The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that has evolved to take advantage of the eukaryotic host cells it inhabits during infection. It uses bacterial effectors translocated into the host cell cytosol to manipulate host cell machinery and establish a replicative niche. In this thesis I study the function of two of these effectors, SopD and SopB, which have been shown to act cooperatively to induce phenotypes associated with gastroenteritis (fluid secretion and neutrophil influx into the intestinal lumen). In addition to promoting gastroenteritis, SopD has also been implicated in systemic and persistent infection of mice. Although recently implicated in invasion, the precise function of SopD has remained elusive. Here I show that SopD affects membrane dynamics during S. Typhimurium invasion of epithelial cells. SopD promotes membrane sealing and macropinosome formation, events that may have important consequences for efficiency of bacterial cell entry in vivo. Furthermore, we demonstrate that SopD is recruited to the invasion site membranes through the phosphatase activity of SopB, suggesting a mechanism for their cooperative action during induction of gastroenteritis. Unlike SopD, SopB has been a focus of intense research efforts and its role in invasion as a phosphoinositide phosphatase is well documented. However, we have observed that SopB also inhibits fusion of lysosomes with Salmonella-containing vacuoles (SCVs) following invasion. This ability depends on SopB-mediated reduction of negative membrane charge of the SCV during invasion by hydrolysis of the phosphoinositide PI(4,5)P2. Membrane charge alterations driven by SopB result in removal of Rab GTPases from the SCV that depend on electrostatic interactions for their targeting. Two of these Rabs, Rab23 and Rab35 were previously shown to promote phagosome-lysosome fusion. Therefore their removal from the SCV may promote SCV trafficking away from the degradative endocytic pathway of host cells. This represents a new mechanism by which an invasion associated effector controls SCV maturation. Together, this work advances our knowledge of the interaction between S. Typhimurium and its host. This research also suggests a new mechanism by which pathogens other than S. Typhimurium could promote their intracellular survival.