Manipulation of the eukaryotic cytoskeleton by invasive Salmonella

Mechanical cell fractionation and immunofluorescence microscopy were applied to systematically investigate the subcellular localisation of epitope-tagged effectors in cultured cells after transfection or infection with wildtype Salmonella strains exogenously expressing individual effectors. Although five Salmonella effectors contain no apparent membrane-targeting domains, all six localised to the plasma membrane fraction and were visualised a the cell periphery, from where they induced distinct effects on the actin cytoskeleton. Unexpectedly, no translocated cytoplasmic effector pool was detectable. Parallel experiments with Shigella IpgD demonstrated analogous plasma membrane localisation. In agreement with their cellular location, in vitro reconstitution demonstrated that the prenylated cellular GTPase Cdc42 was necessary and sufficient for SopE and SptP membrane association. Three effector-augmented bacterial strains exhibited increased invasion compared to wildtype bacteria; SopE- and SopB- but not SipC-mediated events were inhibited by dominant negative Rho GTPase expression, demonstrating that effector directed actin rearrangements occur via both redundant and robust mechanisms. These data shoed that the host plasma membrane is a critical interface for effector-target interaction during both Salmonella and Shigella cell entry, and established versatile systems to further dissect effector interplay. Salmonella strains and effector transfected cells were combined in screens to assess potential pairwise interplay between effectors. These identified both synergistic and antagonistic effector protein pairs, namely three novel co-operativitive interactions (SipC:SopB, SipC:SopE, SipA:SopB) in addition to those previously demonstrated (SipA:SipC) or implied (SopE:SopB). A temporal requirement for effector activities was also revealed as SopB/IpgD or SptP transfection inhibited bacterial entry. Circuitry modelling of these data using logical gates was used analyse Salmonella effector activities during bacterial entry. The requirements for effector delivery were also investigated. Together these findings illustrate the complex cross-talk between host and bacterial factors and highlight the central role of the host plasma membrane as an interactive interface during bacterial invasion.EThOS - Electronic Theses Online ServiceGBUnited Kingdo