which has a second FimF donor strand segment fused to its C-terminus that enables intramolecular complementation of the FimF fold. NMR studies on bimolecular complexes between FimFF and donor strand-depleted variants of FimF and FimG support for the intrinsic flexibility of the tip fibrillum and that this flexibility would significantly increase the probability that the adhesin at the distal end of the fibrillum successfully targets host cell receptors. To understand whether the mechanical properties of the fimbrial rod regulate the stability of the FimH–mannose bond, Forero et al. (2006) pulled the fimbriae via a mannosylated tip of an atomic force microscope. Individual fimbriae rapidly elongate for up to 10 µm at forces above 60 pN and rapidly contract again at forces below 25 pN. At intermediate forces, fimbriae change length more slowly, and discrete 5.0±0.3-nm changes in length can be observed, consistent with uncoiling and coiling of the helical quaternary structure of one FimA subunit at a time. The force range at which fimbriae are relatively stable in length is the same as the optimal force range at which FimH–mannose bonds live longest. Higher or lower forces, which cause shorter bond lifetimes, cause rapid length changes in the fimbria that help Page 26 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review maintain force at the optimal range for sustaining the FimH–mannose interaction. The modulation of force and the rate at which it is transmitted from the bacterial cell to the adhesive catch bond present a novel physiological role for the fimbrial rod in bacterial host cell adhesion. This suggests that the mechanical properties of the fimbrial shaft have co-developed to optimize the stability of the terminal adhesion under flow. In the case of FGL chaperone-assembled polyadhesins, all subunits may possess two independent binding sites specific to different host cell receptors (Fig. 9; Anderson et al., 2004a,b; Korotkova et al., 2006a,b, 2008; Pettigrew et al., 2004). Dimensions of the bacterial poly-adhesive fibres Dr, assembling of which is assisted with FGL chaperone, were investigated with negative-stain electron microscopy (Anderson et al., 2004a). Thin flexible fibers (2 nm diameter) were observed. The results are entirely consistent with the model with end-to-end contact between each subunit (Fig. 9) (Anderson et al., 2004a) and are reminiscent of the model of capsular F1 antigen from Y. pestis, Caf1 (Zavialov et al., 2003). Similar thin fibres have been observed for pH6 antigen (Lindler & Tall, 1993). In addition to the predominance of thin fibers Dr, the electron microscopy also revealed thicker morphology with overall dimensions larger than the linear model suggested (Anderson et al., 2004a). Thick fibers are not consistent with end-to-end contact and imply that more extensive intersubunit interactions also exist. This would rigidify the resulting rod by the tighter coiling of a single fiber or formation of a trimeric coiled- coil arrangement of fibers. Recently Runco et al. (2008) examined the ultrastructure of the Y. pestis capsule with whole bacteria and negative-stain transmission electron microscopy. Bacteria were grown to logarithmic phase at 37ºC, pH 7.4. The appearance of the capsule was more clearly visible than reported in previous studies, in which the capsule generally appeared as an amorphous haze or dense mass surrounding the bacteria (Chen & Elberg. 1977; Du et al., 2002; Liu et al., 2006). The Y. pestis KIM6+ strain consistently produced an extended halo composed of thin fibrils and denser aggregates. This denser capsular material, likely composed of aggregates of the thin Page 27 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review fibrils, sometimes extended out from the bacterial surface in long strands. The thin, fibrillar appearance of the F1 capsule resembles structures previously reported for other members of the FGL family of chaperone/usher pathways, including the pH6 antigen of Yersinia (Iriarte et al., 1993; Lindler & Tall, 1993), and the CS3 and CS6 pili of enterotoxigenic E. coli (Knutton et al., 1989; Levine et al., 1984). This supports a common structure and assembly mechanism for members of the FGL family. Salih et al. (2008) used negative-stain electron microscopy and single-particle image analysis to determine the three-dimensional structure of the S. typhimurium Saf polyadhesin. Saf polyadhesin comprises of highly flexible linear multi-subunit fibers that are formed by globular subunits connected to each other by short links giving a “beads on a string”-like appearance. Quantitative fitting of the atomic structure of the SafA polyadhesin subunit into the electron density maps, in combination with linker modelling and energy minimization, has enabled analysis of subunit arrangement and intersubunit interactions in the Saf polyadhesin. Short intersubunit linker regions provide means for flexibility of the Saf polyadhesin by acting as molecular hinges allowing a large range of movement between consecutive subunits in the fibre. Structure of Outer Membrane Molecular Usher Proteins 3D structure of outer membrane molecular usher protein. Recently, the 3.4 Å crystal structure of the 130-640 amino acid fragment of the PapC usher was solved. The results help to understand adhesive fiber biogenesis at the outer membrane (Fronzes et al., 2008; Remaut et al., 2008). It encompasses the full translocation pore consisting of a kidney-shaped, 24-stranded β- barrel (residues 146–635), 45 Ǻ in height and with outer and inner dimensions of 65 - 45 Ǻ and 45 - 25 Ǻ, respectively (Figs 10a and b). The β-barrel closes in an end-to-end fashion and positions the N- and C-termini on the periplasmic side of the outer membrane. The N- and C- terminal globular domains will thus be juxtaposed and reside in the periplasm, consistent with Page 28 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review their role in chaperone-subunit recruitment and adhesin-induced pore activation (Ng et al., 2004; Nishiyama et al., 2003; Saulino et al., 1998; Thanassi et al., 2002). The predicted middle domain (residues 257–332) (Capitani et al. 2006) is formed by a long sequence between strands β6 and β7 and consists of a six-stranded, β sandwich fold (strands βA–βF). The domain is positioned laterally inside the β-barrel pore (Figs 10a and b). As a result, the middle domain, referred to as the plug domain, completely occludes the luminal volume of the translocation pore, preventing passage of solutes or periplasmic proteins across the channel in its non-activated form. The PapC plug domain is inserted into the loop connecting two β strands (strands 6 and 7). The plug domain is held in place by a β-hairpin (strands β5 and β6, hereafter referred to as the β5-6 hairpin) that is folded in from the barrel wall into the channel lumen. The inward curvature of the β5-6 hairpin creates a large gap in the side of the β-barrel extending well into the part submerged in the outer membrane bilayer (Figs 10a and b). The luminal part of the β5-6 hairpin is capped from the extracellular side by the only helix in the structure, the α1 helix (residues 448–465). Inside the β-barrel, the β5-6 hairpin interacts with the inner surface of the channel and helix α1 through a patch of hydrophobic interactions. In addition, the β5-6 hairpin forms two electrostatic interaction networks that bridge the plug domain with the channel wall and α1 helix and help position the plug domain laterally inside the translocation channel (Figs 10a and b). Mechanism of channel gating. In its non-activated form, the PapC channel is obstructed by the plug domain (Figs 2 and 10). Therefore, the adhesive subunit-induced activation of the usher (Saulino et al., 1998) must include the displacement of the plug domain from the translocation channel. A rotation of the plug domain out of the pore lumen and into the periplasm would create a channel of 37Ǻ×25Ǻ or 45Ǻ×25Ǻ when the β5-6 hairpin and α1 helix are displaced from the channel as well (Fig. 2, position P'). Alternatively, a conformational change in the β5-6 hairpin and α1 helix could allow an upward rotation of the plug domain inside the pore, thereby liberating a translocation channel of approximately 27 Ǻ×25Ǻ (Fig. 2, position P"). Outer membrane ushers function independently of a hydrolyzable energy source or a proton gradient Page 29 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (Jacob-Dubuisson et al., 1994). Gating of the plug domain in the usher therefore relies solely on conformational changes induced by the binding of the chaperone-adhesin complex. Powering the large conformational rotation of the plug domain must therefore come from the energy emanating from the binding of the chaperone-adhesive subunit complex and/or must be stored as structural strains in the non-activated PapC channel. One such area of apparent structural strain is seen in the β5-6 hairpin that breaks out of the β-barrel lining. The exposed part of the adjacent strand β4 represents a large open-edged β sheet structure. Such exposed edges form highly aggregative surfaces for the edge-to-edge docking of β strands or β sheets (Richardson & Richardson, 2002). As part of the activation process, the β5-6 hairpin could line up with β4 into the barrel wall. This would break the interaction with the plug domain and allow its upward rotation or displacement out of the barrel lumen. Another area of structural strain could reside in the interaction of the plug domain with the barrel lumen, which includes a number of like-charge residues. It seems plausible that during activation, these repulsive forces will aid in expelling the plug domain from the barrel lumen. Comparison of isolated Caf1A plug domain with the structure of the corresponding domain of PapC usher. Yu et al. (2009) reported isolation and structural-functional characterization of the plug domain of Caf1A usher from Y. pestis. The isolated Caf1A plug domain is a highly soluble monomeric protein capable of autonomous folding. A 2.8 Å resolution crystal structure of the Caf1A plug domain reveals that this domain has an immunoglobulin-like fold similar to that of donor strand complemented Caf1 fibre subunit. Moreover these proteins display significant structural similarity. Although the Caf1A plug domain is in the middle of the predicted amphipathic β-barrel of Caf1A, the usher is still assembled in the membrane in the absence of this domain. The Caf1A plug domain does not bind Caf1M-Caf1 complexes, but its presence shows to be essential for Caf1-fibre secretion. The study suggests that Caf1A plug domain may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Page 30 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Comparison of isolated Caf1A plug domain with the structure of the corresponding domain of PapC usher (Fig. 10c) shows a high similarity of the core structures suggesting a universal adaptation of FGL and FGS chaperone/usher pathways for the secretion of different types of fibres. Mechanism of Function of Chaperone/Usher Machinery N-terminal periplasmic domain of the usher protein binds chaperone-subunit complex. Nishiyama et al. (2003) identified N-terminal periplasmic domain of FimD usher protein (FimDN) comprising the N-terminal 139 residues of mature FimD. Purified FimDN usher domain is a monomeric, soluble protein that specifically recognizes complexes between FimC chaperone and individual Type 1 pilus subunits, but does not bind the isolated FimC chaperone, or isolated subunits. In addition, FimDN usher domain retains the ability of FimD usher protein to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the chaperone FimC–FimH adhesion complex. Overexpression of FimDN usher domain in the periplasm of wild-type E. coli cells diminished incorporation of FimH adhesion at the tip of Type 1 pili, while pilus assembly itself was not affected. Nishiyama et al. (2003) reported nuclear magnetic resonance and X-ray protein structures of the FimDN usher domain before and after binding of a chaperone FimC–FimHP pilin domain complex. FimDN usher domain consists of a flexible N-terminal segment of 24 residues (N-terminal "tail"), a structured core with a novel fold, and a C-terminal hinge segment (Fig. 11). In the ternary complex, residues 1–24 of FimDN usher domain specifically interact with both FimC chaperone and the FimHP pilin domain. The structures of FimC chaperone and FimHP pilin domain in the ternary complex are closely similar to those in the previously published chaperone FimC–FimH adhesion binary complex (Choudhury et al., 1999). The residues 1–24 of the N-terminal tail, which are completely unstructured in free FimDN usher domain, become ordered upon complex formation and Page 31 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review specifically interact with both FimC chaperone and the bound FimHP pilin domain (Fig. 11; Nishiyama et al., 2005). The interactions formed by the N-terminal FimDN usher domain tail comprise 60% of the total interface area of 1260Å2 between FimDN (1–125) usher domain and the chaperone FimC–FimHP pilin domain complex. The other 40% of the contact area is contributed by the folded core FimDN (25–125) usher domain, which exhibits a complementary surface to FimC chaperone. The N-terminal tail 1–24 of FimDN usher domain thus serves as a sensor that selectively detects loaded FimC chaperone molecules. As the tail is the only FimDN usher domain region that forms contacts with the chaperone-bound subunit, it may be exclusively responsible for the discrimination of the different chaperone FimC–subunit complexes by the assembly platform (Saulino et al., 1998). FimD usher protein binds to different chaperone FimC–subunit complexes with different affinities, which is a key element for correct initiation of pilus assembly and for the correct ordering of the subunit incorporation into the pilus (Saulino et al., 1998; Nishiyama et al., 2003). In the case of the chaperone FimC–FimH adhesion complex, which is bound by FimD usher protein with highest affinity (Saulino et al., 1998), additional contacts between the FimH lectin domain and other FimD usher regions could contribute to binding, as FimD usher protein has been shown to recognize the isolated FimH lectin domain (Barnhart et al., 2003), which is not bound by FimDN usher domain (Nishiyama et al., 2003). The X-ray structure of the FimDN (1–125) usher domain–FimC chaperone–FimHP pilin domain complex thus predicts that the common element of the interactions of FimDN usher domain with the four different chaperone FimC–subunit complexes is the contact area between the N-terminal FimC chaperone domain and the structured FimDN (25–125) usher domain. Nishiyama et al. (2005) assumed that this contact area alone is, however, neither sufficient for binding of chaperone FimC–subunit complexes to FimDN usher domain, nor for stable binding of the free chaperone to the assembly platform (Saulino et al., 1998; Nishiyama et al., 2003). The fact that FimC chaperone alone is not bound by FimD usher ensures that FimC chaperone is Page 32 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review released to the periplasm for another reaction cycle as soon as the bound subunit dissociates from the ternary complex and is delivered to the translocation pore. Twinned-pore model of the translocation machinery functioning. The Type 1 pilus tip complex was analyzed by cryo-electrone microscopy (Fronzes et al., 2008; Remaut et al., 2008). The available structural and biochemical data on the purified Type 1 tip complex and PapC firmly establish the outer membrane ushers function as dimers organized in twinned pores that is the functional unit for chaperone/usher pilus biogenesis (Li et al., 2004; So & Thanassi, 2006). The dimer interface in the 3D crystals is formed by the flat side of the kidney-shaped β-barrel, encompassing strands β11–β20 (residues 397–573; Fig. 10). The 3D cryo-electron microscopy reconstruction reveals translocation of the polymerized subunits occurs asymmetrically and through only a single pore (usher 1; Fig. 2). The usher mediates the translocation of folded, polymerized protein units across the outer membrane (Sauer et al., 2002; Remaut et al., 2006; Vetsch et al., 2006). The width of the PapC translocation pore (inner diameter of 45Ǻ×25Ǻ) corresponds well with the passage of P pilus subunits in an upright orientation, along the length of the polymer (P pilus subunit dimensions are 55Ǻ×30Ǻ×25Ǻ, 57Ǻ×32Ǻ×23Ǻ and 60Ǻ×27Ǻ×22Ǻ for PapE (Protein Data Bank accession number 1N12), PapK (Protein Data Bank accession number 1PDK), and PapA (Protein Data Bank accession number 2UY6), respectively (Sauer et al., 1999, 2002; Verger et al., 2007). Integrated model for fiber assembly at the outer membrane molecular usher protein. Based on the cryo-electron microscopy data, Remaut et al. (2008) proposed a mechanism where the two ushers in the twin pores cooperate in pilus polymerization by alternately recruiting new chaperone-subunit complexes through their N-terminal domains (Fig. 2). This mechanism provides a rationale for the known requirement of a dimeric usher complex: one usher provides the secretion channel, but two ushers are needed for successive rounds of subunit binding and fiber assembly. The structural information presented and the available biochemical background can be combined into an integrated model for subunit recruitment, subunit polymerization, and Page 33 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review fiber translocation during pilus biogenesis at the outer membrane usher (Fig. 2). The structure of the Fim(D×2):C:F:G:H tip complex captures the fiber assembly process after FimH, FimG, and FimF have assembled into a pilus tip. In this complex, the last incorporated chaperone-subunit complex (FimC:F) is bound to the N-terminal domain of usher pore 1. In the model, the next subunit to be incorporated into the fiber is recruited to the twinned pores through the N-terminal domain of usher 2 (step 1; Fig. 2). The N-terminal domain of the usher resides in the periplasm, tethered by a 20-residue flexible linker to the translocation pore. This spacer between the usher translocation pore and its N-terminal domain allows reorientation of the chaperone-subunit complex to a position where the N-terminal donor strand Gd of the newly recruited subunit is in proximity with the pilin domain of the previously recruited chaperone-subunit complex (bound to usher 1). This strand exchange reaction results in the release of the chaperone from the previously recruited chaperone-subunit complex and its dissociation from the N-terminal domain of usher 1 (the usher lacks any detectable affinity for the chaperone when not in complex with the adhesin or a pilus subunit [Dodson et al., 1993; Nishiyama et al., 2003; Saulino et al., 1998]) (step 3; Fig. 2). Upon release of the chaperone, the subunit can now enter the translocating pore of usher 1 and the N-terminal domain of usher 1 is free to bind a new chaperone-subunit complex from the periplasmic pool and bring it within proximity of the previously recruited chaperone-subunit complex for donor strand exchange (steps 4 and 5; Fig. 2). In this way, iterations of the alternating recruitments of new chaperone-subunit complexes at either usher’s N-terminal domain, followed by donor strand exchange with the previously assembled subunit, allow the stepwise polymerization and translocation of the pilus fiber (steps 5 and 6; Fig. 2). The model for chaperone/usher pilus assembly presented above incorporates all available biochemical data into the new structural framework. It does, however, lack the usher C-terminal domain. Earlier studies in the P pilus system showed that the C-terminal domain of PapC is involved in the activation of the usher (So and Thanassi, 2006). From studies in type 1 pili, it is known that FimH recruitment triggers a conformational change in the usher required for its Page 34 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review activation (Saulino et al., 1998). This activation step depends on the presence of the FimH adhesin domain (Munera et al., 2007). Remaut et al. (2008) proposed that, in its inactive state, the C-terminal domain (not shown in Fig. 2) is positioned at least partially under the PapC or FimD channel and is in contact with the β5-6 hairpin and plug domain. This is consistent with cryo-electron microscopy data showing that in the absence of the C-terminal domain, the electron density within the channel is weaker (Li et al., 2004). When the chaperone-adhesive subunit complex is recruited to the usher via the usher’s N-terminal domain, a putative additional interaction between the adhesive subunit and the usher’s C-terminal domain may relay a conformational change to the plug domain and the β5-6 hairpin and result in opening of the channel. Such an interaction has been observed between the PapC C-terminal domain and the PapD:G chaperone-adhesive subunit complex (So & Thanassi, 2006). As the FimD:tip complex captures a later stage of pilus biogenesis, a stage at which the twinned pores are already activated and the C-terminal domains may no longer be involved, it is not surprising that the C-terminal domains should not be seen. The C-terminal domains may lay idle, possibly tethered to a long linker (as for the N-terminal domain). However, further functional and structural work is needed to elucidate the role of the usher C-terminal domain and the mechanism of channel gating. FUNCTIONS OF ADHESIVE ORGANELLES ASSEMBLED WITH CHAPERONE/USHER MACHINERY Binding of Bacterial Adhesive Organelles to Host Cell Receptors and Serum Proteins Binding of polyadhesins to host cell receptors and serum proteins. Afa/Dr polyadhesins. Dr, F1845 (DaaE), NFA-I, and AfaE-III adhesins allow binding to the Dra blood-group antigen presented on the CD55/decay-accelerating factor (DAF), a complement regulatory and signaling molecule (Nowicki et al., 1988). Under physiological conditions, CD55/DAF plays a central role Page 35 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review in preventing