title: SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins.
abstract: 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.
authors.affiliation: Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College School of Medicine, Armstrong Road, London SW7 2AZ, UK.
authors.forename: Xiu-Jun
authors.initials: XJ
authors.lastname: Yu
authors.forename: Javier
authors.initials: J
authors.lastname: Ruiz-Albert
authors.forename: Kate E
authors.initials: KE
authors.lastname: Unsworth
authors.forename: Steven
authors.initials: S
authors.lastname: Garvis
authors.forename: Mei
authors.initials: M
authors.lastname: Liu
authors.forename: David W
authors.initials: DW
authors.lastname: Holden
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C120280:SpiC protein, Salmonella; D002402:Cathepsin D