The enteropathogenic bacteria Salmonella are the main cause of food borne gastroenteritis worldwide. The activation of Toll-like receptor 4 (TLR4) by LPS triggers an immune response to counter infection. Signaling by TLR4 requires the adaptor proteins, TIRAP and TRAM. Recruitment and activation of these molecules is dependent on the membrane lipid, PIP2. The Salmonella effector, SigD, is a 4-phosphatase that depletes PIP2 from the host plasma membrane during invasion. Thus, we investigated if SigD could lead to the interruption of the TLR4 pathway. We observed that SigD expression caused the disappearance of TIRAP from the Salmonella containing vacuoles (SCVs) in HeLa cells. Furthermore, we demonstrated that SigD attenuates NF-κB activation, implicating SigD in the disruption of the MyD88 dependent pathway. In addition, the observed inhibition of PKCε phosphorylation suggests SigD may also block the other branch of the TLR4 signaling cascade, the MyD88 independent pathway.

The enteropathogenic bacteria Salmonella are the main cause of food borne gastroenteritis worldwide. The activation of Toll-like receptor 4 (TLR4) by LPS triggers an immune response to counter infection. Signaling by TLR4 requires the adaptor proteins, TIRAP and TRAM. Recruitment and activation of these molecules is dependent on the membrane lipid, PIP2. The Salmonella effector, SigD, is a 4-phosphatase that depletes PIP2 from the host plasma membrane during invasion. Thus, we investigated if SigD could lead to the interruption of the TLR4 pathway. We observed that SigD expression caused the disappearance of TIRAP from the Salmonella containing vacuoles (SCVs) in HeLa cells. Furthermore, we demonstrated that SigD attenuates NF-κB activation, implicating SigD in the disruption of the MyD88 dependent pathway. In addition, the observed inhibition of PKCε phosphorylation suggests SigD may also block the other branch of the TLR4 signaling cascade, the MyD88 independent pathway.

Chez B. subtilis, le régulateur Mta régule l'expression de deux gènes codant pour des pompes d'efflux de drogues, bmr et blt. Ce régulateur joue un rôle physiologique beaucoup plus large que la régulation des gènes impliqués dans la résistance aux drogues puisqu'il régule l'expression d'au moins 18 autres gènes. Dans un mutant mta la quantité de protéine CpgA est très diminuée. Cette protéine est une GTPase impliquée dans l'assemblage du ribosome et dans la formation de la paroi. Cette diminution est supprimée par une mutation secondaire qui restaure un niveau intracellulaire de CpgA supérieur à celui de la souche sauvage. Cette mutation confère également un avantage de croissance, sur la souche sauvage et sur le mutant mta, au double mutant résultant. L'augmentation de la quantité de CpgA est corrélée avec l'augmentation du niveau d'expression du gène cpgA ainsi que de celui des gènes def et prpC avec lesquels cpgA est en opéron. Le gène def code pour une déformylase, une protéine impliquée dans la traduction, et prpC pour une Ser/Thr phosphatase capable de déphosphoryler CpgA. Dans le mutant supprimé, la surexpression de l'opéron def-cpgA est accompagnée d'un phénotype drastique de cellules enchaînées et immobiles en phase exponentielle de croissance (phénotype Mhp). Ces cellules contiennent le facteur alternatif de transcription SigD nécessaire à l'expression des gènes dont les produits sont impliqués dans la mobilité cellulaire et dans le clivage des septa lors de la division cellulaire, mais SigD est inactif dans ces cellules (SigD OFF). / In B. subtilis, the regulator Mta regulates the expression of two genes encoding drug efflux pumps, bmr and blt. This regulator has a physiological role far wider than the regulation of genes involved in resistance to drugs since it regulates the expression of at least 18 other genes. In a mta mutant, the amount of the CpgA protein is markedly reduced. This protein is a GTPase involved in ribosome assembly and in cell wall expansion. This decrease is suppressed by a secondary mutation that restores a higher level of intracellular CpgA than that of the wild type strain. This mutation also confers a growth advantage to the resulting double mutant over the wild type and the mta mutant strains. The increased amount of CpgA is correlated with increased level of cpgA gene expression and of the prpC and def genes with which cpgA is in operon. The def gene encodes a deformylase, a protein involved in translation, and prpC for a Ser / Thr phosphatase able to dephosphorylate CpgA. In the double mutant, overexpression of the def-cpgA operon is accompanied by a drastic phenotype of chained non-motile cells in the exponential phase of growth (Mhp phenotype). These cells contain SigD, an alternative sigma factor, necessary for the expression of genes whose products are involved in cell motility and in the cleavage of septa during cell division, but SigD is inactive in these cells (SigD OFF). In exponential growth phase, a wild type strain of B. subtilis shows a heterogeneous population consisting of cells SigD ON, motile and isolated, and cells SigD OFF, chained and non-motile. In the mta strain, the transition from one state to another is reversible as in the wild type strain.

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.