the amplification of the complement cascade on host cell surfaces (Fujita et al., 1987; Lublin & Atkinson, 1989). CD55/DAF interacts directly with membrane-bound C3b or C4b and prevents the subsequent uptake of C2 and factor B. Human CD55/DAF is a cell-associated protein with Mr of 55,000 to 70,000, depending on its glycosylation level. Membrane-bound CD55/DAF is attached to the cell surface membrane by a glycosylphosphatidylinositol anchor (Caras et al., 1987; Davitz et al., 1986), attached to a serine- threonine-proline-rich region followed by four repeating domains (Carroll et al., 1988; Rey- Campos et al., 1988). They are currently named complement control protein repeat (CCP) domains, originally known as short consensus repeats (Le Bouguénec & Servin, 2006; Servin, 2005). Removal of CCP-1 has no effect on CD55/DAF function, but individual deletion of CCP- 2 and CCP-3 or CCP-4 totally abolished it (Brodbeck et al., 1996; Coyne et al., 1992). Afa/Dr adhesins recognize CCP-3 on CD55/DAF (Nowicki et al., 1993). Indeed, a single point substitution in CCP-3 (Ser155 to Leu) causes complete abolition of adhesin binding to CD55/DAF (Nowicki et al., 1993). Dr adhesin-binding and complement-regulating epitopes of CD55/DAF appear to be distinct and are approximately 20Å apart (Hasan et al., 2002). The amino acids Gly159, Tyr160, and Leu162 also aid in binding adhesin Dr, while residues Phe123 and Phe148 at the interface of CCP-2 and CCP-3, and also Phe154 in CCP-3 cavity, are important in complement regulation. An atomic resolution model for functions of AfaE-III adhesin reveals the pivotal role of CCP- 2 and CCP-3 in binding of adhesins onto CD55/DAF (Anderson et al., 2004a). Simultaneously the residues of AfaE-III adhesin involved in CD55/DAF binding were localized (Fig. 9; Anderson et al., 2004a). Like DraE, AfaE-III binds to CCP-2 and CCP-3, but CCP-3 contributes most to the free energy of binding. The binding regions for AfaE-III and the complement pathway convertases lie in close proximity to each others on CD55/DAF. Binding of adhesin Dr to CD55/DAF is inhibited by chloramphenicol, whereas binding of AfaE-III is unaffected (Nowicki et al., 1988; Westerlund et al., 1989). This was used for Page 36 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review localization of DraE adhesive site. 3-D structure of strand-swapped trimer of wild-type DraE in complex with chloramphenicol was solved. NMR data supported the binding position of chloramphenicol within the crystal (Anderson et al., 2004b; Pettigrew et al., 2004). Chloramphenicol binds to a surface pocket between the N-terminal portion of strand B and C- terminal portion of strand E and lies within the recently identified CD55/DAF-binding site (Fig. 9; Anderson et al., 2004a). Recently Pettigrew et al. (2009) reported X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N- acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent haemagglutinin Dr inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM. Korotkova et al. (2006b) solved the 3D structure of DaaE at resolution 1.48 Å. Trimers of the protein were found in the crystal, as has been the case for other adhesins Dr. Naturally occurring variants and directed mutations in DaaE have been generated and analyzed for their ability to bind CD55/DAF. Mapping of the mutation sites onto the DaaE molecular structure shows that several of them contribute to a contiguous surface that is likely the primary CD55/DAF binding site (Fig. 9). Dr, F1845 (DaaE), and AfaE-III adhesins interacts also with carcinoembryonic antigen (CEA)-related cellular adhesion molecules, CEACAM1, CEACAM5, and CEACAM6 (Berger et al., 2004). This recognition is followed by activation of CEACAMS-associated signaling by pathogens triggering the cellular events. CEACAM1, CEACAM5, and CEACAM6 belong to the immunoglobulin superfamily of adhesion molecules (Grunert & Kuroki, 1998; Hammarstrom, 1999; Öbrink, 1997; Thompson et al., 1991). They share a conserved N-terminal immunoglobulin variable-like domain that is followed by 3, 6 and 2 immunoglobulin constant- like domains, respectively. CEACAM1 is inserted into the cellular membrane via C-terminal Page 37 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review transmembrane and cytoplasmic domains, whereas CEACAM5 and CEACAM6 have a glycosylphosphatidylinositol anchor. CEACAMS family generally functions as intercellular adhesion molecules (Benchimol et al., 1989), and could play a role in innate immunity (Fahlgren et al., 2003). CEACAM1 has been shown to be expressed in leukocytes, including granulocytes, activated T cells, B cells, and natural killer cells (Grunert & Kuroki, 1998). CEACAM1 acts as a novel class of immunoreceptor tyrosine-based inhibition motif-bearing regulatory molecules on T cells that are active during the early phases of the immune response in mice (Benchimol et al., 1989; Fahlgren et al., 2003; Kammerer et al., 1998, 2001; Nakajima et al., 2002). The intracytoplasmic domain, which contains two immunoreceptor tyrosine-based inhibition motif- like domains, is required for activation of a fraction of T cells in Lamina propria that express CEACAM1 by IL-7 and IL-15, indicating that CEACAM1 amplifies T-cell activation and thus could facilitate cross talk between epithelial cells and T lymphocytes in the intestinal immune response (Donda et al., 2002). The particular role of CEACAM1 in Neisseria pathogenicity has been documented. N. gonorrhoeae evades host immunity by switching off T lymphocytes (Bradbury, 2002). In N. gonorrhoeae, the Opa52 protein is able to bind CEACAM1 expressed by primary CD4+ T lymphocytes and to suppress their activation and proliferation after the Opa gonococcal protein associates with the tyrosine phosphatases SHP-1 and SHP-2 in the ITIM of CEACAM1 (Boulton & Gray-Owen, 2002; Chen et al., 2001). Rougeaux et al. (2008) found that, as Opa, the adhesin Dr induces the Tyr-phosphorylation of ITIM and ITSM and the recruitment of Shp-2. The recent review by Nouvion & Beauchemin (2009) summarized multiple functions of CEACAM1. It was shown that this multifunctional protein plays a role in intercellular adhesion, as an inhibitor of tumor development, as a bacterial adhesin, and as a receptor for the mouse hepatitis virus. Moreover, CEACAM1 is an active regulator of cell signaling, modulating the insulin or EGF receptor pathways in epithelial cells or the Zap-70 pathway in hematopoietic cells. The recent development of genetically modified mouse models altering the Ceacam1 gene corroborates most of these data, but also highlights CEACAM1's Page 38 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review functional complexity. Thus, in addition to the functions identified previously, CEACAM1 is an important regulator of lipid metabolism, of tumor progression as a regulator of the Wnt signaling pathway, of normal and tumor neo-angiogenesis and of immunity (Nouvion & Beauchemin, 2009). Random mutagenesis with functional analysis and chemical shift mapping by NMR show a clear-cut CEACAMS binding site located primarily in the A, B, E and D strands of adhesin Dr subunit (Fig. 9; Korotkova et al., 2006a). This site is located opposite to the β-sheet encompassing the previously determined binding-site for CD55/DAF, which implies that the polyadhesin Dr can bind simultaneously to both receptors on the epithelial cell surface. Recently, the structure of the CEA/Dr adhesin complex was proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization (Korotkova et al., 2008). The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that adhesins Dr can dissociate CEA dimers prior to the binding of monomeric forms (Korotkova et al., 2008). Hemagglutinin Dr is unique in Afa/Dr adhesin family since it binds specifically to the 7S domain (tetramer) of the basement membrane protein type IV collagen that is inhibited by the presence of chloramphenicol (Nowicki et al., 1988; Westerlund et al., 1989; Westerlund & Korhonen, 1993). Site-directed mutagenesis has been used to show that a negatively charged amino acid is required at position 54 of adhesive subunit Dr to confer chloramphenicol sensitivity of binding and that mutations at positions 32, 40, 54, 90, and 113 have different effects on type IV collagen binding and the chloramphenicol sensitivity of binding (Carnoy & Moseley, 1997). In particular, replacement of a single amino acid at position 113 of the DraE subunit results in loss of type IV collagen binding. Moreover, the two conserved Cys of the Afa/Dr family structural subunits form a disulfide bond, and mutations of these residues abolish both hemagglutination and binding to type IV collagen. Together with fibronectin, laminin, Page 39 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review tenascin, and heparin sulfate proteoglycans, type IV collagen is a component of the basement membrane, which is involved in complex interactions at the epithelial-mesenchymal interface. In particular, type IV collagen interacts with integrins expressed at the basal domain of polarized cells (Beaulieu, 1999), to form a link between the basement membrane and epithelial cells (Louvard et al., 1992). However, during inflammation, deregulated expression of membrane- bound molecules that are normally segregated in the basolateral domain of polarized intestinal cells occurs, and it is possible that in this context type IV collagen binding may contribute to the pathogenic action of Afa/Dr adhesins (Selvarangan et al., 2004; Servin, 2005). pH6 antigen. It was found that pH6 antigen of Y. pestis is a novel bacterial IgG-binding receptor (Zav’yalov et al., 1996). A pseudo-immune complex with human IgG1, IgG2 and IgG3 was formed. No binding to human IgG4, rabbit, mouse and sheep IgG was found. Antigen pH6 binds human IgG1 Fc subunit and does not bind Fab and pFc' subunits. This finding may be explained by pH6 antigen binding to β1-linked galactosyl residue (Payne et al., 1998) in a carbohydrate moiety of human IgG1, IgG2 and IgG3 that is linked to CH2 domains of their Fc subunit (Deisenhofer, 1981). Binding of purified recombinant pH6 antigen to gangliotetraosylceramide, gangliotriaosylceramide, and lactosylceramide was indicated by an enzyme-linked immunosorbent assay (Payne et al., 1998). The binding was saturable, with 50% of maximal binding occurring at 498, 390, and 196 nM, respectively. Intact E. coli cells that expressed pH6 antigen had specificity similar to purified pH6 antigen of Y. pestis on a thin-layer chromatography, except that nonhydroxylated galactosylceramide was also bound. The binding pattern indicates that the presence of β1-linked galactosyl residue in glycosphingolipids is the minimum determinant required for binding of pH6 antigen. Purified pH6 antigen selectively binds to apolipoprotein B-containing lipoproteins (low density lipoproteins) in human plasma (Makoveichuk et al., 2003). Low density lipoproteins at normal physiological concentration in human blood (equal to ~250 µg/ml) nearly abolish the Page 40 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review interaction of purified pH6 antigen with macrophages. This process could prevent recognition of a pathogen by the host immune defense system (Makoveichuk et al., 2003). Such immune masking might be important for the ability of the pathogen to cause the disease (Makoveichuk et al., 2003). Liu et al. (2006) found by flow cytometry that individual Y. pestis cells can express the capsular F1 antigen concomitantly with pH6 antigen (Psa) on their surface when analyzed. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties of Y. pestis, isogenic ∆caf (F1 genes), ∆psa, and ∆caf ∆psa mutants were constructed and studied with the three respiratory tract epithelial cells. The ∆psa mutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the ∆caf and ∆caf ∆psa mutants, indicating that pH6 antigen acts as an adhesin for respiratory tract epithelial cells. An anti- adhesive effect of F1 antigen was clearly detectable only in the absence of pH6 antigen, underlining the dominance of the Psa+ phenotype. Both F1 and pH6 antigens inhibited the intracellular uptake of Y. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas pH6 antigen seems to block bacterial uptake by interacting with a host receptor which controls the direct internalization. The ∆caf ∆psa double mutant bound and invaded all three epithelial cell types well, indicating to the presence of undefined adhesin(s) and invasin(s). It was found that pH6 antigen (Psa) fimbriae mediate bacterial binding to human alveolar epithelial cells (Galván et al., 2006). The pH6 fimbriae bound mostly to one component present in the total lipid extract from type II alveolar epithelial cells A549. Receptor of pH6 antigen was identified as phosphatidylcholine with thin-layer chromatography, molybdenium blue staining and pH6 antigen overlays. The pH6 antigen fimbriae bound to phosphatidylcholine in a dose- dependent manner while the binding was inhibited with phosphorylcholine and choline. Antigen pH6 also bound to pulmonary surfactant, which covers the alveolar surface as a product of type II alveolar epithelial cells and includes phosphatidylcholine as the major component. The Page 41 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review observed dose-dependent interaction of pH6 antigen with pulmonary surfactant was blocked by phosphorylcholine. Interestingly, surfactant did not inhibit pH6 antigen-mediated bacterial binding to alveolar cells, suggesting that both surfactant and cell membrane phosphatidylcholine retain pH6 antigen -fimbriated bacteria on the alveolar surface. Altogether, the results indicate that pH6 antigen uses the phosphorylcholine moiety of phosphatidylcholine as a receptor to mediate bacterial binding to pulmonary surfactant and alveolar epithelial cells. F1 antigen. It was found that human IL-1β specifically binds to a protein of caf operon, expressed on the surface of recombinant E. coli strain (Zav’yalov et al., 1995a). The binding was specifically inhibited by the Caf1M–Caf1 complex but not by the free Caf1M chaperone. Partially purified Caf1A also demonstrated binding with human IL-1β. The contradiction between the results can be explained by a presence of an admixture of Caf1M–Caf1 complex in Caf1A sample. Indeed, it has been demonstrated that chaperone-subunit complex is co-purifying in complex with usher protein (Nishiyama et al., 2005). A surprise is the absence of IL-1β binding with the fragments of F1 antigen that are scattering into cultural media (Chen & Elberg, 1977). This implies that only short non-aggregated F1 fibers, expressed during the early stages of cultivation/infection, possess the IL-1β binding activity. Binding of monoadhesins to host cell receptors and serum host proteins. FGS chaperone- assembled mono-adhesive fimbriae or pili are cell-surface fibers that project a specialized bacterial lectin, or adhesin subunit, away from the bacterial surface, to reach out for specific glycan receptors on the host cell (De Greve et al., 2007). Mono-adhesive fimbriae/pili can also mediate interbacterial interactions, thereby facilitating biofilm formation (De Greve et al., 2007). Monoadhesins are important virulence factors that exploit the diversity and virtually unlimited combinatorial potential of their carbohydrate receptors to ensure selective and fine-tuned pathogen–host interactions (De Greve et al., 2007). In recent years, it was shown that mono- adhesive fimbriae/pili allow bacterial pathogens to colonize, multiply, disseminate, and, in some Page 42 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review cases, persist for weeks to months within their animal hosts (Bower et al., 2005). Among these invasive bacteria, there are strains of uropathogenic E. coli which were previously characterized as strictly extracellular microbes but have been now shown to behave as opportunistic intracellular pathogens. Worldwide, uropathogenic E. coli account for the majority of urinary tract infections, including both cystitis (bladder infection) and pyelonephritis (kidney infection) (Foxman & Brown, 2003). These infections are exceedingly common in females, suffered by 11% of women each year (Foxman et al., 2000). FimH adhesin. Uropathogenic E. coli typically expresses filamentous adhesive organelles, called Type 1 pili, which mediate both bacterial attachment to and invasion of bladder urothelial cells. Type 1 pili or fimbriae possess a lectin-like component, FimH that is commonly thought to cause binding to mannose-containing oligosaccharides of host receptors. Since adhesion of Type 1 fimbriated organisms are inhibited by mannose, the reactions are described as mannose sensitive. Sokurenko et al. (1992) studied the adhesion of the Type 1 fimbriated CSH-50 strain of E. coli (which expresses only Type 1 fimbriae) to fibronectin (FN). E. coli CSH-50 does not bind detectable amounts of soluble FN but adheres well to immobilized plasma or cellular FN. This adhesion was inhibited by mannose-containing saccharides. By using purified domains of FN, it was found that E. coil CSH-50 adheres primarily to the N-terminal and gelatin-binding domains, only one of which is glycosylated, in mannose sensitive (MS) way. Binding of the mannose- specific lectin concanavalin A to FN and ovalbumin was eliminated or reduced, respectively, by incubation with periodate or endoglycosidase. Adhesion of E. coli CSH-50 to ovalbumin was reduced by these treatments, but adhesion to FN was unaffected. E. coli CSH-50 also adheres in an MS way to a synthetic peptide copying a portion of the amino-terminal FN domain (FNspl). Purified CSH-50 fimbriae bound to immobilized FN and FNspl in an MS way and inhibited adhesion of intact organisms. However, fimbriae purified from HB101(pPKL4), a recombinant strain harboring the entire Type l fim gene locus and expressing functional Type 1 fimbriae, Page 43 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review neither bound to FN or FNspl, nor inhibited E. coil adhesion to immobilized FN or FNspl. These findings suggest that there are two forms of Type 1 MS fimbriae. One form exhibits only the well-known MS lectin-like activity that requires mannose-containing glycoproteins. The other form exhibits not only the MS lectin-like activity but also binds to nonglycosylated regions of proteins in an MS manner. Sokurenko et al. (1994) provided evidence that this functional heterogeneity is due to variations in the fimH genes. They also investigated functional heterogeneity among clinical isolates and whether variation in fimH genes accounts for differences in receptor specificity. Twelve isolates obtained from human urine were tested for their ability to adhere to mannan, fibronectin, periodate-treated fibronectin, and a synthetic peptide copying 30 amino-terminal residues of fibronectin. CSH-50 and HB101 (pPKLA) were tested for comparison. Selected isolates were also tested for adhesion to purified fragments spanning the entire fibronectin molecule. Three distinct functional classes, designated M, MF, and MFP, were observed. The fimH genes were amplified by PCR from chromosomal DNA obtained from representative strains and expressed in a fim- strain (AAEC191A) transformed with a recombinant plasmid containing the entire fim gene cluster but with a translational stop- linker inserted into the fimH gene (pPKL114). Cloned fimH genes conferred on AAEC191A (pPKL114) receptor specificities mimicking those of the parent strains from which the fimH genes were obtained, demonstrating that the FimH subunits are responsible for the functional heterogeneity. Representative fimH genes were sequenced, and the deduced amino acid sequences were compared with the previously published FimH sequence. Allelic variants exhibiting >98% homology and encoding proteins differing by as little as a single amino acid substitution confer distinct adhesive phenotypes. This unexpected adhesive diversity within the FimH family broadens the scope of potential receptors for enterobacterial adhesion and may lead to a fundamental change in the understanding of the role(s) that Type 1 fimbriae may play in enterobacterial ecology or pathogenesis. Page 44 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review To further study the relationship between allelic variation of the fimH gene and adhesive properties of Type 1 fimbriae, Sokurenko et al. (1995) cloned the fimH genes from five strains and used to complement the FimH deletion in E. coli KB18. The parental and recombinant strains showed a wide quantitative range in the ability of bacteria to adhere to immobilized mannan. The differences in adhesion are due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences showed two sequence homology groups, based on the presence of Asn70 and Ser78 or Ser70 and Asn78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from different host niches in the urinary tract, expressed Type 1 fimbriae which conferred an increased level of adhesion. The results suggest that the quantitative variations in MS adhesion are primarily due to structural differences in the FimH adhesin. The differences in MS adhesion among of E. coli isolates makes possible that phenotypic variants of FimH play a functional role in population dynamics. Sokurenko et al. (1997) analyzed in more detail the ability of isogenic, recombinant strains of E. coli expressing fimH genes of the predominant fecal and urinary tract infection (UTI) phenotypes to adhere to glycoproteins and to uroepithelial cells. Type 1 fimbriae differ in their ability to recognize various mannosides by utilizing at least two different mechanisms. All FimH subunits studied to date are capable of mediating adhesion via trimannosyl residues, but only certain variants are capable of mediating high levels of adhesion via monomannosyl residues. The ability of the FimH lectins to interact with monomannosyl residues strongly correlates with their ability to mediate E. coli adhesion to uroepithelial cells. It would be possible for certain phenotypic variants of Type 1 fimbriae to contribute more than others to virulence of E. coli in the urinary Page 45 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review tract. Sokurenko et al. (1998) showed that genetic variation in FimH lectin of Type 1 fimbriae, can change the tropism of E. coli, shifting it toward the urovirulent phenotype. Random point mutations in fimH genes that increase binding of an adhesin to mono-mannose residues, structures abundant in the oligosaccharide moieties of urothelial glycoproteins, confer increased virulence. These mutant FimH variants, however, owe increased sensitivity to soluble inhibitors bathing the oropharyngeal mucosa, the physiological portal of E. coli. This functional trade-off seems to be detrimental for the intestinal ecology of the urovirulent E. coli. Thus, bacterial virulence can be increased by random functional mutations in a commensal trait that are adaptive for a pathologic environment, even at the cost of reduced physiological fitness in the nonpathologic habitat. Schembri et al. (2000) used random mutagenesis to specifically identify nonselective mutations in the FimH adhesin which modify its binding phenotype. Isogenic E. coli clones expressing FimH variants were tested for their ability to bind yeast cells and model glycoproteins which contain oligosaccharide moieties rich in either terminal monomannose, oligomannose, or nonmannose residues. Type 1 fimbriae were altered for amino acids in the FimH protein. The monomannose-binding phenotype was particularly sensitive to the changes, with extensive differences in binding being observed in comparison to wild-type FimH levels. Different structural alterations caused similar functional changes in FimH, suggesting to a high degree of flexibility to target recognition by this adhesin. Alteration of residue Pro49 of the carbohydrate-binding pocket of FimH, completely abolished its function. Amino acid changes which increased the binding capacity of FimH were located outside receptor-interacting residues, indicating that functional changes relevant to pathogenicity are likely to be due to conformational changes of the adhesin. Weissman et al. (2006) analysed the variability of fimA and fimH in strains of E. coli O1:K1-, O2:K1- and O18:K1 serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains are identical at eight housekeeping loci around the genome and belong to the ST95 complex. