SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins

Summary Replication 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 spiC – mutant 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.