Extracellular secretion of the virulence plasmid-encoded ADP-ribosyltransferase SpvB in Salmonella.
Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.
Gotoh
Hideo
H
Department of Microbiology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Okada
Nobuhiko
N
Kim
Yun Gi
YG
Shiraishi
Kouya
K
Hirami
Naoko
N
Haneda
Takeshi
T
Kurita
Ai
A
Kikuchi
Yuji
Y
Danbara
Hirofumi
H
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D004847:Epithelial Cells; D015964:Gene Expression Regulation, Bacterial; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D012475:Salmonella / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D037521:Virulence Factors; D036002:ADP Ribose Transferases; C415223:spvB protein, Salmonella enterica virulence plasmid