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal Page 46 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintes-tinal E. coli are under strong positive selection (Dn/ Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements. Thomas et al. (2002) showed that bacterial attachment to target cells switches from loose to firm upon a 10- fold increase when a shear stress is applied. Steered molecular dynamics simulations of tertiary structure of the FimH receptor binding domain and subsequent site-directed mutagenesis studies indicate that shear-enhancement of the FimH-receptor interactions involves extension of the interdomain linker chain under mechanical force. The ability of FimH to function as a force sensor provides a molecular mechanism for discrimination between surface-exposed and soluble receptor molecules. Thomas et al. (2004) demonstrated that raising the shear stress (within the physiologically relevant range) increased accumulation of Type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti-FimH antibody-coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force-activated; that is, that FimH mediates 'catch-bonds' with mannose that are strengthened by tensile mechanical force. As a result, on monomannose-coated surfaces, bacteria displayed a complex 'stick-and-roll' adhesion in which they tend to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Page 47 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Mutations in FimH that were predicted earlier to increase or decrease force-induced conformational changes in FimH were furthermore shown to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This 'stick-and-roll' adhesion could allow Type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions. Nilsson et al. (2008) described two distinctively different conformations of the mannose- bound FimH binding site. Force-induced dissociation was slowed when the mannose ring rotated such that additional force-bearing hydrogen bonds formed with the base of the FimH binding pocket. The lifetime of the complex was further significantly enhanced by rigidifying this base. It was shown how even sub-Å spatial alterations of the hydrogen bonding pattern within the base can lead to significantly decreased bond lifetimes (Nilsson et al., 2008). Pereverzev et al. (2005) developed a physical model that explains how the ligand escapes the receptor binding site via two alternative routes, a catch-pathway that is opposed by the applied force and a slip-pathway that is promoted by force. The model predicts under what conditions and at what critical force the catch-to-slip transition would be observed, as well as the degree to which the bond lifetime is enhanced at the critical force. Nilsson et al. (2006) discovered that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment was shown for the fimbrial FimH-mannose-mediated surface adhesion of E. coli. Shear stress- enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs. Nilsson et al. (2006) showed that the oligosaccharide-specific interaction of FimH with trimannose (3M), lacks a shear threshold for binding, since the number of bacteria bind under static conditions stronger than under any flow. However, similar to 1M, the binding strength of surface-interacting bacteria is enhanced by shear. Bacteria change from rolling into firm Page 48 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review stationary surface adhesion as the shear increases. The shear-enhanced bacterial binding on 3M is mediated by catch bond properties of the 1M-binding subsite within the extended oligosaccharide-binding pocket of FimH, since structural mutations in the putative force- responsive region and in the binding site affect 1M- and 3M-specific binding in an identical manner. A shear-dependent conversion of the adhesion mode is also exhibited by P-fimbriated E. coli adhering to digalactose surfaces. Anderson et al. (2007) compared levels of surface colonization by E. coli strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. They showed that the weak rolling mode of surface adhesion allows a more rapid spreading during growth on a surface in the presence of fluid flow. An attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) an advantage of a weak adhesion is a rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have unintended effect of enhancing the rate of surface colonization. Nilsson et al. (2007) showed that removal of the cysteine bond in the mannose-binding domain of FimH did not affect FimH-mannose binding under static or low shear conditions (< or = 0.2 dyne cm-2). However, the adhesion level was substantially decreased under increased fluid flow. Under intermediate shear (2 dynes cm-2), the ON-rate of bacterial attachment was significantly decreased for disulphide-free mutants. Molecular dynamics simulations demonstrated that the lower ON-rate of cysteine bond-free FimH could be due to destabilization of the mannose-free binding pocket of FimH. In contrast, mutant and wild-type FimH had similar conformation when bound to mannose, explaining their similar binding strength to mannose under intermediate shear. The stabilizing effect of mannose on disulphide-free FimH was also confirmed by protection of the FimH from thermal and chemical inactivation in the presence of mannose. However, this stabilizing effect could not protect the integrity of FimH Page 49 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review structure under high shear (> 20 dynes cm cm-2), where lack of the disulphide significantly increased adhesion OFF-rates. Thus, the cysteine bonds in bacterial adhesins could be adapted to enable bacteria to bind target surfaces under increased shear conditions. Yakovenko et al. (2008) applied force to single isolated FimH bonds with an atomic force microscope in order to test this directly. If force was loaded slowly, most of the bonds broke up at low force (<60 pN of rupture force). However, when force was loaded rapidly, all bonds survived until much higher force (140-180 pN of rupture force), behavior that indicates to a catch bond. Structural mutations or pretreatment with a monoclonal antibody both of which allosterically stabilize a high affinity conformation of FimH cause all bonds to survive until high forces regardless of the rate at which force is applied. Pretreatment of FimH bonds with intermediate force has the same strengthening effect on the bonds. This demonstrates that FimH forms catch bonds and that tensile force induces an allosteric switch to the high affinity, strong binding conformation of the adhesin. The catch bond behavior of FimH, the amount of force needed to regulate FimH, and the allosteric mechanism, all provide insight into how bacteria bind and form biofilms in fluid flow. Additionally, these observations may provide a means for designing antiadhesive mechanisms. Thomas et al. (2008) reviewed experimental data and biophysical theory to analyze why mechanical force prolongs the lifetime of these bonds rather than shortens the lifetime by pulling the ligand out of the binding pocket. Although many mathematical models can explain catch bonds, experiments using structural variants have been more helpful in determining how catch bonds work. The underlying mechanism has been worked out so far only for the bacterial adhesive protein FimH. This protein forms catch bonds because it is allosterically activated when mechanical force pulls an inhibitory domain away from the ligand-binding domain. Other catch bond-forming proteins, including blood cell adhesion proteins called selectins and the motor protein myosin, show evidence of allosteric regulation between two domains, but it remains unclear if this is related to their catch bond behavior. Page 50 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review FimH adhesin consists of a fimbria-associated pilin domain and a mannose-binding lectin or adhesin domain, with the binding pocket positioned opposite the interdomain interface (Fig. 9; Choudhury et al., 1999). By using the yeast two-hybrid system, purified lectin and pilin domains, and docking simulations, it was showed that the FimH domains interact with one another (Aprikian et al., 2007). The affinity for mannose is greatly enhanced (up to 300-fold) in FimH variants in which the interdomain interaction is disrupted by structural mutations in either the pilin or lectin domains. Also, affinity to mannose is dramatically enhanced in isolated lectin domains or in FimH complexed with the chaperone molecule that is wedged between the domains. Furthermore, FimH with native structure mediates weak binding at low shear stress but shifts to strong binding at high shear, whereas FimH with disrupted interdomain contacts (or the isolated lectin domain) mediates strong binding to mannose-coated surfaces even under low shear. Interactions between lectin and pilin domains decrease the affinity of the mannose-binding pocket via an allosteric mechanism (Choudhury et al., 1999). Mechanical force at high shear separates the two domains, allowing the lectin domain to switch from a low affinity to a high affinity state. This shift provides a mechanism for FimH-mediated shear-enhanced adhesion by enabling the adhesin to form catch bond-like interactions that are longer lived at high tensile force. FimH lectin domain possesses a ligand-induced binding site - a type of allosterically regulated epitopes characterized in integrins (Tchesnokova et al., 2008). Analogous to integrins, in FimH the ligand-induced binding site epitope becomes exposed in the presence of the ligand (or 'activating' mutations) and is located far from the ligand-binding site, close to the interdomain interface. Also, the antibody binding to the ligand-induced binding site shifts adhesin from the low- to high-affinity state. Binding of streptavidin to the biotinylated residue within the ligand- induced binding site also locks FimH in the high-affinity state, suggesting that the allosteric perturbations in FimH are sustained by the interdomain wedging. In the presence of antibodies, the strength of bacterial adhesion to mannose is increased similar to the increase observed under shear force, suggesting the same allosteric mechanism - a shift in the interdomain configuration. Page 51 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Thus, an integrin-like allosteric link between the binding pocket and the interdomain conformation can serve as the basis for the catch bond property of FimH and, possibly, of other adhesive proteins. Pereverzev et al. (2009) found that the Type 1 fimbrial adhesive protein (FimH)/mannose bond is governed by the interface between the lectin and pilin domains of FimH. Catch-binding occurs in these systems when the external force stretches the receptor proteins and increases the interdomain distance. The proposed model accurately describes the experimentally observed anomalous behavior of the lifetimes of the FimH/mannose complexes as a function of applied force and provides valuable insights into the mechanism of catch- binding. Recently Pereverzev et al. (2009) reviewed the proposed model that demonstrates the allosteric role of the two-domain region of the receptor protein in the increased lifetimes of biological receptor/ligand bonds subjected to an external force. Interaction between the domains is represented by a bounded potential, containing two minima corresponding to the attached and separated conformations of the two protein domains. The dissociative potential with a single minimum, describing receptor/ligand binding, fluctuates between deep and shallow states, depending on whether the domains are attached or separated. A number of valuable analytic expressions are derived and are used to interpret experimental data for two catch bonds. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, β1 and α3 integrins were identified as key host receptors for uropathogenic E. coli (Eto et al., 2007). FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, β1 and α3 integrin receptors co- localize with invading type 1–piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by β1 and α3 integrin–specific antibodies and by disruption of the β1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of β1 integrin alter integrin signaling. They also variably affect uropathogenic E. coli entry into host cells, by either attenuating or boosting invasion frequencies (Eto et al., 2007). Page 52 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signalling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that β1 and α3 integrins are functionally important receptors for type 1 pili–expressing bacterium within the urinary tract and possibly at other sites within the host (Eto et al., 2007). GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2’s closest homologue is uromodulin, a protein expressed by the kidney that shows 52% identity and 67% conservation in amino acid sequence. Uromodulin is secreted into the urine and binds E. coli with Type 1 fimbriae. A role in host defense has been proposed in which uromodulin serves as a molecular decoy that prevents bacteria from binding to uroplakin, the host receptor in uroepithelia (Mo et al., 2004; Pak et al., 2001). In addition, two independent laboratories (Mo et al., 2004; Pak et al., 2001) have produced uromodulin null mice that showed increase sensitivity to urinary tract infections. Yu S & Lowe (2009) examined whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. It was demonstrated that GP2 binds E. coli which expresses Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Thus, GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family. PapG adhesin. The most extensively studied adhesin, and also the first virulence-associated factor identified for uropathogenic E. coli is P fimbria (Lane & Mobley, 2007), encoded by the pap (pyelonephritis-associated pili) genes. P fimbria are prevalent among strains of Page 53 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review uropathogenic E. coli causing pyelonephritis4 and are characterized by their mannose-resistant adherence to Gal(a1–4)Galb moieties present in the globoseries of membrane glycolipids on human erythrocytes of the P blood group and on uroepithelial cells (Johnson, 1991; Jones et al., 1996; Leffler & Svanborg-Edén, 1980, 1981). Three major and well-studied classes of papG alleles exist, which encode the molecular variants of adhesin PapGI, -II, and -III. Each PapG variant is known to have a distinct isoreceptor specificity, which in turn results in altered host tissue tropism. PapGII, which is clinically associated with acute pyelonephritis in humans, binds preferentially globoside, or GbO4, the predominant glycolipid isoreceptor of the human kidney. Both the solution structure of the PapGII adhesin domain and the crystal structure of the PapGII receptor bound to GbO4 as well as the unbound form of the adhesin have been determined using NMR and the multiwavelength anomalous dispersion phasing method (Fig. 9; Dodson et al., 2001; Sung et al., 2001). F17G/GafD adhesin. Bacterial adhesion to intestinal surfaces is important for successful colonization, and a number of fimbrial adhesins expressing differing receptor-binding specificities and serological properties have been detected on enterotoxigenic E. coli from different hosts (Nataro & Kaper, 1998). The G fimbria is most closely related to the F17c fimbriae that are common on bovine septicemic and diarrhea-associated E. coli (Saarela et al., 1995) and they occur in human E. coli infections as well (Le Bouguénec & Bertin, 1999). The G fimbriae bind to the terminal N-acetyl-D-glucosamine (GlcNAc) residues of glycoproteins at calf intestinal brush borders as well as mammalian basement membrane (Saarela et al., 1996; Sanchez et al., 1993). The latter is thought to potentiate translocation of the enterotoxigenic E. coli into circulation. G fimbrial binding to GlcNAc receptors is mediated by the 321 amino acid residue GafD lectin subunit (Saarela et al., 1995) present mainly at the G-fimbrial tip (Saarela, 1999). The structure of the ligand-binding domain, GafD1-178, has been determined at 1.7Å resolution in the presence of the receptor sugar N-acetyl-D-glucosamine (Fig. 9; Merckel et al., 2003). As in N-terminal adhesin PapG and FimH domains the overall fold of GafD1-178 is a β-barrel jelly- Page 54 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. MrkD adhesin. Li et al. (2009) expressed in E. coli and purified to homogeneity the recombinant adhesin MrkD of K. pneumoniae. The adhesive activity of MrkD was examined and the binding site was studied with the laser confocal microscopy. The adherent activity of K. pneumoniae was significantly inhibited by MrkD showing that MrkD putative adhesin contains adhesion epitopes. Anti-Immune and Pro-Inflammatory Activities of Adhesive Organelles Features of anti-immune and pro-inflammatory activities of poly- and mono-adhesive organelles. In contrast to mono-adhesive pili, which possess only one binding domain on the tip of pilus (Fig. 12a), each poly-adhesive fiber potentially might (Fig. 12b): (1) Ensure a powerful polyvalent fastening of a bacterial pathogen to a host target cell (Galván et al., 2006); (2) Aggregate host cell receptors and trigger transduction of signals causing immunosuppressive and pro-inflammatory responses (Galván et al., 2006; Sharma et al., 2005a,b; Sodhi et al., 2004); (3) Pull a bacterium to a host cell by a zipper-like mechanism that increases tightness of the contact. It was directly demonstrated that Psa fimbriae (pH6 antigen) of Y. pestis are functioning as polyadhesins (Galván et al., 2006). The Psa fimbriae bound to phosphatidylcholine in a dose- dependent manner and binding was inhibited by phosphorylcholine and choline. Binding inhibition was dose-dependent, although only high concentrations of phosphorylcholine Page 55 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review completely blocked Psa binding to phosphatidylcholine. In contrast, less than 1 µM of a phosphorylcholine-polylysine polymer inhibited specifically the adhesion of Psa-fimbriated E. coli to phosphatidylcholine, and type I (WI-26 VA4) and type II alveolar epithelial cells. A tight contact between interacting cells hampers diffusion of Ca+2 in the site of contact, and, consequently, triggers Ca+2-dependent Type III secretion system (encoded by the pCD1 virulence plasmid) that destroys defense activity of the host cell (Cornelis & Wolf-Watz, 1997; Viboud & Bliska, 2005). This is extremely important for the bacterial virulence. In particular, Y. pestis appears to utilize the Type III secretion pathway to destroy cells with innate immune functions (macrophages, dendritic cells and neutrophils) that represent the first line of defence, thereby preventing adaptive responses and precipitating the fatal outcome of plague (Marketon et al., 2005). It was found that dendritic cells infected with Y. pestis failed to adhere to solid surfaces and to migrate toward the chemokine CCL19, in an in vitro transmembrane assay. Both effects were dependent on presence of a pCD1 plasmid, and on bacterial growth shift to 37°C, prior to infection (Velan et al., 2006). Moreover, while instillation of a pCD1-cured Y. pestis strain into mice airways triggered effective transport of alveolar dendritic cells to the mediastinal lymph node, instillation of Y. pestis harboring the plasmid failed to do so. Taken together, these results suggest that pCD1 virulence-plasmid dependent impairment of dendritic cell migration is the major mechanism utilized by Y. pestis to subvert dendritic cell function. Contribution of polyadhesins to virulence and anti-immune activity of bacteria. The most important structure-functional information by now was obtained