title: Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton.
abstract: A family of nine Salmonella typhimurium type III secretion effectors with a conserved amino-terminus have been defined. Three family members (SifA, SifB and SseJ) have previously been demonstrated to localize to the Salmonella-containing vacuole and to Salmonella-induced filaments. In contrast, we demonstrate that two other family members, SspH2 and SseI, co-localized with the polymerizing actin cytoskeleton. These proteins also interacted with the mammalian actin cross-linking protein filamin in the yeast two-hybrid assay through their highly conserved amino-terminal domains. This amino-terminus was sufficient to direct localization to the polymerizing actin cytoskeleton, suggesting that the interaction with filamin is important for this subcellular localization. In addition, SspH2 co-localized with vacuole-associated actin polymerizations (VAP) induced by intracellular bacteria through the Salmonella pathogenicity island (SPI)-2 type III secretion system (TTSS). SspH2 interacted with the actin-binding protein profilin in the yeast two-hybrid assay and by affinity chromatography. This interaction was highly specific to SspH2 and was mediated by its carboxy-terminus. Furthermore, SspH2 inhibited the rate of actin polymerization in vitro, suggesting that it functions to reduce or remodel VAP. Strains with mutations in sspH2 and sseI retained the ability to form VAP. However, a third intracellular virulence factor, spvB, which ADP-ribosylates actin, strongly inhibited VAP formation in HeLa cells, suggesting a more subtle effect for SspH2 and SseI on the actin cytoskeleton.
authors.affiliation: Department of Microbiology, University of Washington, HSB K-140, Box 357710, Seattle, WA 98195-7710, USA.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Miao
authors.forename: Mitchell
authors.initials: M
authors.lastname: Brittnacher
authors.forename: Andrea
authors.initials: A
authors.lastname: Haraga
authors.forename: Robert L
authors.initials: RL
authors.lastname: Jeng
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Welch
authors.forename: Samuel I
authors.initials: SI
authors.lastname: Miller
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D064448:Filamins; D005456:Fluorescent Dyes / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D051304:Profilins; D011721:Pyrenes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D003285:Contractile Proteins; D064448:Filamins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D051304:Profilins; D011721:Pyrenes; D011993:Recombinant Fusion Proteins; C421862:SifA protein, Salmonella; C030984:pyrene