Imaging salmonella at a single cell resolution
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Salmonella is a rod-shaped bacterium that infects many cells including macrophages in
the gastrointestinal tract and causes salmonellosis. Single-cell technologies are being
utilised to study the heterogeneity of host-pathogen interactions, which focus on
unravelling the different experiences Salmonella goes through during infection within the
same host. ADP-ribosylation is the process of transferring of ADP-ribose from NAD⁺
onto a target molecule and is catalysed by PARPs. PARPs have many roles including
inflammation and host–pathogen interactions, yet it is hardly studied when comes to
bacterial infections. It is involved in M1/M2 polarisation of macrophages, but on the other
hand, Salmonella is known to favour M2 polarised macrophages for long term
persistence. In an ongoing study in the host laboratory (TCML), PARP14 was found to
be upregulated in Salmonella infected macrophages in vitro. The aim of this study is to
optimise in vitro conditions to monitor Salmonella-macrophage interaction at a single cell
resolution and the possible heterogeneity within. Subsequent aim is to determine the
functional importance of ADP-ribosylation-mediated cell signalling processes to
Salmonella-macrophage interaction using pharmaceutical inhibition of PARP-enzymes.
Flow cytometry was employed to optimise the MOI of Salmonella SL1344-GFP and
PARP inhibitor PJ34 concentration. For single-cell visualisation, epifluorescence
microscopy was used. Automated batch image analysis was done using ImageJ macro
that calculated the number of bacteria and cells per coverslip. A manual approach was
implemented to calculate the number of bacteria per cell. After optimising the experiment
conditions, the chosen MOI was 100 to be able to visualise Salmonella-macrophage
interaction. For the functional importance of PARPs, a concentration of 50 µM was
chosen for PJ34 because higher concentrations were causing interference in
flowcytometry with the SL-1344-GFP signal. Results from flowcytometry revealed that
during salmonellosis, PJ34 treatment showed more cells associated with bacteria than
non-treated cells at 6 hours, yet this difference was not significant indication that PJ34
has no major effects on salmonellosis. Epifluorescence microscopy showed the
heterogenous nature of infection that is characterised by the different number of bacteria
per infected cell.