for the Afa/Dr adhesins. Nowicki et al. (1994) found gestational age-dependent distribution of E. coli fimbriae in pregnant patients with pyelonephritis. Later Hart et al. (1996) indicated that it is likely that E. coli associated with acute pyelonephritis during different trimesters of pregnancy represents non- random closely related isolates, and some of these strains may be characteristic in pregnant patients only. Nowicki et al. (1997) demonstrated that the rate of uterine infection in pregnant rats was about 10-fold higher than in non-pregnant animals. It was proposed that infectious Page 56 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review complications of pregnancy may be related to gestation-dependent sensitivity to the pathogenic microorganism and the host nitric oxide, NO, status. Fang et al. (1999) with immunofluorescence studies indicated that macrophages and natural killer cells, located in the endometrial layer clustering around epithelial cells, expressed type II protein. They suggested that localized increase in type II NO synthase (NOS) expression and NO production occurs in response to intrauterine infection while the NO system may play a role in host response to restrict the infection. Moreover, Fang et al. (2001) demonstrated that intrauterine infection induced an elevated expression of tumor necrosis factor (TNF)-α in both non-pregnant and pregnant rats. The sequential stimulation of NOS expression, especially the inducible isoform, and generation of uterine NO may be lacking during pregnancy despite of an elevated TNF-α after infection. Fang et al. (2001) indicated that NO synthesis response may be maximal at pregnancy, and infection may not further induce the NO system. These studies, together with the previous report (Fang et al., 1999) suggest that intrauterine infection-induced lethality in pregnant rats is amplified with the inhibition of NO and that pregnancy is a state predisposed for increased complications associated with intrauterine infection. The constitutively elevated uterine NO during pregnancy may help reduce the risk of infection-related complications. Kaul et al. (1999) reported that expression of CD55/DAF protein, recognized by adhesin Dr of diffusially adhering E. coli as the host tissue receptor, is increased during pregnancy. Induction of pathogenesis is a cumulative process of the host-pathogen relationship involving specific host factors and virulence characteristics of the invading organism. Kaul et al. (1999) developed an experimental model of chronic pyelonephritis with E. coli bearing adhesin Dr (E. coli Dr1) in non-pregnant lipopolysaccharide-hyporesponder C3H/HeJ mice. With this model the role of E. coli Dr1 was investigated on the outcome of pregnancy in C3H/HeJ mice. Groups of pregnant mice were infected with E. coli Dr1 or its isogenic mutant which does not bear the adhesin Dr (E. coli Dr2) by urethral catheterization. Nearly 90% of pregnant mice infected with E. coli Dr1 delivered preterm (before 90% gestation) compared to 10% of mice infected with E. Page 57 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review coli Dr2 but none of the mice treated with phosphate-buffered saline (PBS). There was a significant reduction in fetal birth weight in the E. coli Dr1-infected group compared to the E. coli Dr2- and PBS-treated groups (P = 0.003) (Kaul et al., 1999). Goluszko et al. (2001) used a gentamycin protection assay to assess the ability of gestational pyelonephritis isolates of E. coli to invade HeLa cells. The ability to enter HeLa cells was strongly associated with the presence of dra gene clusters coding adhesins Dr. In contrast, the nonivasive isolates predominantly expressed papG, coding P fimbriae. Hart et al. (2001) found significant increase of ampicillin resistance among gestational pyelonephritis E. coli and the association with the dra gene cluster encoding colonization and invasive capacity. It was found that the family of adhesins Dr, like Type 1 fimbriae, mediated concentration- dependent adherence to human neutrophils (PMNs) (Johnson et al., 1995). Adherence to human neutrophils was mannose sensitive for Type 1 fimbriae but mannose resistant for Dr family adhesins. Chloramphenicol inhibited PMNs adherence for the hemagglutinin Dr with the same potency as that with which it inhibited hemagglutination, but it was inactive against PMN adherence and hemagglutination mediated by other members of the adhesin Dr family. In contrast to PMN adherence, mediated by type 1 fimbriae, the adherence, mediated by the hemagglutinin Dr, did not lead to significantly increased bacterial killing. These data suggest that family of adhesins Dr mediate a novel pattern of adherence to PMNs, probably by recognizing CD55/DAF, with minimal consequent bacterial killing. Peiffer et al. (1998) studied F-actin rearrangements in the host cells expessing CD55/DAF protein as a result of attachment of E. coli strain bearing adhesin Dr. Infection of INT407 cells by the diffusely adhering strain E. coli C1845 (DAEC C1845) can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845- CD55/DAF F interaction involves recruitment of signal transduction molecules. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cg, phosphatidylinositol 3-kinase, protein kinase C, and Page 58 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Ca21 inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. coli recombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. Thus, DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the CD55/DAF -GPI signal cascade without bacterial cell entry. Later Peiffer et al. (2000a) provided evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [3H]-mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction- associated occludin and ZO-1 protein were observed, whereas the distribution of the zonula adherens-associated E-cadherin was not affected. Using the recombinant E. coli strains HB101- (pSSS1) and - (pSSS1C) expressing the F1845 fimbrial adhesin, it was demonstrated that the adhesin- CD55/DAF interaction is not sufficient for the induction of structural and functional tight-junction lesions (Peiffer et al., 2000). Moreover, using the actin filament-stabilizing agent Jasplakinolide, Peiffer et al., 2000 demonstrated that the C1845-induced functional alterations in tight-junctions are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845. Infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 causes clustering of CD55/DAF around adhering bacteria (Guignot et al., 2000). Mapping of CD55/DAF epitopes involved in CD55/DAF clustering by Afa/Dr DAEC was conducted with CD55/DAF deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55/DAF clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC- induced CD55/DAF clustering. It was shown that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e/CEA (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by Page 59 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review clustering of CD66e/CEA around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e/CEA, accompanied by CD66e/CEA clustering around adhering bacteria. Inhibition assay with monoclonal antibodies directed against CD55/DAF SCR domains, and polyclonal anti- CD55/DAF and anti- CD66e/CEA antibodies demonstrate that CD55/DAF and CD66e/CEA function as receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, Guignot et al. (2000) found that a mutant in which cysteine replaces aspartic acid at position 54 displayed conserved the binding capacity but failed to induce CD55/DAF and CD66e/CEA clustering. Peiffer et al. (2000b) further characterized cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and hemagglutinin Dr, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin; proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Peiffer et al. (2000b) found that a mutant in which cysteine replaces aspartic acid at position 54 conserved the binding capacity but failed to induce F-actin disassembly. Distribution of brush border-associated functional proteins sucrase-isomaltase, dipeptidylpeptidase IV, glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, sucrase-isomaltase and dipeptidylpeptidase IV enzyme activity decreased. Selvarangan et al. (2004) constructed an isogenic mutant in the DraE adhesin subunit that was unable to bind type IV collagen but retained binding to CD55/DAF and examined its virulence in the mouse model. The collagen-binding mutant DrI113T was eliminated from the mouse renal tissues in 6 to 8 weeks, while the parent strain caused persistent renal infection which lasted at least for 14 weeks. Trans-complementation with the intact operon dra restored collagen-binding activity, interstitial tropism, and the ability to cause persistent renal infection. It Page 60 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review was concluded that type IV collagen binding mediated by DraE adhesin is a critical step for the development of the infection in murine model of E. coli pyelonephritis. Brest et al. (2004) found that infection of polymorphonuclear leukocytes by Afa/Dr DAEC strains induced PMNL apoptosis characterized by morphological nuclear changes, DNA fragmentation, caspase activation, and a high level of annexin V expression. PMNL apoptosis depended on their agglutination, induced by Afa/Dr DAEC, and was still observed after preincubation of PMNs with anti-CD55/DAF and/or anti-CD66e/CEA antibodies. Low levels of phagocytosis of Afa/Dr DAEC strains were observed both in nontransmigrated and in transmigrated polymorphonuclear leukocytes compared to that observed with the control E. coli DH5α strain. Interaction of Afa/Dr DAEC with polymorphonuclear leukocytes may increase the bacterial virulence both by inducing apoptosis of polymorphonuclear leukocytes through an agglutination process and by diminishing their phagocytic capacity. Wroblewska-Seniuk et al. (2005) investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection, using afaE and/or afaD mutants. The highest maternal mortality was observed in the group infected with the afaE+ afaD+ strain, followed by the group infected with the afaE+ afaD strain. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE+ strains were the most invasive (afaE+ afaD strain > afaE+ afaD+ strain), while the afaE mutant strains (afaE afaD+ and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE+ E. coli strains in vitro. Korotkova et al. (2008b) demonstrated that CD55/DAF or CEACAM receptors independently promote DraE mediated internalization of E. coli by CHO cell transfectants expressing these receptors. They also found that DraE-positive recombinant bacteria adhere to and are internalized by primary human bladder epithelial cells which express CD55/DAF and Page 61 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review CEACAMs. DraE-mediated bacterial internalization by bladder cells was inhibited by agents, which disrupt lipid rafts, microtubules, and phosphatidylinositol 3-kinase (PI3K) activity. Immunofluorescence confocal microscopic examination of epithelial cells detected considerable caveolin, β1 integrin, phosphorylated ezrin, phosphorylated PI3K, and tubulin, but not F-actin, by cell-associated bacteria. DraD subunit, previously implicated as an “invasin,” is not required for β1 integrin recruitment or bacterial internalization. Guignot et al. (2009) also provided evidence that AfaD or DraD putative invasin subunits do not participate in the cell-association and -entry of bacteria, whereas DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing CD55/DAF, CEACAM1, CD66e/CEA, or CEACAM6. They confirmed independently data of Korotkova et al. (2008b) that internalization of Dr fimbriae-positive E. coli within CHO- CD55/DAF, -CEACAM1, - CD66e/CEA, or -CEACAM6 cells occurs through microtubule- and lipid rafts-dependent mechanism. Wild-type Dr fimbriae-positive bacteria survived more than within cells expressing CD55/DAF as compared with bacteria internalized within CHO- CEACAM1, - CD66e/CEA, or -CEACAM6 cells (Guignot et al., 2009). Korotkova et al. (2007) supposed that immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the family Dr of E. coli adhesins, structural and adhesive functions are carried out by the same subunit. Adhesins Dr have been shown to bind CD55/DAF, collagen IV, and CEACAMs. They showed that genes encoding adhesins Dr from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the haemagglutinin Dr (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity Page 62 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review to CD55/DAF, decreased sensitivity of CD55/DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification. F1 antigen. Y. pestis is the etiologic agent of bubonic and pneumonic plague, one of the most deadly diseases known to man (Cleri et al., 1997; Li et al., 2009; Perry & Fetherston, 1997; Smiley, 2008a, b). Electron micrographs of Y. pestis demonstrate that F1 antigen forming capsule is maximally expressed at 37°C after 72 hours of cultivation in vitro (Chen & Elberg, 1977). The expression of F1 antigen at 22°C is negligible (Chen & Elberg, 1977). During the early stages of infection, when the F1 capsule is not yet formed, type III secretion system protects Y. pestis from phagocytosis (Cornelis & Wolf-Watz, 1997; Viboud & Bliska, 2005). This system is encoded on a virulence plasmid of 70 kb in size that is common to Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. After 24-h of cultivation in vitro at 37°C, Y. pestis expresses enough large capsule-like structure composed of aggregating F1 antigen (Chen & Elberg, 1977). The capsule material is readily soluble and dissociates from the bacterium during in vitro cultivation. Association of F1 antigen with virulence is evident from recent studies since all F1‒ mutants were of low virulence to mice compared with the wild types (Welkos et al., 2004). Similar to other capsules or capsule-like antigens, F1 seems to be involved in the antiphagocytic activity reported for Y. pestis, but the contribution of F1 to this activity was not understood until recently. Y. pestis strain EV76 is highly resistant to uptake by J774 cells (Du et al., 2002). Y. pestis strain EV76 with an in-frame deletion of the caf1M gene fails to express F1 polymer on the bacterial surface. This strain had somewhat lowered ability to prevent uptake by J774 cells. Strain EV76C, cured with the virulence plasmids, was much reduced in its ability to resist uptake. A strain lacking both the virulence plasmid and caf1M was almost totally phagocytosed (95%; Du et al., 2002). It was concluded that F1 and the type III secretion system act in concert to make Y. pestis highly resistant to phagocytosis. Type III secretion system of Y. pestis may function optimally Page 63 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review only during early stages of infection, when the contact-dependent delivery of Yop effector proteins is the highest. Later on, when the surface of Y. pestis is covered with the F1 capsule, the delivery of Yop effector proteins may be lower. While the expression of F1 capsule reduces the number of bacteria that interacts with the macrophages, it does not influence on the general phagocytic ability of J774 cells (Du et al., 2002). This suggests that F1 capsule prevents uptake by interfering at the level of receptor interaction in the phagocytosis process. Sebbane et al. (2009) found that a caf ‒ Y. pestis mutant was neither impaired in flea colonization nor in virulence in mice after intradermal inoculation of cultured bacteria. In contrast, absence of the caf operon decreased bubonic plague incidence after fleabite. Successful development of plague in mice infected by fleabite with the caf ‒ mutant required a higher number of infective bites per challenge. In addition, the mutant displayed a highly autoaggregative phenotype in infected liver and spleen. The results suggest that acquisition of the caf locus via horizontal transfer by an ancestral Y. pestis increased transmissibility and the potential for epidemic spread. Sebbane et al. (2009) suggested a model in which atypical caf ‒ strains could emerge during climatic conditions that favor a high flea burden. Human infection with such strains would not be diagnosed by the standard clinical tests that detect F1 antibody or antigen, suggesting that more comprehensive surveillance for atypical Y. pestis strains in plague foci may be necessary. Y. pestis survives and replicates in phagosomes of murine macrophages. Y. pestis -containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with IFN-γ. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies on mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages were undertaken to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages (Pujol et al. 2009). Some YCVs contained double membranes Page 64 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that approximately 40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3- II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (approximately 30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, showing that this pathogen does not require autophagy for intracellular replication. Pujol et al. (2009) concluded that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification. pH6 antigen. The pH6 antigen was first described more than 40 years ago and was initially identified as an antigen expressed only at pH below 6 at 37°C (Ben-Efraim et al., 1961). The electron micrographs of highly virulent phenotype Y. pestis grown at 37°C, pH6, indicate the expression both the F1 capsule and thin filaments of pH6 (PsaA) antigen on the bacterial surface (Lindler & Tall, 1993). The pH6 antigen is essential for full virulence of Y. pestis (Lindler et al., 1991; Lindler & Tall, 1993). A ∆psaA mutant had a significant dissemination defect after subcutaneous infection but only slight attenuation by the pneumonic-disease model indicating different roles of the pH6 antigen in bubonic and pneumonic plague (Cathelyn et al., 2006). The expression of pH6 antigen adds to the antiphagocytic armament of the bacterium (Huang & Lindler, 2004). Y. pestis psaA isogenic strains do not show any significant difference in their association with mouse macrophage cells. However, expression of psaA appeared to reduce significantly phagocytosis of both Y. pestis and E. coli by mouse macrophages (P < 0.05). Furthermore, complementation of psaA mutant of Y. pestis strains could completely restore the bacterial resistance to phagocytosis. Fluorescence microscopy following differential labeling of intracellular and extracellular portion of Y. pestis revealed that significantly lower numbers of Page 65 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review psaA-expressing bacteria were located inside the macrophages. Enhanced phagocytosis resistance was specific for bacteria expressing psaA and did not influence the ability of the macrophages to engulf other bacteria. This shows that Y. pestis pH6 antigen does not enhance adhesion to macrophages but rather promotes resistance to phagocytosis helping the bacteria to escape host immune defence mechanisms (Huang & Lindler, 2004). Recently Anisimov et al. (2009) generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans two isogenic sets of Y. pestis strains, composed of wild-type strains 231 and I-1996, their non-polar pH6‒ mutants with deletions in the psaA gene or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion fimbriae or constitutive production of pH6 antigen. It was shown that the loss of synthesis or constitutive production of pH6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen. SefD putative invasin. The translocation of the minor putative invasin SefD subunit is a prerequisite for the export of the major structural SefA subunit across the outer membrane and formation of the SEF14 fimbriae (Edwards, Matlock & Maloy, unpublished observations); thus, SefD is probably located at the tip of the fimbrial shaft (Edwards et al., 2000). The LD50 values for the wild-type strain and mutants lacking SefA are comparable, but both the oral and intra- peritoneal virulence of mutants lacking SefD are greatly reduced. It means that major SEF14 subunit SefA is not required for the virulence of S. enteritidis, indicating that the tip of the fimbrial structure composed of SefD subunits is probably sufficient for successful interactions with phagocytes (Edwards et al., 2000). SefD may bind to a receptor on the macrophage surface and alter the uptake of S. enteritidis into the phagocyte so that S. enteritidis can survive in the intracellular environment. Polyadhesin Ral. Hart et al. (2009) investigated the contribution of a fimbrial polyadhesin, Ral, of rabbit-specific EPEC (REPEC) to host specificity by introducing Ral into derivatives of Page 66 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review human-specific EPEC (hEPEC) strain, E2348/69, in which expression of the fimbrial adhesin, Bfp, had been interrupted. Although unable to cause diarrhoeal disease in rabbits, Ral-bearing hEPEC strains colonised rabbit intestine more efficiently and showed altered intestinal localisation when compared to an isogenic Ral‒ strain. These findings suggest that Ral enhances the initial interaction between a ∆bfpA mutant of hEPEC and rabbit intestine and may influence tissue specificity, but is not sufficient on its own to transform hEPEC into a rabbit pathogen. Contribution of monoadhesins to virulence. Type 1 fimbriae. Urinary tract infection is the second most common infectious disease and is caused predominantly by Type 1-fimbriated uropathogenic E. coli (UPEC). UPEC initiates infection by attaching to uroplakin (UP) Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. UP Ia, together with UP Ib, UP II, and UP IIIa, forms a 16-nm receptor complex that is assembled into hexagonally packed, two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the UP IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the UP receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. Wang et al. (2009) showed by cryo-electron