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Salmonella enterica serovar Typhimurium (S.Tm) causes enterocolitis with significant worldwide morbidity and mortality. The general aim of this thesis is to investigate variation in host cell invasion mechanisms used by S.Tm across different host cell contexts, as well as the influence of bacterial cell-cell heterogeneity on invasion-relevant S.Tm behaviours. The thesis is divided into four sub-projects, each a section in the presented work.

First, a genetic barcoding technique for tracking bacteria in mixed consortium infections was developed and applied to evaluate the dependency on the type three secretion system 1 (T3SS-1) and its effectors for host cell entry. It was found that S.Tm invasion of cultured epithelial cells and monocytes is mainly mediated by T3SS-1, or by cooperative uptake of bystander bacteria. T3SS-1-independent entry was possible in cultured macrophages, although T3SS-1-dependent entry was predominant also there. In fact, active invasion was promoted by the same T3SS-1 effectors in all three cell types. 

Second, an in-depth comparison of S.Tm infections in cell line cultures and in the mouse gut mucosa in vivo highlighted a “discreet-invasion” modality in vivo, in sharp contrast to the prevailing “ruffle” model for host cell invasion. While ruffle-mediated entry into epithelial cell lines was driven by the T3SS-1 effectors SopBEE2, discreet-invasion into the murine gut absorptive epithelium is driven predominantly by the SipA effector, as well as the SiiE adhesin. Furthermore, discreet-invasion targeted apicolateral “hot spots” near cell-cell junctions, dependent on the local cell neighbourhood, which was further charted in the final two sub-projects.    

Third, single-bacterium characteristics among S.Tm populations were studied using time-lapse microscopy. The indistinct nature of the shift from growth to virulence induction spawned a transient subpopulation of S.Tm “doublets”, cell division intermediates also exhibiting pronounced swimming and host cell invasion aptitude. The longer doublets also displayed a different search pattern during near-surface swimming, highlighting bacterial cell length heterogeneity as a key determinant of target search atop epithelia.   

Fourth, the morphogenic impact of clinically relevant antibiotics were explored, in context of the previous data. Even S.Tm bacteria with the most extreme morphological abnormalities (e.g. highly filamentous or coccoid individuals), induced by chloramphenicol, ciprofloxacin, nitrofurantoin, and meropenem, could robustly swim and invade epithelial host cells. While high concentrations of these antibiotics were effective at suppressing growth and virulence, a range of low, sub-inhibitory concentrations even enhanced host cell invasion capacity and affected the near-surface swimming behaviour among surviving bacteria.   

In summary, the present investigation highlights the pivotal importance of taking both host cell features and bacterial heterogeneity into account when studying infection processes.