A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA ± mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR ± mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA ± bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

One sentence summary: A physiological signal triggering intracellular Salmonella virulence protein delivery across the vacuolar membrane is shown to be cytosolic pH, which causes disassembly of a regulatory complex.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI‐2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F‐actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G‐actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI‐2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin‐depolymerizing agents significantly inhibited intramacrophage replication of wild‐type Salmonella typhimurium. Furthermore, after this treatment, wild‐type bacteria were released into the host cell cytoplasm, whereas SPI‐2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

SummarySalmonella enterica serovar Typhimurium ( S. typhimurium ) induces actin assembly both during invasion of host cells and during the course of intracellular bacterial replication. In this study, we investigated the involvement in these processes of host cell signalling pathways that are frequently utilized by bacterial pathogens to manipulate the eukaryotic actin cytoskeleton. We confirmed that Cdc42, Rac, and Arp3 are involved in S. typhimurium invasion of HeLa cells, and found that N-WASP and Scar/WAVE also play a role in this process. However, we found no evidence for the involvement of these proteins in actin assembly during intracellular replication. Cortactin was recruited by Salmonella during both invasion and intracellular replication. However, RNA interference directed against cortactin did not inhibit either invasion or intracellular actin assembly, although it resulted in increased cell spreading and a greater number of lamellipodia. We also found no role for either the GTPase dynamin or the formin family member mDia1 in actin assembly by intracellular bacteria. Collectively, these data provide evidence that signalling pathways leading to Arp2/3-dependent actin nucleation play an important role in S. typhimurium invasion, but are not involved in intracellular Salmonella-induced actin assembly, and suggest that actin assembly by intracellular S. typhimurium may proceed by a novel mechanism.

SummaryReplication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella -containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB -, sseC -and sseD -mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC -mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiCmutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC -mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.