microscopy that FimH binding to the extracellular domain of UP Ia induces global conformational changes in the entire UP receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the UP cytoplasmic tails, which can be sufficient to trigger downstream signaling events. The results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor- mediated bacterial invasion in general. Page 67 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Enteropathogenic E. coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPECs are sub-grouped into typical (tEPEC) and atypical (aEPEC). aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. Yamamoto et al. (2009) showed that invasion of HeLa cells by aEPEC 1551-2 depends on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface. It was concluded that some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type. FimH, the mannose-specific, Type 1 fimbrial adhesin of E. coli, acquires amino acid replacements adaptive in extraintestinal niches (the genitourinary tract) but detrimental in the main habitat (the large intestine). This microevolutionary dynamics is reminiscent of an ecological "source-sink" model of continuous species spread from a stable primary habitat (source) into transient secondary niches (sink), with eventual extinction of the sink-evolved populations. Chattopadhyay et al. (2007) adapted two ecological analytical tools - diversity indexes DS and alpha - to compare size and frequency distributions of fimH haplotypes between evolutionarily conserved FimH variants ("source" haplotypes) and FimH variants with adaptive mutations (putative "sink" haplotypes). Both indexes show two- to three-fold increased diversity of the sink fimH haplotypes relative to the source haplotypes, a pattern that ran opposite to those seen with nonstructural fimbrial genes (fimC and fimI) and housekeeping loci (adk and fumC) but similar to that seen with another fimbrial adhesin of E. coli, papG-II, also implicated in extraintestinal infections. The increased diversity of the sink pool of adhesin genes is due to the increased richness of the number of unique haplotypes, rather than their extent of similarity in relative abundances. Taken together, this pattern supports a continuous emergence and extinction of the gene alleles adaptive to virulence sink habitats of E. coli, rather than a one-time change in the habitat conditions. Thus, ecological methods of species diversity analysis can be successfully Page 68 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review adapted to characterize the emergence of microbial virulence in bacterial pathogens subject to source-sink dynamics. The enteric bacterium K. pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI) and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. Stahlhut et al. (2009) showed that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity, but are prone to frequent horizontal transfer events between different bacterial clones. Addition of the fimH locus to Multiple Locus Sequence Typing significantly improved resolution of the clonal structure of pathogenic strains, including the K1-encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic E. coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of Type 1 fimbriae in Enterobacteriaceae, the finding, presented by Stahlhut et al. (2009), suggests that fimH genes are subject to adaptive microevolution substantiates and the importance of Type 1 fimbriae-mediated adhesion in K. pneumoniae. Long Polar Fimbriae.The Long Polar Fimbriae (Lpf) is one of few adhesive factors of enterohemorrhagic E. coli O157:H7 associated with colonization of the intestine. E. coli O157:H7 strains possess two lpf loci encoding highly regulated fimbrial structures. Database analysis of the genes encoding the major fimbrial subunits demonstrated that they are present in pathogenic E. coli (including commensal as well as intestinal and extra-intestinal pathogenic E. Page 69 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review coli isolates), and Salmonella strains; and that the lpfA1 and lpfA2 genes are highly prevalent among LEE-positive E. coli strains associated with severe and/or epidemic disease (Torres et al., 2009). Further DNA sequence analysis of the lpfA1 and lpfA2 genes from different "Attaching and Effacing" E. coli strains has led to the identification of several polymorphisms and the classification of the major fimbrial subunits in distinct variants (Torres et al., 2009). Using collections of pathogenic E. coli isolates from Europe and Latin America, Torres et al. (2009) demonstrated that the different lpfA types are associated with the presence of specific intimin (eae) adhesin variants, and most importantly, they are found in specific E. coli pathotypes. Their results showed that the use of these fimbrial genes as markers, in combination with the different intimin types, resulted in a specific test to identify E. coli O157:H7 from other pathogenic E. coli strains. Induction of pro-inflammatory responses by polyadhesins. Afa/Dr polyadhesins. Afa/Dr diffusely adhering E. coli strains in polarized monolayers of intestinal T84 cells, were able to promote the basolateral secretion of IL-8 through the activation of the mitogen-activated protein kinases (MAP kinases), including ERK1/2, p38, and SAPK/JNK (stress-activated protein kinase/c-Jun NH2-terminal kinases) kinases (Betis et al., 2003a). IL-8 induced in turn the transmigration across the epithelial monolayer of polymorphonuclear leukocytes (Betis et al., 2003a). The polymorphonuclear leukocytes transepithelial migration induced epithelial synthesis of TNF-α and IL-1β, which in turn promoted the upregulation of DAF, increasing the adhesion of Afa/Dr diffusely adhering E. coli bacteria (Betis et al., 2003b). Moreover, upregulation of the inflammation-associated molecule, MICA, has been found in intestinal Caco-2 cells infected by AfaE-III-positive bacteria, an effect mediated by the specific interaction between bacterial adhesin and DAF (Tieng et al., 2002). Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor has been found increased in Crohn’s disease and ulcerative colitis mucosa. To question whether a pro-inflammatory E. coli Page 70 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review could regulate the expression of vascular endothelial growth factor in human intestinal epithelial cells, Cane et al. (2007) examined the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). Vascular endothelial growth factor mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in vascular endothelial growth factor induction was identified by recombinant E. coli expressing adhesin Dr, purified adhesin Dr and lipopolysaccharide. The signaling pathway activated for the up-regulation of vascular endothelial growth factor was identified by a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. C1845 bacteria induced the production of vascular endothelial growth factor protein which is bioactive. Vascular endothelial growth factor was induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon was not cell line dependent since Cane et al. (2007) reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of vascular endothelial growth factor production requires: (1) The interaction of the bacterial F1845 adhesin with the brush border-associated CD55/DAF acting as a bacterial receptor; (2) The activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. Results demonstrate that Afa/Dr diffusely adhering E. coli strain induces an adhesin- dependent activation of CD55/DAF signaling that leads to the upregulation of bioactive vascular endothelial growth factor in cultured human intestinal cells. Thus, these results suggest a link between an enteroadherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of inflammatory bowel disease pathogenesis. Diard et al. (2006) showed that fragments of polyadhesin Dr are released in response to multiple environmental signals. Production and secretion of fragments of polyadhesin Dr are Page 71 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review clearly regulated by temperature. Secretion of fragments of polyadhesin Dr is drastically increased during anaerobic growth in minimal medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total fimbriae Dr produced by bacteria grown in mineral medium with glucose and LB broth, respectively. Controlled release of fragments of polyadhesin Dr, which are carried out in the absence of cellular lysis, appears independent of the action of proteases or process of maturation. The fragments of polyadhesin Dr secreted into environmental medium by diffusely adhering E. coli strains can provoke unproductive pro-inflammatory responses like the fragments of F1 capsule scattered into cultural media by Y. pestis (see below the chapter "F1 antigen"). F1 antigen. Fragments of recombinant F1 capsule of Y. pestis scattered into cultural media activate mice peritoneal macrophages in vitro (Sodhi et al., 2004). The fragments of F1 capsule induce the production of pro-inflammatory cytokines, TNF-α, IL-1 and IL-6. The activation suggests the involvement of NF-κB and MAPK pathways (Sharma et al., 2005a, b; Sodhi et al., 2004). While IL-1β and F1 stimulate macrophages to produce various pro-inflammatory mediators via the same pathway (Kida et al., 2005), Yersinia virulence factor YopJ, that is essential for the death of infected macrophages, can block host pro-inflammatory responses by inhibiting both NF-κB and MAPK pathways (Zhou et al., 2005). Lemaitre et al. (2006) confirmed that YopJ suppresses TNF-α induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague. Thus, during early stage of infection, the type III secretion system and short non-aggregated F1 Ag act in concert: the former inhibits production of pro-inflammatory cytokines and the latter inhibits binding of IL-1β to the host cell receptors. However, at the final stage of systemic infection, fragments of F1 capsule from the disseminated bacteria can provoke unproductive pro- inflammatory response contributing to a toxic shock and death of the host. Sebbane et al. (2006) demonstrated that high NO levels induced during plague may also influence the developing adaptive immune response and contribute to septic shock. Page 72 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Induction of pro-inflammatory responses by monoadhesins. P pili. Bergsten et al. (2005) uncovered a molecular crosstalk between innate immune Toll-like receptor 4 binds bacterial lipopolysaccharide signaling and P-fimbrial-mediated attachment, which is lipopolysaccharide- independent. Upon P-fimbrial attachment to its glycosphingolipid receptor, ceramide is released from the lipid part of the receptor; in particular, it has recently been shown that ceramide acts as an agonist of Toll-like receptor 4 and potentially acts as a signaling intermediate between Toll- like receptor 4 and the glycosphingolipid receptor (Fischer et al., 2007). Activation of the Toll- like receptor 4 receptor by P-fimbrial attachment subsequently leads to the production of pro- inflammatory cytokines and chemokines (interleukin-6 and CXCL8, respectively) and recruitment of neutrophils (Bergsten et al., 2005). Although this proinflammatory response is beneficial in initiating bacterial clearance, it also causes damage to the surrounding tissue and is associated with renal complications. Since P fimbriae are implicated in triggering inflammation, it can be deduced that they may also contribute to the pathology and symptoms of acute pyelonephritis. FUNCTION-STRUCTURAL CLASSIFICATION OF ADHESIVE ORGANELLES ASSEMBLED WITH CHAPERONE-USHER MACHINERY In fact, the first classification of periplasmic chaperones and organelles assembled by them was suggested by Zav'yalov et al. (1995b). For this aim the 3D structure of the CaflM chaperone was reconstructed by computer modeling, using a primary structure homology between CaflM and PapD proteins, and the atomic coordinates obtained by the X-ray crystallography for PapD (Holmgren & Branden, 1989). In the 3D model of CaflM an accessory sequence between Fl and Gl β-strands (as compared to PapD) was recognized. The sequences of 17 periplasmic chaperones known at that time were aligned and two families with specific structural properties were identified. It was found that the characteristic structural feature of the family of periplasmic Page 73 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review chaperones with the accessory sequence (CaflM family) is the existence of solvent exposed Cys residues in Fl and Gl β-strands which can form disulfide bond in the putative binding site for organelle subunits. The specific functional property of CaflM family is the assisting in assembly of nonfimbrial surface structures or thin fibrillae of a simple composition (i.e., F1 and pH6 antigens of Y. pestis consist of only one subunit) in comparison with the chaperones of PapD family which assist in assembling of more complex thick fimbriae/pili (i.e., P pili are composed of 7 different subinits). Hung et al. (1996) studied 26 chaperones and defined proteins containing a relatively long F1-G1 loop as the FGL chaperone family and proteins with a short F1-G1 loop as the FGS chaperone family. Hung et al. (1996) also revealed that the FGL chaperone family assembles nonfimbrial surface structures or thin fibrillae, composed of 1-2 subunits, while the FGS chaperone family assembles thick fimbriae/ pili, composed of up to 7 subunits. Sequence comparison of 31 chaperones by the neighbor-joining method suggests that the classical chaperone/usher superfamily can be divided into several clades, each including members that apparently share a common ancestor that is not shared by another protein outside of the clade (Bonci et al., 1997). This phylogenetic tree suggests that members of the FGL chaperone family (i.e., MyfB, PsaB, Caf1M, CS3-1, AggD, AfaB, NfaE, SefB, and CssC) share a common ancestor. However, this analysis also shows that the FGS chaperone subfamily cannot be defined by a single node or branch on the phylogenetic tree, suggesting that further subdivision is needed to explicitly categorize the respective fimbriae into clades on the basis of common ancestry. The next principal step in development of function/structural nomenclature of adhesive fimbrial organelles, assembled with the classical chaperone/usher machinery, was the discovery that FGL chaperone-assembled organelles possess polyadhesive function (Zavialov et al., 2007) in distinction to FGS chaperone-assembled monoadhesive thick fimbriae/pili with one adhesive domain on the tip of fibre. Page 74 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Analysis of the currently available data suggests that the classical chaperone/usher machinery involves three distinct families and a few subfamilies of surface-exposed adhesive organelles, which have unique functional and structural properties (Table 2). The FGL chaperone-assembled adhesive organelles represent linear polymers of one or in some cases two distinct types of protein subunits. The characteristic feature of the organelles is that each protein subunit in the fiber possesses two independent binding sites, which are specific to different receptors on cells of the host. Such architecture enables a single fiber to establish polyvalent contacts with receptors on the host cells. The FGL chaperone-assembled polyadhesins may be subdivided into three subfamilies (Table 2): (1) FGL chaperone-assembled polyadhesins-1-1 where the first numeral shows that organelles of this subfamily are homopolymers composed of only one subunit. The second numeral displays that assembly of the organelles is assisted by one chaperone; (2) FGL chaperone-assembled polyadhesins-(1+1)-1 where the numerals in parentheses indicate that organelles of this subfamily are heteropolymers composed of two distinct subunits secreted via different pathways. One subunit is secreted with the classical chaperone/usher machinery, and another subunit is displayed on the tip of fibre with type II secretion system. The numeral out the parentheses indicates that assembly of the organelles is assisted with one chaperone; (3) FGL chaperone-assembled polyadhesins-2-1 where the first numeral shows that organelles of this subfamily are heteropolymers composed of two distinct subunits both of which are secreted with the chaperone/usher machinery. The second numeral shows that assembly of the organelles is assisted by one chaperone. The majority of FGS chaperone-assembled adhesive fimbriae/pili are monoadhesins, which display only one adhesin domain on the tip of the pilus. The FGS chaperone-assembled monoadhesins may be subdivided into six subfamilies (Table 2): (1) The subfamily of FGS chaperone-assembled monoadhesins-2-1 collects the structurally simplest mono-adhesive organelles composed of two subunits one of which is structural and Page 75 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review another contains adhesive domain exposed on the tip of pili. Assembly of the organelles is assisted with one chaperone. (2) The subfamily of FGS chaperone-assembled monoadhesins-3-1 includes the mono-adhesive organelles composed of three subunits two of which are structural and one contains adhesive domain exposed on the tip of pili. Assembly of the organelles also is assisted with one chaperone. (3) The subfamily of FGS chaperone-assembled monoadhesins-3-3 also represents the mono- adhesive organelles composed of three subunits two of which are structural and one is specific adhesin exposed on the tip of pili. However, assembly of these organelles is assisted by three distinct chaperones. (4) The subfamily of FGS chaperone-assembled monoadhesins-4-1 collects the mono-adhesive organelles composed of four subunits three of which are structural and one contains adhesive domain exposed on the tip of pili. Assembly of the organelles is assisted with one chaperone. (5) The subfamily of FGS chaperone-assembled monoadhesins-5-1 represents the mono- adhesive organelles composed of five subunits four of which are structural and one is specialized adhesin exposed on the tip of pili. Assembly of the organelles is assisted with one chaperone. (6) The subfamily of FGS chaperone-assembled monoadhesins-7-1 is the most complex subfamily of mono-adhesive organelles composed of seven subunits six of which are structural and one is specialized adhesin exposed on the tip of pili. Assembly of the organelles is assisted by one chaperone. The FGS chaperone-assembled thin flexible pili F4 (K88), F5 (K99) and Lda of E. coli, PE (plasmid-encoded) fimbriae of S. typhimurium, atypical fimbriae ACIAD of Acinetobacter sp. and ATF of P. mirabilis, however are an exception, as they do not display specialized adhesive domains on the tip of the pilus, but carry binding site on their main structural subunit (FaeH, FanH and LdaH) or are composed of only one structural subunit that is functioning as an Page 76 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review adhesin subunit (ACIAD122, AtfA and PefA). Such architecture enables a single fiber to establish polyvalent contacts with receptors on the host cells. Therefore we called this family as FGS chaperone-assembled polyadhesins. These polyadhesins may be subdivided into five subfamilies (Table 2): (1) The subfamily of FGS chaperone-assembled polyadhesins-1-1 collects the poly-adhesive homopolymers. Assembly of the organelles is assisted by one chaperone; (2) The subfamily of FGS chaperone-assembled polyadhesins-1-2 also represents the poly- adhesive homopolymer. However, assembly of the fiber is assisted with two distinct chaperones; (3) The subfamily of FGS chaperone-assembled polyadhesins-3-1 includes the heteropolymer composed of three distinct subunits. The organelle carries binding site on its main structural subunit. Assembly of the polyadhesin is assisted by one chaperone; (4) The subfamily of FGS chaperone-assembled polyadhesins-4-1 includes the heteropolymer composed of four distinct subunits. The organelle carries binding site on its main structural subunit. Assembly of the polyadhesin is assisted by one chaperone; (5) The subfamily of FGS chaperone-assembled polyadhesins-5-1 includes the heteropolymers composed of five distinct subunits. The organelles carry binding site on their main structural subunit. Assembly of the polyadhesins is assisted with one chaperone. PHYLOGENESIS OF THE CLASSICAL CHAPERONE/ USHER GENE CLUSTERS It is very interesting to compare the suggested function-structural nomenclature of adhesive organelles, assembled with the classical chaperone/usher machinery, and their phylogenetic classification. Sequence comparison of chaperones (Bonci et al., 1997) may not be ideally suited for developing of a phylogenetic subdivision because some fimbrial operons encode more than one Page 77 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review chaperone, thus raising the question as to which protein should be used to assign the respective operon to a phylogenetic group. Therefore comparison of usher sequences has been used to derive phylogenetic trees of members of the chaperone/usher assembly class (Anantha et al., 2004; Yen et al., 2002). This approach has the advantage that the resulting definition of phylogenetic groups is unambiguous, because all fimbrial operons belonging to the chaperone/usher assembly class contain only a single usher gene. An initial comparison of 58 members of the fimbrial usher protein (FUP) superfamily distinguished 10 clusters on the basis of common ancestry (Yen et al., 2002). A revised phylogenetic tree of the FUP superfamily constructed by comparing 189 proteins is shown in Fig. 13 (Nuccio & Bäumler, 2007). The phylogenetic analysis of 189 usher proteins suggests a classification into clades, which is similar but not identical to that proposed based on analysis of 58 usher proteins (Yen et al., 2002). Nuccio & Bäumler (2007) proposed a nomenclature using Greek letters to refer to individual clades. Using a node-based definition, the FUP superfamily can be divided into six clades, designated