| Salmonellae are Gram-negative pathogens that are important causes of human disease worldwide. To promote internalization and intracellular replication during infection, salmonellae utilize two differentially expressed type III secretion systems (TTSS) encoded on Salmonella pathogenicity-island (SPI) 1 and SPI2, respectively, which transfer virulence proteins, called effectors, to the host cytosol in order to manipulate host cellular processes. Following internalization, SPI2 TTSS effectors are translocated across the phagosomal membrane, however the mechanism by which these effectors promote intracellular replication is not yet understood. Salmonella infection of cultured epithelial cells induces elongation of the Salmonella phagosomal membrane, a phenotype called Sif, for Salmonella-induced filaments. Sif formation requires the SPI2 effector SifA, and sifA-null strains are attenuated in mice, indicating that Sif formation is important for pathogenesis. SifA contains a WxxxE motif, which has been shown in other bacterial TTSS effectors to confer the ability to mimic activated mammalian GTPases. SseJ, a SPI2 effector with homology to glycerophospholipid-cholesterol acyltransferases, is also important for virulence in the mouse. In this work, evidence is presented that demonstrates that SseJ has deacylase activity, which contributes to Salmonella virulence in mice. In addition, SseJ binds to the mammalian small GTPase RhoA, and co-expression of SseJ and RhoA in cultured cells induces elongation and tubulation of SseJ-coated membranous compartments, dramatic structures that resemble Sif. This novel membrane tubulation phenotype is induced when SseJ is co-expressed with RhoA, RhoB, or RhoC, but not Cdc42, Rac1, or Rab7, indicating that it is a Rho-GTPase specific phenotype. SseJ also interacts with and induces membrane tubulation with SifA, suggesting that SifA has Rho-GTPase mimicry activity. Tubulated membranes induced by SseJ and SifA have all the known characteristics of Sif, and the conserved motifs of SifA and SseJ are important for membrane tubulation. These results suggest that the intracellular function of SifA is to mimic activated Rho-GTPases, and supports the hypothesis that the deaylase activity of SseJ and the GTPase mimicry activity of SifA cooperate during infection to induce extension of the Salmonella phagosomal membrane. |