α, β, γ, κ, π, and σ-fimbriae, each stemming from a common ancestor represented by a node in the phylogenetic tree (Fig. 13). The γ-fimbrial clade is further subdivided into four clades, termed γ1, γ2, γ3, and γ4-fimbriae. Nuccio & Bäumler (2007) assigned arbitrarily α, β, γ, κ, π, and σ-fimbrial clade names to recall a particular characteristic of the clade or a prominent member as follows: α-fimbriae, alternate chaperone/usher family; κ-fimbriae, K88 (F4) fimbriae; π-fimbriae, pyelonephritis-associated fimbriae (P fimbriae); and σ-fimbriae, spore coat protein U from Myxococcus xanthus. The β- and γ-fimbriae were assigned names alphabetically. This subdivision of the FUP superfamily largely confirms the subdivisions proposed initially (Yen et al., 2002), but former FUP clusters 4 and 5 now form a single clade (γ3-fimbriae). The subdivision of the chaperone/usher class into six FUP clades confirms that the alternate chaperone/usher family (α-fimbriae) (Anantha et al., 2004) contains operons that stem from a common ancestor (Fig. 13; Nuccio & Bäumler, 2007). The FGL chaperone assembled family of polyadhesins forms a monophyletic group (γ3-fimbriae) within the classical chaperone/usher Page 78 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review family (Hung et al., 1996; Zav'yalov et al., 1995b). However, the FGS chaperone assembled family of adhesive organelles is composed of several clades (β-, γ1-, γ2-, γ4-, κ-, and π-fimbriae) that are not more closely related to each other than to the FGL chaperone assembled family of polyadhesins (γ3-fimbriae). The analysis also reveals the existence of a major FUP clade (σ- fimbriae) that was represented only by two usher proteins in a previous analysis (Yen et al., 2002) and whose members share limited or no sequence homology to members of the alternate chaperone/usher family (α-fimbriae) or the classical chaperone/usher family (β-, γ-, κ-, and π - fimbriae). The utility of delineating the genealogy of gene clusters that encode fimbrial adhesins lies in its value for predicting their evolutionary relationship. For instance, gene clusters of the γ3- and κ-fimbrial clades, identified by Nuccio & Bäumler (2007), encode exclusively FGL and FGS chaperone assembled polyadhesins, respectively, while gene clusters belonging to the α-, γ1-, γ2- , γ4-, and π-fimbrial clades exclusively encode monoadhesins. When the most common gene clusters within each clade are placed at the end of each of the corresponding branches on the FUP tree, an evolutionary scenario explaining the divergence of gene clusters from a common precursor can be derived. The exact relationship between major clusters in the FUP tree (Fig. 13; Nuccio & Bäumler, 2007) is currently not clear, since the nodes connecting α, β, γ, κ, π, and σ- fimbrial clades are supported by low bootstrapping values. However, the gene clusters of κ- and π-fimbriae are related to each other, as both share a core structure composed of genes encoding a major subunit, an usher, and a chaperone. Furthermore, a close relationship of the γ-fimbriae to the κ- and π-fimbriae is indicated by the presence of a PFAM00419 domain exclusively in subunits of gene clusters belonging to these three clades (Nuccio & Bäumler, 2007). These data suggest that members of the γ-, κ-, and π-fimbrial clades form a monophyletic group, which will be referred to as the γκπ cluster from here on. The γκπ cluster and the β-fimbriae together comprise the previously defined classical chaperone/usher superfamily. Page 79 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review The FGL chaperone-assembled polyadhesins that correspond to γ3-fimbriae (Fig. 14; Nuccio & Bäumler, 2007) comprise the adhesive organelles, which consist of only one or two distinct types of subunits and at low resolution typically have non-pilus, amorphous or capsule-like morphology (Anderson et al., 2004a,b; Hung et al., 1996; Korotkova et al., 2006a,b, 2008; Li et al., 2007; Pettigrew et al., 2004; Remaut et al., 2006; Salih et al., 2008; Soto & Hultgren, 1999; Verger et al., 2007; Westerlund-Wikström & Korhonen, 2005; Zavialov et al., 2003, 2005, 2007). Their notable property is that all subunits possess two independent binding sites specific to different host cell receptors. In particular, DraE/AfaE/DaaE subunits of the Dr/Afa polyadhesins have two independent binding sites to CD55/DAF and CEACAMs (Fig. 9; Anderson et al., 2004a, b; Korotkova et al., 2006a, b; 2008a; Pettigrew et al., 2004). The PsaA subunit of pH6 antigen binds to β1-linked galactosyl residue of glycosphingolipids (Payne et al., 1998) and to phosphorylcholine moiety of phosphatidylcholine (Galván et al., 2006) as the host cell receptors. The κ-clade (Fig. 15; Nuccio & Bäumler, 2007) comprises all subfamilies of the FGS chaperone-assembled polyadhesins, in particular, the FGS chaperone-assembled polyadhesins-1- 1 (Pef pili), -3-1 (AF/R1 pili), -4-1 (K88 pili), and -5-1 (K99 pili, REPEC fimbriae, and afimbrial adhesin, encoded by the locus for diffuse adherence, lda). Like the FGL polyadhesive fibers, the FGS polyadhesins carry binding site on their main structural subunit (FaeH, FanH and LdaH) or are composed of only one structural subunit that is functioning as an adhesin subunit (ACIAD122 and PefA). In contrast to mono-adhesive pili, which possess only one binding domain on the tip of pilus (Fig. 12a), each poly-adhesive fiber potentially might (Fig. 12b) ensuring powerful polyvalent fastening of a bacterial pathogen to a host target cell (Galván et al., 2006), and aggregating host cell receptors by a zipper-like mechanism that trigger transduction of signals causing immunosuppressive and pro-inflammatory responses (Galván et al., 2006; Sharma et al., 2005a,b; Sodhi et al., 2004). Page 80 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review The γ2-clade (Fig. 14; Nuccio & Bäumler, 2007), corresponds to the FGS chaperone- assembled monoadhesins-3-3 which are representing the mono-adhesive organelles composed of three subunits two of which are structural and one is specific adhesin exposed on the tip of pili. Assembly of these organelles is assisted by three distinct chaperones. Although the FasB, CswB and FotB chaperones belong to the FGS family, they possess the same length of F1-G1 loop as the CssC1 and CssC2 chaperones of the FGL family (see Fig. 4). The γ1-clade (Fig. 14; Nuccio & Bäumler, 2007) comprises some of the members of the FGS chaperone-assembled monoadhesins-3-1, 4-1 and 5-1, however, the Ambient-temperature fimbriae (Atf) of Pr. mirabilis related to the FGS chaperone-assembled polyadhesins-1-1, is the exception. Like the most of FGL chaperone assembled polyadhesins, Atf fimbriae are polyadhesive homopolymers. The γ4-clade (Fig. 14; Nuccio & Bäumler, 2007) includes some of the members of the FGS chaperone-assembled monoadhesins-2-1, -3-1, 4-1 and 5-1. The π-clade (Fig. 15; Nuccio & Bäumler, 2007) consists of some of the members of the FGS chaperone-assembled monoadhesins-3-1, -4-1, -5-1, and -7-1. The remarkable feature of the κ- and π-gene clusters is the location of gene encoding chaperone after gene encoding usher, while in the γ-clusters gene encoding chaperone, precedes gene for usher. This finding underlines more close phylogenetic relationships between κ- and π- clusters of genes than with the γ-clusters. APPLICATIONS OF ADHESIVE ORGANELLES ASSEMBLED WITH CHAPERONE/USHER MACHINERY Applications of Polyadhesins Page 81 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Applications of polyadhesins for vaccine design. Anti-plague vaccines based on the recombinant F1 antigen or on peptides from the F1 antigen. Y. pestis, the causative agent of pneumonic plague, is a rapidly progressing and exceptionally virulent disease (reviewed by Cleri et al., 1997; Perry & Fetherston, 1997; Smiley, 2008a, b). Extensively antibiotic-resistant strains of Y. pestis exist and a safe and effective pneumonic plague vaccine is currently absent (Smiley, 2008a, b). These facts raise concern that Y. pestis may be exploited as a biological weapon. F1 antigen is the major or single protective component of the current human whole-cell vaccines against plague (Li et al., 2005; Li & Yang, 2008; Smiley, 2008a, b). This vaccine is, however, ineffective against pneumonic plague caused by typical F1+ strains of Y. pestis (Heath et al., 1998). It is also ineffective against F1‒ Y. pestis strains, which have been isolated from one human patient and from several rodents. For these reasons, new recombinant plague vaccines comprising Caf1 and V antigens of Y. pestis are under development. Heath et al. (1998) developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. V antigen is an essential virulence factor and mediator of immunity common for Yersinia. It plays a crucial role in the functioning of the type III secretion system. This protein forms a distinct structure at the tip of injectisome needle (Mueller et al., 2005). Derewenda et al. (2004) solved the three-dimensional structure of V antigen with high resolution X-ray analysis. The developed recombinant F1-V fusion vaccine protected mice against pneumonic as well as bubonic plague produced by either an F1+ or F1‒ strain of Y. pestis and provided a better protection than recombinant F1 or recombinant V alone against the F1+ strain. Therefore, the recombinant proteins serve as the basis of an improved human anti-plague vaccine (Heath et al., 1998). Powell et al. (2005) re-engineered a two-component F1-V fusion protein antigen and tested as a medical countermeasure against the possible biological threat of aerosolized Y. pestis. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V fusion protein also protected mice from wild-type and non-encapsulated Y. pestis Page 82 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review challenge strains, modeling prophylaxis against pneumonic and bubonic plague. Jones et al. (2006) found that the rF1‒V antigen given intramuscularly with Alhydrogel adjuvant protects mice against the challenge, but is less effective in nonhuman primates against high-dose aerosolized Y. pestis, perhaps because the antigen fails to induce respiratory immunity. Mice immunized intranasally with rF1‒V formulated with a proteosome-based adjuvant (ProtollinTM) were 100% protected against aerosol challenge with 170 LD50 of Y. pestis and 80% protected against 255 LD50 (Jones et al., 2006). Indeed, the examination of different prime- boost regimens with rF1‒V demonstrated that inclusion of an appropriate adjuvant is critical for nonparenteral immunization (Glynn et al., 2005). In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, Honko et al. (2006) evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in subunit antiplague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen and flagellin exhibited dramatic increases in anti-F1 plasma IgG titers that remained stable over time. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93–100% survival of immunized mice. Vaccination of cynomolgus monkeys with flagellin and the rF1‒V fusion protein induced a robust antigen-specific IgG antibody response. Alvarez et al. (2006) developed a novel production and delivery system for an anti-plague vaccine of the rF1‒V fusion protein expressed in tomato. The immunogenicity of the rF1‒V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially- produced rF1‒V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit made it possible to produce an oral vaccine candidate without protein purification and with minimal processing technology. The recombinant plague antigens F1, V, and fusion protein F1-V were produced by transient expression in Nicotiana benthamiana using a reconstructed tobacco mosaic virus-based system that allowed very rapid and extremely high levels of expression (Santi et al., 2006). All of the plant-derived purified antigens, administered Page 83 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review subcutaneously to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis. Chichester et al. (2009) reported a plague vaccine consisting of the F1 and LcrV antigens fused to a single carrier molecule, the thermostable enzyme lichenase from Clostridium thermocellum, and expressed in and purified from Nicotiana benthamiana plants. When administered to Cynomolgus Macaques this purified plant-produced vaccine induced high titers of serum IgG, mainly of the IgG1 isotype, against both F1 and LcrV. These immunized animals were subsequently challenged and the LcrV-F1 plant-produced vaccine conferred complete protection against aerosolized Y. pestis. Del Prete et al. (2009) produced in Nicotiana benthamiana F1 and V antigens, and F1-V fusion protein and administered them to guinea pigs resulted in immunity and protection against an aerosol challenge of virulent Y. pestis. They examined the effects of plant-derived F1, V, and F1-V on human cells of the innate immunity. F1, V, and F1-V proteins engaged TLR2 signalling and activated IL-6 and CXCL-8 production by monocytes, without affecting the expression of TNF- α, IL-12, IL-10, IL-1β, and CXCL10. Native F1 antigen and plant-derived rF1 and rF1-V all induced similar specific T-cell responses, as shown by their recognition by T-cells from subjects who recovered from Y. pestis infection. Native F1 and rF1 were equally well recognized by serum antibodies of Y. pestis-primed donors, whereas serological reactivity to rF1-V hybrid was lower, and that to rV was virtually absent. In conclusion, plant-derived F1, V, and F1-V antigens are weakly reactogenic for human monocytes and elicit cell-mediated and humoral responses similar to those raised by Y. pestis infection. The subunit vaccine involving a mixture of recombinant F1 and V antigens protects mice against exposure to 4104 CFUs of virulent plague organisms (100 LD50 doses), whereas the whole cell vaccine provided only 50% protection against 1.8-103 CFUs (Williamson et al., 1997). The enhanced protective efficacy of this subunit vaccine over existing vaccines has been demonstrated in an animal model of pneumonic plague. Bronchopulmonary administration of the combined subunits (1 mg V plus 5 mg F1) entrapped within microspheres composed of a Page 84 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review biodegradable polyester (poly-L-lactide) elicits a similar level of protective immunity against systemic plague infection as that evoked by injecting coencapsulated subunits into the muscle (Eyles et al., 2000). Such findings indicate that introduction of appropriately formulated F1 and V subunits into the respiratory tract may be an alternative to parenteral immunization schedules for protecting individuals from plague (Eyles et al., 2000). Elvin et al. (2006) individually encapsulated the recombinant F1 and V antigens in polymeric microspheres; the same antigen was adsorbed to the surface of these microspheres. Virulent challenge experiments showed that noninvasive immunization by intranasal instillation can provide strong systemic and local immune responses and protect against high-level challenge. Recently Thomas et al. (2009) reported that the pathogenesis patterns of plague infections caused by the deposition of 1- and 12-µm-particle aerosols of Y. pestis in the lower and upper respiratory tracts (URTs) of mice are different. The median lethal dose for 12-µm particles was 4.9-fold higher than that for 1-µm particles. The 12-µm-particle infection resulted in the degradation of the nasal mucosa and nasal- associated lymphoid tissue (NALT) plus cervical lymphadenopathy prior to bacteremic dissemination. Lung involvement was limited to secondary pneumonia. In contrast, the 1-µm- particle infection resulted in primary pneumonia; in 40% of mice, the involvement of NALT and cervical lymphadenopathy were observed, indicating entry via both URT lymphoid tissues and lungs. Despite bacterial deposition in the gastrointestinal tract, the involvement of Peyer's patches was not observed in either infection. Although there were major differences in pathogenesis, the recombinant F1 and V antigen vaccine and ciprofloxacin protected against plague infections caused by small- and large-particle aerosols. Human immune response to the recombinant plague vaccine comprising F1 and V antigens was assessed during a phase 1 safety and immunogenicity trial in healthy volunteers (Williamson et al., 2005). All the subjects produced specific IgG in serum after the priming dose, which peaked in value after the booster dose (day 21). However, no significant vaccination-related change in activation of peripheral blood mononuclear cells was detected at any time. Thus, any Page 85 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review evidence on the cell immune response to recombinant F1 and V antigens is missing. Williamson et al. (2005) suppose that it may be associated with the immunosuppressive action of these antigens. DeBord et al. (2006) therefore designed the recombinant V10 (rV10) variant lacking residues 271–300. This variant does not suppress the release of proinflammatory cytokines by immune cells. In contrast to Y. pestis LcrV, the immunization with rV10 generates robust antibody-induced protective responses against bubonic plague and pneumonic plague, suggesting that rV10 may serve as an improved component of anti-plague vaccine. The antigen structure may critically influence on the protective immune responses (Watts, 2004). The data on the structural and thermodynamic properties of Caf1 (Zavialov et al., 2003, 2005) can explain the failure to induce the cell immune response to this antige. The structurally observed complete collapse of the donor-strand-complemented fiber Caf1 subunit results in a dramatic increase in the enthalpy and transition temperature for the melting of the fiber module (Zavialov et al., 2005). The collapse of the hydrophobic core of subunits shifts the equilibrium towards fiber formation (Zavialov et al., 2003, 2005). As a result, the temperature of melting of the fiber subunit increases as high as to 90°C (Zavialov et al., 2005). The subunit preserves practically the same stability at pH 2.4 (Fooks et al., personal communication). It can be deduced that such a high stability can reduce the processing of Caf1 antigen in macrophages to CD4 T cell epitopes and therefore abolish cellular immune responses. Indeed, Musson et al. (2006) found that optimal T cell responses required significantly extended exposure of antigen presenting cells to highly stable polymeric Caf1 compared with Caf1 that was depolymerized and destabilized by heating. Destabilization of Caf1 caused a shift toward presentation by mature MHC class II and toward independence of low pH and proteolytic processing. An overcoming of low proteolytic processing of highly stable native Caf1 may be the attempt to develop an anti-plague vaccine based upon the peptide conjugates made between different B- and T-cell epitopes of F1 antigen of Y. pestis (Sabhnani & Rao, 2000; Sabhnani et al., 2003; Tripathi et al., 2006). Intranasal immunization generated consistently high titers and a Page 86 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review longlasting immune response for both IgG and IgA in sera and secreted IgA in washes, whereas the intramuscular route generated peak IgG levels in sera only. In vivo protective studies showed that B1–T1 and B2–T1 peptide conjugates protected the mice till day 15. Anti-plague passive immunization with monoclonal antibodies against the F1 antigen. The newest anti-plague vaccines based on the F1 and V antigens provide a high degree of protection. However, they must be administered several weeks before exposure to prevent plague. It is unlikely that vaccines will provide post-exposure protection against plague. As an alternative to vaccines, the passive immunization with monoclonal antibodies against the F1 protein has been demonstrated to be effective in mice for protection from fatal bubonic and pneumonic plague (Anderson et al., 1997). Moreover, Hill et al. (2003) showed that intraperitoneal injection of monoclonal antibodies that target the F1 and LcrV proteins protected mice in a synergistic manner as either a pretreatment or a post-exposure therapy. Recently, Hill et al. (2006) demonstrated that intratracheal delivery of aerosolized monoclonal antibodies with specificity for LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting post-exposure treatment for plague. The efficacy of passive immunization with monoclonal IgG antibodies specific for Caf1 suggests that opsonization is a major mechanism to overcome the resistance of Y. pestis to phagocytosis conferred by the Caf1 capsule. Application of polyadhesin Saf for design of Salmonella vaccine. Typhoid fever caused by S. enterica serovar Typhi (S. Typhi), which is a predominantly human pathogen, remains a burden problem in India and worldwide (Crump et al., 2004; Hamid & Jain, 2007). The mortality rates in untreated typhoid fever infections can be 10–15%. There are estimated 20 million cases and 200,000 deaths worldwide each year (Crump et al., 2004). In some instances patients recover but remain carriers of the bacteria for many years. Another clinical syndrome associated with Salmonella infection is nontyphoidal salmonellosis – a gastrointestinal disease also known as enteritis. Components of the Salmonella atypical fimbriae (Saf) were investigated for Page 87 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review inclusion in a vaccine (Strindelius et al., 2004). A complex of recombinant SafB chaperone with SafD adhesin was expressed in E. coli and purified. Starch microparticles were used as the adjuvant. The recombinant cholera toxin B subunit (rCTB) was included as mucosal antigen- uptake enhancer. BALB/c mice were immunized orally or subcutaneously with SafB/D- and rCTB-conjugated microparticles and intranasally or subcutaneously with SafB/D mixed with rCTB. The systemic and mucosal immune responses were studied. An oral challenge with S. enteritidis was performed. All the immunized groups, except the group receiving oral immunization, responded with high IgM–IgG titers to SafB/D. Analysis of the subclass ratio (IgG1/IgG2a1IgG2b) indicated a mixed Th1 and Th2 response, with Th1 predominating. Only the group receiving intranasal immunization got the mucosal response, measured as specific IgA/total IgA (from fecal samples), significantly higher than that in the untreated control group (P < 0.05). Spleens were removed 6 days after oral challenge and Salmonella CFU were counted. The group immunized subcutaneously with SafB/D- and rCTB-conjugated microparticles had significantly lower CFU counts than the untreated control group (P < 0.05). Application of sefA gene for design of a live recombinant Salmonella vaccine. Lopes et al. (2006) cloned the sefA gene, which encodes the main subunit of the SEF14 fimbrial protein, into a temperature-sensitive expression vector and transformed it into a nonpathogenic, avirulent strain of E. coli. The recombinant strain was used as a vaccine to elicit specific immune response against the SefA protein of S. enteritidis in 1-day-old chickens. The recombinant strain was reisolated from the intestines of treated birds for up to 21 days after treatment, demonstrating its ability to colonize the intestinal tracts of 1-day-old chickens. In addition, IgA against the SefA protein was detected by ELISA in intestinal secretions from treated birds 7 days after treatment and in bile samples 14–21 days after treatment. Non-treated birds did not show any evidence of intestinal colonization by the recombinant strain or anti-SefA IgA response in their bile or intestinal secretions. Thus, the preliminary evaluation of the recombinant strain showed a potential use of this strain to elicit protection against S. enteritidis infection in chickens. Page 88 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Vaccination with E. coli polyadhesin Dr against chronic urinary tract infection. E. coli expressing Dr fimbria and related adhesins are associated with urinary tract infection, including cystitis and/or pyelonephritis and diarrhea. Children and pregnant women are prone to recurrent or persistent infections caused by these organisms (Garcia et al., 1996). Goluszko et al. (2005) used purified E. coli Dr fimbrial antigen to vaccinate C3H/HeJ mice against an experimental urinary tract infection due to a homologous strain bearing Dr polyadhesins. They demonstrated reduced mortality in the vaccinated animals. Immune sera with high titers of anti-Dr antibody inhibited bacterial binding to bladders and kidneys but did not affect the rate of renal colonization. Oral vaccination with E. coli F4 (K88) poly-adhesive fimbriae against intestinal infection. Enterotoxigenic E. coli (ETEC) is the leading cause of diarrhea in piglets and newborn calves. Massive efforts have therefore been made to develop a vaccine for the induction of protective mucosal immunity against ETEC. Verdonck et al. (2009) showed that as a result of oral immunization of piglets with F4 fimbriae purified from pathogenic enterotoxigenic E. coli, the fimbriae bind to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune response. F4 fimbriae are very stable polymeric structures composed of some minor subunits and a major subunit FaeG that is also the fimbrial adhesin. Verdonck et al. (2009) identified with the mutagenesis experiments FaeG amino acids 97 (N to K) and 201 (I to V) as determinants for F4 polymeric stability. The interaction between the FaeG subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae behaved identically in F4R binding and showed equal stability in the gastro-intestinal lumen. Oral immunization experiments indicated that a higher degree of polymerisation of the fimbriae in the intestine was correlated with a better F4-specific mucosal immunogenicity. Hu et al. (2009) developed recombinant Lactococcus lactis which expresses K88 (F4) fimbrial polyadhesin FaeG for oral vaccination. They demonstrated protective immune response in mice to FaeG. Recently Remer et al. (2009) constructed the recombinant strain EcN pMut2-kanK88 (EcN-K88) stably expressing the Page 89 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review determinant for the K88 fimbrial adhesin of ETEC on the bacterial surface. After oral application of EcN-K88 to mice for one week, EcN-K88 as well as wild-type EcN and EcN mock- transformed with the plasmid vector only could be detected in faecal samples for a minimum of 7 days after the last feeding, indicating that EcN can transiently colonise the murine intestine. Oral application of EcN-K88 resulted in significant IgG serum titres against K88 as early as 7 days after the initial feeding with EcN-K88, but no significant IgA titres. In contrast, Remer et al. (2009) failed to detect any specific T cell responses towards the K88 antigen both in spleen and mesenteric lymph nodes. Although dendritic cells readily upregulated maturation and activation markers in response to K88 stimulation, accompanied by secretion of interleukin (IL)- 12, IL-6, IL-10, and tumour necrosis factor, restimulation of T cells from mice having received EcN-K88 with K88-loaded dendritic cells did not result in detectable T cell proliferation and IL- 2 secretion, but rather induced an IL-10 bias. While the serum antibody responses clearly demonstrate that K88 is recognized by the humoral immune system, the findings of Remer et al. (2009) indicate that oral application of probiotic EcN expressing the K88 fimbrial adhesin does not induce a selective T cell response towards the antigen. Applications of polyadhesins for expression of heterologous proteins. Application of polyadhesins for design of antiviral vaccines. The potential of the major structural subunit DraE of E. coli Dr fimbriae has been used to display a peptide of glycoprotein D derived from Herpes simplex virus (HSV) type 1 (Zalewska et al., 2003). The heterologous sequence mimicking an epitope from glycoprotein D was inserted in one copy into the draE gene in place of a predicted 11-amino acid sequence in the N-terminal region of surface-exposed domain 2 within the conserved disulfide loop (from Cys21 to Cys53). The inserted epitope was displayed on the surface of the chimeric DraE protein as evidenced by immunofluorescence and was recognized by monoclonal antibodies to the target HSV glycoprotein D antigen. Conversely, immunization of rabbits with purified chimeric Dr-HSV fimbriae resulted in a serum that specifically Page 90 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review recognized the 11-amino acid epitope of HSV glycoprotein D, indicating the utility of the strategy employed (Zalewska et al., 2003). Application of polyadhesins for expression of cytokines. The ability of the Caf1M chaperone/Caf1A usher pathway to express large amounts of F1 antigen (Caf1) in E. coli was investigated to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit (Zavialov et al., 2001). Despite correct processing of chimeric protein composed of a modified Caf1 signal peptide, mature human IL-1β (hIL-1β), and mature Caf1, the processed product (hIL-1β–Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1β–Caf1 in the periplasm. Soluble hIL-1β– Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1β. The results indicate that the Caf1M-induced release of hIL-1 β–Caf1 from the inner membrane promotes folding of the hIL-1β domain. Similar results were obtained with the fusion of Caf1 to hIL-1ra or to human GM-CSF. Following co-expression of the hIL-1β: Caf1 precursor with the Caf1M chaperone and Caf1A outer membrane protein, hIL-1β–Caf1 could be detected on the cell surface of E. coli (Zavialov et al., 2001). These results demonstrated for the first time the potential of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represented a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains (Korpela et al., 1999). Applications of Monoadhesins Applications of monoadhesins for vaccine design. Vaccination with FimH adhesin against infection by uropathogenic E. coli. Virtually all uropathogenic strains of E. coli, the primary cause of cystitis, assemble adhesive surface organelles called type 1 pili that contain the FimH adhesin. Langermann et al. (1997) demonstrated that sera from animals vaccinated with Page 91 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review candidate FimH vaccines inhibited uropathogenic E. coli from binding to human bladder cells in vitro. They found that immunization with FimH reduced in vivo colonization of the bladder mucosa by more than 99% in a murine cystitis model, and immunoglobulin G to FimH was detected in urinary samples from protected mice. Furthermore, passive systemic administration of immune sera to FimH also resulted in reduced bladder colonization by uropathogenic E. coli. Later Langermann et al. (2000) studied 4 monkeys inoculated with 100 µg of FimCH adhesin- chaperone complex mixed with MF59 adjuvant, and 4 monkeys given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 ml of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans. Monoadhesins as targets for specific inhibition of adhesion. Mannose-binding type 1 pili are important virulence factors for the establishment of E. coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Bouckaert et al. (2005) provided the binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from an uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Their results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl α-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd=0.15 µM) than mannose (Kd=2.3 µM). Exploration of the binding affinities of α-D-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with Page 92 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. Wellens et al. (2008) demonstrated that α-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl α-D-mannosides was elucidated in the crystal structure of the FimH receptor- binding domain in complex with oligomannose-3. FimH interacts with Man1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The interactions along the α1,3 glycosidic bond and the first β1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl α-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted with an aromatic group. Chaperone/usher assembly-translocation machinery as target for a new generation of antimicrobials interrupting assembly of adhesive organelles. Pinkner et al. (2006) rationally designed small compounds that specifically inhibit biogenesis of adhesive pili assembled by the chaperone–usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2- pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic E. coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by ≈90% in laboratory and clinical E. coli strains. Fig. 16 shows stereoimage of the PapD–pilicide complex (Pinkner et al., 2006) in overlay with the FimD1–125 N-terminal usher domain in complex with the FimC-FimH158–279 chaperone–adhesion complex (Fig. 16; Nishiyama et al., 2005). The Page 93 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review conserved hydrophobic patch across the back of the F1-C1-D1 β-sheet formed by residues I90/I93, L32/L32, and L54/V56 (FimC/PapD) forms part of both the usher-interaction site and the pilicide-binding site. In the chaperone–pilicide interaction, the plane of the 2-pyridone system and the R1-cyclopropyl and R2-CH2-naphthyl substituents coincide with and mimic the interactions made by the F4, L19, and F22 side chains from the usher N-terminal domain. The overlay demonstrates the steric clash between the pilicide and the usher N-terminal domain. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding (Pinkner et al., 2006). Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone–subunit complexes to the usher (Pinkner et al., 2006). These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors. CONCLUSIONS AND FUTURE PERSPECTIVES Extensively antibiotic-resistant strains of Gram-negative pathogens emerged during the last dozen of years whereas safe and effective vaccines against many of them currently are absent. There are now a growing number of reports of cases of infections caused by Gram-negative organisms for which no adequate therapeutic options exist (de Jong & Ekdahl, 2006; Giske et al., 2008). This return to the pre-antibiotic era has become a reality both in Europe as in other parts of the world. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Two general strategies exist to inhibit fimbrial adhesion mediated functions (Cegelski et al., 2008; Cusumano & Hultgren, 2009): (1) The specific inhibition of adhesion, which involves physically precluding pathogen binding to host cells, for example, with carbohydrate derivatives of host ligands. Page 94 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (2) The interruption of fimbrial adhesion assembly, which also blocks adhesion as well as invasion and intracellular biofilm formation. The first strategy is most effective for specific inhibition of monoadhesins. It was demonstrated that α-D-mannose based inhibitors of FimH adhesin not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation (Bouckaert et al., 2005; Wellens et al., 2008). The second general strategy to inhibit fimbrial adhesion mediated functions is targeted to the classical chaperone/usher assembly-translocation machinery (Pinkner et al., 2006; Aberg & Almqvist, 2007). The recently solved structure of the usher translocator pore of the twinned-pore translocation machinery created a ground for a rational design of a new generation of antimicrobials interrupting assembly of adhesive organelles. However, chaperone/usher machinery contains a few crucial details that are not studied well yet. They are likely to be the major direction for future studies. The revealed strong correlation between a number of residues in a F1-G1 loop of chaperones and a number of subunits operating by them was as the basis for the novel function- structural classification of the fimbrial adhesins. The FGS chaperone-assembled polyadhesive fimbriae were discovered in addition to the previously found family of the FGL chaperone- assembled polyadhesins. The FGL and FGS chaperone-assembled polyadhesins are encoded exclusively by the gene clusters of the γ3- and κ-monophyletic groups, respectively, while gene clusters belonging to the γ1-, γ2-, γ4-, and π-fimbrial clades exclusively encode monoadhesins (Nuccio & Bäumler, 2007). Poly-adhesive binding possesses an advantage over mono-adhesive binding because it would result in formation of more powerful and tight contact between the pathogen and the host cell and it may lead to a massive aggregation of the receptors. This subsequently would trigger subversive signals directed to mislead functions of host cells, in particular, the cells of immune system. Anti-immune function is likely to be common for all fimbrial polyadhesins including FGS chaperone-assembled poly-adhesive fimbriae/pili. Hence, Page 95 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review a search for binding to immune system associated receptors could be a starting point for functional characterization of known and newly revealed members of both FGL and FGS chaperone-assembled families of polyadhesins. The fimbrial polyadhesins that are represented by the linear homopolymers of hundreds to thousands of subunits have very high potential for cross-linking of B cell receptors and stimulation of antibody production. They are specific and very powerful surface antigens typical only for pathogenic strains. Therefore they are promising candidates for development of recombinant vaccines against Gram-negative infections and for medical diagnostics of them. The exploitation of these extraordinary properties of fimbrial polyadhesins is of great importance because Gram-negative infections are a burden problem worldwide with considerable negative economic impact and health risk to people. In particular, the outbreak of pneumonic plague in Surat in 1994 and its spread to other cities in India, lasted only a little over 2 weeks, but it created an unprecedented panic that had global repercussions (Dutt et al., 2006). At first, the Surat hospital doctors could not diagnose the disease, but when they did, immediate intervention, in the form of prevention and treatment (administration of antibodies) ceased the disease spreading beyond Surat, Delhi, Calcutta, Bombay and their vicinities. Fewer than 1,200 people were diagnosed with plague. A DNA- based study in 2000 decisively concluded that the Surat episode was a plague, but the Indian isolates were genetically more heterogeneous compared to others in the world. The last outbreak of primary pneumonic plague took place in the Shimla District of Himachal Pradesh State in northern India during February 2002 (Gupta & Sharma, 2007). Sixteen cases of plague were reported with a case-fatality rate of 25%. The infections caused by two other representatives of Yersinia genus, Y. enterocolitica and Y. pseudotuberculosis, also have a significant health concern in India and worldwide. Y. enterocolitica is the important food-borne enteropathogen that causes a variety of syndromes (Virdi & Sachdeva, 2005). Most commonly, it causes gastroenteritis, terminal ileitis and Page 96 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review mesenteric lymphadenitis. Postinfectious sequalae includes reactive arthritis and erythema nodosum. Y. pseudotuberculosis also causes a variety of gastrointestinal and extraintestinal infections in humans (Kumar et al., 2009). Encoded by the caf gene cluster (Fig. 1) F1 capsular antigen from Y. pestis is the most specific and potent antigen for medical diagnostics of bubonic and pneumonic plague. The identical psa gene clusters present in Y. pestis and Y. pseudotuberculosis (Fig. 1) that encodes for proteins for expression and assembly of the fimbrial pH6 antigen. Positive detection of pH6 antigen without any traces of F1 antigen is indicator of infection caused by Y. pseudotuberculosis or evidence of F1‒ strain of Y. pestis. Y. enterocolitica contains a closely related to psa gene cluster myf (Fig. 1) encoding the Myf fimbriae, which is built up of MyfA subunits. Y. enterocolitica is very heterogeneous (Virdi & Sachdeva, 2005). Therefore specific medical diagnostics of pathogenic strains of Y. enterocolitica is still unsolved problem.The Myf polyadhesin is promising conservative antigen for indication of infection caused by Y. enterocolitica. Salmonella spp. is the extremely heterogeneous species (Layton & Galyov, 2007). There are over 2500 serotypes of Salmonella spp. Specific medical diagnostics of pathogenic strains from Salmonella is still unsolved problem. The Salmonella spp. gene clusters saf and sef (Fig. 1) encode for proteins for expression and assembly of the atypical fimbriae Saf and the filamentous fimbriae-like structures SEF14/18. These gene clusters encode two distinct adhesin subunits: the variable major polyadhesin subunits SefA or SafA and the conservative minor SefD or SafD subunits (Fig. 1). The SefB chaperone of S. enteritidis assists in the assembly of two distinct cell-surface structures, SEF14 and SEF18, which are homopolymers of SefA and SefD subunits, respectively (Clouthier et al., 1994). The SafD subunit is identical for S. enteritidis, S. Typhi, S. Paratyphi A, S. choleraesuis and S. typhimurium, and the SefD subunit is identical for S. Paratyphi A and S. enteritidis. Therefore SefD and SafD subunits and mAbs to them can be used for medical diagnostics of main Salmonella infections. Page 97 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Acknowledgements This work was supported by grants from the European Comission/Research Executive Agency under a Marie Curie International Incoming Fellowship (235538) and a grant from the Academy of Finland (112900) to V. Z., and the FORMAS (221-2007-1057) and Swedish Research Council (K2008-58X-20689-01-3) to A. Z. References Aberg V & Almqvist F (2007) Pilicides-small molecules targeting bacterial virulence. Org Biomol Chem 5: 1827‒1834. Adams LM, Simmons CP, Rezmann L, Strugnell RA & Robins-Browne RM (1997) Identification and characterization of a K88- and CS31A-like operon of a rabbit enteropathogenic Escherichia coli strain which encodes fimbriae involved in the colonization of rabbit intestine. 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Zavialov AV, Tischenko VM, Fooks LJ, Brandsdal BO, Aquist J, Zav'yalov VP, MacIntyre S & Knight SD (2005) Resolving the energy paradox of chaperone-mediated fibre assembly. Biochem J 389: 685–694. Page 135 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Zav’yalov VP, Abramov VM, Cherepanov PG, Spirina GV, Chernovskaya TV, Vasiliev AM & Zav’yalova GA (1996) pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc- receptor. FEMS Immun Med Microbiol 14: 53–57. 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Zhou H, Monack DM, Kayagaki N, Wertz I, Yin J, Wolf B & Dixit VM (2005) Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kB activation. J Exp Med 202: 1327–1332. Page 136 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Table 1. Adhesive fimbrial organelles assembled on the Gram negative pathogen cell surface via classical chaperone-usher pathwaya. Organelle Chaperone/usher Bacterium Disease Reference(s) Aggregative adherence fimbria II, AAF/II AafD/C Escherichia coli Diarrhea Elias et al., 1999 n/db ACIAD0120/0121 Acinetobacter sp. strain ADP1 n/d Barbe et al., 2004; Gohl et al., 2006 Thin pili, an external diameter of 2 to 3 nm AcuD/C Acinetobacter sp. strain BD413 n/d Barbe et al., 2004; Gohl et al., 2006 Afimbrial adhesin, AFA-III Afa-3B/C E. coli Diarrhea or cystitis Garcia et al., 1994 Afimbrial adhesin, AfaE-VIII Afa-8B/C E. coli Diarrhea/ septicaemia Lalioui & Le Bouguénec, 2001 AF/R1 pili AfrC/B E. coli Diarrhea in rabbits Cantey et al., 1999 Aggregative adherence fimbria type I, AAF-I AggD/C E. coli Diarrhea Savarino et al., 1994 Aggregative adhesion fimbria type III, AAF-III Agg-3D/C E. coli Diarrhea Bernier et al., 2002 Ambient-temperature fimbriae AtfB/C Proteus mirabilis Urinary tract infections Massad et al., 1996 Capsular F1 antigen Caf1M/A Yersinia pestis Plague Galyov et al., 1990, 1991; Karlyshev et al., 1992a,b, 1994 Colonization factor-3, CS-3 fimbriae CS3-E/D E. coli Diarrhea Jalajakumar et al., 1989 Colonization factor, CS6 CssC/D E. coli Diarrhea Wolf et al., 1997 CS12 fimbria CswB/C E. coli Diarrhea EMBL accession number Q9ALL0 CS31A capsule-like antigen ClpE/D E. coli Diarrhea Bertin et al., 1993 F1845 (DaaE) fimbrial adhesin DaaB/C E. coli Diarrhea Bilge et al., 1989 Diffuse adherence fibrillar adhesin DafaB/C E. coli Diarrhea Keller et al., 2002 Dr hemagglutinin flexible fimbriae DraB/C E. coli Pyelonephritis Piątek et al., 2005; Servin, 2005; Van Loy et al., 2002 F17 pili F17D/C E. coli Diarrhea Lintermans et al., 1988 987P fimbriae FasB/D E. coli Diarrhea in piglets Emmerth et al., 1999 K99 pili FaeE/D E. coli Neonatal diarrhea in calves, lambs, and piglets Bakker et al., 1991 K88 pili FanE/D E. coli Neonatal diarrhea in piglets Bakker et al., 1991 Type 2 and 3 pili FimB/C Bordetella pertussis Whooping cough Willems et al., 1992 Type 1 pili FimC/D E. coli Cystitis Jones et al., 1993 F1C pili FocC/D E. coli Cystitis Riegman et al., 1990 CS18 fimbriae FotB/D E. coli Diarrhea Honarvar et al., 2003 Haemophilus influenzae biogroup aegyptius fimbriae HafB/E H. influenzae Meningitis, Brazilian purpuric fever Read et al., 1996 Page 137 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review H. influenzae fimbriae HifB/C H. influenzae type b Otitis media van Ham et al., 1994 Afimbrial adhesin LdaE E. coli Diarrhea Scaletsky et al., 2005 Long polar fimbriae LpfB/C Salmonella typhimurium Gastroentiritis Bäumler & Heffron, 1995 Mannose-resistant fimbriae MrfD/C Photorhabdus temperata Insect pathogen Meslet-Cladiere et al., 2004 Type 3 fimbriae MrkB Klebsiella pneumoniae Pneumonia Allen et al., 1991 Mannose- resistant/Proteus-like MR/P pili MrpD/C P. mirabilis Nosocomial urinary tract infections Bahrani & Mobley, 1994 Mucoid Yersinia factor, Myf fimbriae MyfB/C Y. enterocolitica Enterocolitis Iriarte & Cornelis, 1995 Nonfimbrial adhesin, NFA-I NfaE/C E. coli Urinary tract infections Ahrens et al., 1993; Servin, 2005 P pili PapD/C E. coli Pyelonephritis or cystitis Marklund et al., 1992 Pef pili PefD/C S. typhimurium Gastroenteritis Bäumler et al., 1996 PMF pili PmfC/D P. mirabilis Nosocomial urinary tract infections Massad & Mobley, 1994 Fimbrial pH6 antigen PsaB/C Y. pestis, Y. pseudotuberculosis Plague Gastroenteritis Massad & Mobley, 1994 Putative adhesin PSPPH_ A0063/ A0064 Pseudomonas syringae Halo blight of bean Joardar et al., 2005 REPEC fimbriae RalE/D E. coli Diarrhea in rabbits Adams et al., 1997 Atypical fimbriae Saf SafB/C S. typhimurium Gastroenteritis Folkesson et al., 1999; McClelland et al., 2001 Putative atypical fimbriae SafB/C S. typhi Enteric typhoid fever Deng et al., 2003 Putative atypical fimbriae SafB/C S. choleraesuis Sepsis, extraintestinal focal infections Chiu et al., 2005 Putative atypical fimbriae SafB/C S. paratyphi A Enteric typhoid fever McClelland et al., 2004 Filamentous fimbriae-like structures SEF14/18 SefB/C S. enteritidis Gastroenteritis Clouthier et al., 1993, 1994 Putative fimbriae SefB/C S. typhi Enteric typhoid fever EMBL accession number A212T7 Putative fimbriae SefB/C S. paratyphi A Enteric typhoid fever McClelland et al., 2004 S pili SfaE/F E. coli Urinary tract infections Dobrindt et al., 2001 Sfp fimbriae SfpD/C E. coli Diarrhea Brunder et al., 2001 Stf fimbriae StfD/C S. typhimurium Systemic and fatal infection in inbred mice Emmerth et al., 1999 aThe information is placed in alphabetical order of the names of chaperone/usher proteins. bnon-detected. Page 138 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Table 2. Function-structure classification of superfamily of adhesive fimbrial organelles assembled on the Gram negative pathogen cell surface via classical chaperone/usher pathway. SUBFAMILIES FUNCTION MORPHOLOGY CHAPERONES NUMBER OF SUBUNITS ADHESIN SUBUNITS Family 1: Polyadhesins Subamily 1.1: FGL chaperone- assembled polyadhesins-1a-1b Poly-adhesive binding From thin flexible fibers (2 nm diameter) observed for Psa fimbriae to amorphous or capsule-like morphology for F1 antigen (by electron microscopy) Caf1M, CS3-E, MyfB, NfaE, PsaB 1 The main structural subunit: Caf1, CS3, MyfA, NfaA, PsaA. Subfamily 1.2: FGL chaperone- assembled polyadhesins- (1+1c)-1 Poly-adhesive binding Thin flexible fibres (2 nm diameter) were observed for Dr adhesin by electron microscopy AafD, Afa-3B, Afa- 8B, AggD, Agg-3D, DaaB, DafaB, DraB 2 The main structural subunit: AafA, Afa-3E, Afa-8E, AggA, Agg-3A, DaaE, DafaE, DraE. Additional subunit (AafD, Afa-3D, Afa-8D, AggD, Agg-3D, DaaD, DafaD, DraD) is displayed on the tip of fibre with type II secretion system. Subfamily 1.3: FGL chaperone- assembled polyadhesins-2-1 Poly-adhesive binding Atypical fimbrial structures SafB, SefB, CssC 2 The main structural subunits: CssA, CssB; SafA, SefA. SafD and SefD subunits might be displayed on the tip of fibre. Subfamily 1.4: FGS chaperone- assembled polyadhesins-1-1 Poly-adhesive binding Thin flexible pili with a poorly defined diameter (2-4 nm, Pef pili) or atypical fimbria (Atf) AtfB, PefD 1 The main structural subunit: AtfA, PefA. Subfamily 1.5: FGS chaperone- assembled polyadhesins-1-2 Poly-adhesive binding n/dd ACIAD0120, ACIAD0123 1 The main structural subunit ACIAD0122. Page 139 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2 Subfamily 1.6: FGS chaperone- assembled polyadhesins-3-1 Poly-adhesive bindingd AF/R1 pili AfrC 3 The main structural subunit AfrEd. Subfamily 1.7: FGS chaperone- assembled polyadhesins-4-1 Poly-adhesive binding Thin flexible pili with a poorly defined diameter (2-4 nm, K88 pili) FanE 4 The main structural subunit FanH. Subfamily 1.8: FGS chaperone- assembled polyadhesins-5-1 Poly-adhesive binding Thin flexible pili with a poorly defined diameter (2-4 nm, K99 pili) FaeE, LdaE, RalE 5 The main structural subunit FaeH, LdaH, RalGd. Family 2: Monoadhesins Subfamily 2.1: FGS chaperone- assembled monoadhesins-2-1 Mono-adhesive binding Thin pili AcuD, F17D, FimB 2 One domain (adhesin) on the tip: AcuG, F17G, FimD. Subfamily 2.2: FGS chaperone- assembled monoadhesins-3-1 Mono-adhesive binding Fimbriae/pili HifB, HafB, MrkB, LpfB, PmfD 3 One domain (adhesin) on the tip: AfrE, HifE, HafE, MrkD, LpfE, PmfE. Subfamily 2.3: FGS chaperone- assembled monoadhesins-3-3 Mono-adhesive binding Fimbriae/pili FasB, FasC, FasE; CswB, CswC, CswE; FotB, FotC, FotE 3 One domain (adhesin) on the tip: FasG, CswG, FotG. Subfamily 2.4: FGS chaperone- assembled monoadhesins-4-1 Mono-adhesive binding Fimbriae/pili FocC, StfD 4 One domain (adhesin) on the tip: FocH, StfG. Subfamily 2.5: FGS chaperone- assembled monoadhesins-5-1 Mono-adhesive binding Fimbriae/pili FimC, SfpD, SfaE, MrpD 5 One domain (adhesin) on the tip: FimH, SfpG, SfaH, MrpH. Subfamily 2.6: FGS chaperone- assembled monoadhesins-7-1 Mono-adhesive binding Fimbriae/pili MrfD, PapD 7 One domain (adhesin) on the tip: MrfH, PapG. a number of subunits secreted via classical chaperone/usher pathway. b number of periplasmic chaperones assisted assembly of fibre. Page 140 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 3 c additional subunit displayed on the tip of fibre with type II secretion system. d based on high homology of the ral and afr gene clusters with the fae cluster. Page 141 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review General organization of gene clusters encoding adhesive organelles assembled with classical chaperone/usher machinery (elaborated by the authors). The genes encoding periplasmic chaperones and outer membrane ushers are blue and light orange, accordingly. The genes encoding adhesin subunits, structural subunits and subunits with unknown function are red and yellow, correspondingly. The genes encoding regulatory proteins are black. The proteins with putative function are green. The proteins with unknown function are pink. The numbers designate a molecular weight of encoded protein in kDa. 209x297mm (600 x 600 DPI) Page 142 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review General scheme of functioning of the chaperone/usher machinery that drives formation of adhesive protein fibres on the bacterial surface. The subunits are connected in fiber by the "donor strand complementation" (shown by red arrows). The fiber is secreted through the pore (illustrated by split cylinder) in the outer membrane (OM) formed by the "usher protein" (marked by letter U). N1 and N2 indicate N-terminal domains of two different ushers (U1 and U2, accordingly). Letter P indicates position of the plug domain in U2 and P’ and P’’ indicate two different positions of the plug domain in U1 for the two alternative models of gating. The free N1 recruits the C2:A2 complex (step 1), and bring the complex within proximity of the N2-bound C1:A1 complex (step 2). Donor-strand exchange then releases N2 for recruitment of the C3:A3 complex (step 3) and releases C1 (step 4) for recruitment of next A4 subunit (step 5). The redrawing is based on the data published by Remaut et al. (2008). 209x297mm (600 x 600 DPI) Page 143 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Page 144 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Ribbon presentation of the crystal structures of PapD-PapA, SafB–SafA and Caf1M–Caf1 complexes. The chaperones are blue with G1 and A1 edge strands in violet, the subunits are red. The two conserved Cys residues in the whole FGL family which form disulfide bond are shown as ball-and- stick. The hydrophobic residues in G1 strand of the chaperones that interact with the P5–P1 pockets of the subunits are also shown as ball-and-stick. The SafB residue A114, which interacts with the P5 pocket, is in equilibrium between a bound (left, type I structure) and unbound (right, type II structure) state in the P5 pocket. The structures were redrawn based on the co-ordinates of atoms published by Remaut et al. (2006) (PDB accession numbers, 2CO6 and 2CO7), Verger et al. (2007) (PDB accession number, 2UY6) and Zavialov et al. (2003, 2005) (PDB accession number, 1P5V). All figures were prepared with PyMOL (DeLano, 2002). 209x297mm (600 x 600 DPI) Page 145 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Functionally important sequences of the chaperones assembling fimbrial adhesins via chaperone/usher pathway (elaborated by the authors). Numbering in CafM sequence is indicated. Conserved Cys residues involved in disulphide bond formation in the FGL chaperones are yellow. Other residues conserved in the whole superfamily are green, including subunit anchoring Lys that is replaced by Pro in FasB, CswB and FotB operating only with adhesin subunits. Alternating bulky hydrophobic residues (from five to three in the FGL family; three in the FGS family) extending from the beginning of the G1 β-strand are red. The conservative positions, that are typical for the Caf1M- like subfamily of FGL chaperones, are cyan. F1 and G1 β-strands are shown by arrows (see Fig. 3 for details). 209x297mm (600 x 600 DPI) Page 146 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Plot of correlation (elaborated by the authors) between a number of deleted residues in a F1-G1 loop in a chaperone (FG deletion in residues shown on abscissa) in comparison with the F1-G1 loop of the Caf1M chaperone and a number of subunits operating by a chaperone (subunits operated shown on ordinate). The slopes of plots of correlation for FGL and FGS chaperones are different and are shown by green and red, correspondingly. The coefficient of correlation for FGL chaperones is equal to 0.80 and for FGS chaperones it is equal to 0.72. 209x297mm (600 x 600 DPI) Page 147 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (a) Comparison of PapA pilin domain and Caf1 polyadhesin subunit complemented with chaperones. PapA pilin domain and Caf1 polyadhesin subunit are shown red with donor strands from PapD and Caf1M chaperone (blue). Only interacting chaperone-subunit strands are shown. (b) A ribbon diagrams of the native PapD–PapA'–PapA'' and Caf1M–Caf1'–Caf1'' complexes. Caf1M and PapD are blue, except for G1 and A1 β-strands (violet). The chaperone-bound Caf1' and PapA' subunits and N-terminal donor strands (Gd or Nte) are red; the Caf1'' and PapA'' subunits corresponding to the tip of growing fibers are green. The N- and C-termini are labelled in the same colours as the ribbons. The redrawing is based on the co-ordinates of atoms of structures published by Verger et al. (2007) (PDB accession number, 2UY6) and Zavialov et al. (2003, 2005) (PDB accession numbers, 1P5V and 1Z9S). 209x297mm (600 x 600 DPI) Page 148 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Model for the mechanism of donor strand exchange (DSE) in vitro and in vivo. (a) Models for usher- catalyzed assembly proposed by Zavialov et al. (2003). Left image: stepwise DSE in which the entire G1 donor strand is removed before the Gd strand is bound. Right image: sequential concerted DSE in which G1 is gradually replaced by Gd in a zip-in-zip-out mechanism. (1 and 3) correspond to the crystallographically observed chaperone–subunit and subunit–subunit structures, respectively. (2) presents the imaginary structures. DG corresponds to the free energy for fiber formation from chaperone–subunit assembly complex and must necessarily be 0. Values for the different free energy terms are not known, and the figure is not meant to indicate even relative sizes of these terms. (b) Schematic presentation of DSE in vitro based on the experimental data (Remaut et al., 2006). Chaperone and subunit are labeled (Ch, light blue) and (Su, light green), respectively. In the chaperone, strands G1 and F1 are presented as solid dark blue lines. In the subunit, strand F, which directly interacts with the G1 donor strand, is depicted in dark green. An incoming N-terminal Gd Page 149 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review donor strand (depicted in red) forms a ternary complex with the chaperone–subunit complex at the P5 pocket (indicated by a thicker line). DSE then proceeds and terminates by dissociation of the chaperone–subunit complex and insertion of the P1 residue in the P1 pocket. (c) Schematic representation of a single incorporation cycle at the usher. Chaperone and usher are shown light blue and light orange, respectively. For clarity, subunits are differentiated by color with the incoming subunit in light cyan. The N-terminal and C-terminal domains of the usher are indicated. Redrawing based on Zavialov et al. (2003) and Remaut et al. (2006, 2008) and Fronzes et al. (2008). 209x297mm (600 x 600 DPI) Page 150 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Schematic presentation (elaborated by the authors) of the structure of FGS chaperone-assembled thick rigid mono-adhesive fimbriae/pili (a; P pili as example) (Sauer et al., 2000, 2004), FGS chaperone-assembled hetero-polyadhesins (b; F4, K88 pili as example) (van den Broeck et al., 2000), FGS chaperone-assembled homo-polyadhesins (c; PE fimbriae as example) (Chessa et al., 2008) and FGL chaperone-assembled polyadhesins (d; AfaE polyadhesin as example) (Anderson et al., 2004a). Periplasmic chaperones and outer membrane ushers are in blue and light orange, accordingly. Adhesin subunits are in red. Structural subunits are in yellow. Green arrow shows chaperone/usher independent secretion of AfaD subunit (shown in green) via type II secretion system (Zalewska-Piątek et al., 2008) and its potential display on the tip of the AfaE fimbrial polyadhesin (Anderson et al., 2004a). 209x297mm (600 x 600 DPI) Page 151 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Binding sites of FGL chaperone assembled polyadhesins (DaaE and AfaE) and FGS chaperone assembled mono-adhesive fimbriae/pili (GafD/F17G, FimH and PapG). A ribbon diagrams and solvent accessible surface presentations of DaaE subunit of a strand-swapped trimer of wild type DaaE of F1845 adhesin and self-complemented AfaE subunit of AFA-III adhesin with chloramphenicol as a yellow stick presentation. CD55/DAF and CEACAMS binding sites derived from DraE and DaaE mutagenesis are shown in green and red, respectively. Molecular surface rendering of a model for AfaE fiber was generated by assuming the same orientation between successive subunits as observed for Caf1′ and Caf1″ in F1 fiber (Zavialov et al., 2003). The residues involved in binding with CD55/DAF and CEACAMS are in green and red, respectively. Ribbon presentations also are given for adhesin domains of GafD/F17G (PDB accession number, 1OIO), FimH (PDB accession number, 1KLF) and PapG (PDB accession number, 1J8R). Bound ligands, determined crystallographically, for GafD/F17G, FimH and PapG are also shown and labeled. The redrawing is Page 152 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review based on the data and co-ordinates of atoms of the structures published by Anderson et al. (2004a, b), Korotkova et al. (2006a, b), Pettigrew et al. (2004) and Li et al. (2007). 209x297mm (600 x 600 DPI) Page 153 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review (a) Ribbon presentation of the PapC130–640 translocation channel (PDB accession number, 2VQI) viewed from the extracellular side. The β-barrel, plug domain, β5-6 hairpin, and helix α1 are colored blue, magenta, orange, and yellow, respectively. Beta strands are labeled β1 through β24, the labels N and C indicate the N and C termini of the translocation channel. (b) Ribbon presentation of PapC130–640 viewed from the side. Structural elements are colored as in (a). The N and C termini, helix α1, strand β4, and the β5-6 hairpin are labeled. (c) Structural superposition of the Caf1A plug domain (232-320 amino acid fragment of Caf1A; PDB accession number, 3FCG) and the PapC plug domain (252-330 amino acid fragment of PapC; PDB accession number, 2VQI). Caf1A232-320 and PapC252-330 are in magenta and cyan, respectively. Beta strands are labeled according to the immunoglobulin fold classification. N- and C-terminal ends of structured fragments are indicated. The structures are redrawn based on the co-ordinates of atoms published by Remaut et al. (2008) and Yu et al. (2009). Page 154 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 209x297mm (600 x 600 DPI) Page 155 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review X-ray structure of the ternary N-terminal usher domain FimDN(1–125)–FimC chaperone–FimHP pilin complex. The ribbon diagram of the ternary complex is shown, with FimDN(1–125) N-terminal usher domain depicted in green, FimC chaperone in cyan and the pilin domain FimHP in yellow. The G1 donor strand of FimC chaperone is colored in blue. A black dashed line indicates residues 10–18 of FimDN N-terminal usher domain, for which no electron density was observed. The N- and C-termini of FimDN N-terminal usher domain are labeled in green. The structure is redrawn based on the co- ordinates of atoms published by Nishiyama et al. (2005) (PDB accession number, 1ZE3). 209x297mm (600 x 600 DPI) Page 156 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Schematic illustration (elaborated by the authors) a binding of mono-adhesive (a) and poly- adhesive (b) organelles to host cell receptors. Periplasmic chaperones and outer membrane ushers are in blue and orange, accordingly. Adhesin and pilin subunits are in red and yellow, respectively. Host receptors for moadhesins are shown by green. Two different types of host receptors for polyadhesins are shown by violet and greencyan. 209x297mm (600 x 600 DPI) Page 157 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Phylogenetic tree of the fimbrial usher protein family (the redrawn is based on Nuccio & Bäumler, 2007). The graph shows an unrooted phenogram generated using analysis of amino acid sequences of 189 ushers (Nuccio & Bäumler, 2007). Ushers are grouped into six fimbrial clades (highlighted in light blue) termed α, β, γ, κ, π, and σ-fimbriae. Ushers of γ-clade are subdivided into four clades (highlighted in yellow) termed γ1, γ2, γ3, and γ4- fimbriae. Bootstrap values of nodes defining these clades (indicated by open circles at the base of each fimbrial clade) are shown. For the details of generation of the phylogenetic tree and bootstrap values see Nuccio & Bäumler (2007). 209x297mm (600 x 600 DPI) Page 158 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Phylogenetic relationship of operons belonging to the γ1, γ2, γ3, and γ4-fimbriae (the redrawn is based on Nuccio & Bäumler, 2007). The branch of the fimbrial usher protein tree representing γ1, γ2, γ3, and γ4-fimbriae is shown on the left. The bootstrap value for the node defining each subclade is displayed at the top and was generated in the analysis performed for Fig. 14. Numbers at the end of each branch of the phylogenetic tree correspond to the numbers given for each operon on the right. 209x297mm (600 x 600 DPI) Page 159 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Phylogenetic relationship of operons belonging to the β, κ, and π-fimbriae (the redrawing is based on Nuccio & Bäumler, 2007). The branch of the fimbrial usher protein tree representing β, κ, and π- fimbriae is shown on the left. The bootstrap value for the node defining each subclade is displayed at the top and was generated in the analysis performed for Fig. 14. Numbers at the end of each branch of the phylogenetic tree correspond to the numbers given for each operon on the right. 209x297mm (600 x 600 DPI) Page 160 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Stereoimage of the FimD1–125 N-terminal usher domain in complex with the FimC-FimH158–279 chaperone–adhesion complex with the PapD– pilicide complex in overlay. PapD and pilicide are shown in light blue ribbon and ball-and-stick presentation, respectively. FimC and FimH158–279 are shown in magenta and green, with the FimD1–125 N-terminal domain shown in yellow. The redrawing is based on the co-ordinates of atoms of the structures published by Nishiyama et al. (2005) (PDB accession number, 1ZE3). 209x297mm (600 x 600 DPI) Page 161 of 157 ScholarOne Support 1-434/817-2040 ext 167 FEMS Microbiology Reviews 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60