Vibrio vulnificus is a pathogenic bacterium causing primary septicemia, which follows a classical septic shock pathway, including an overwhelming inflammatory cytokine response. In this study, we identified a putative lipoprotein of V. vulnificus, encoded by the ilpA gene, as one of the surface proteins that specifically reacted with the antibodies raised against outer membrane proteins of V. vulnificus. Using a mutant V. vulnificus in which its ilpA gene was knocked out, we found that IlpA is important in the production of interferon-␥ in human peripheral blood mononuclear cells. Production of tumor necrosis factor-␣ and interleukin-6 is also induced by the recombinant IlpA (rIlpA) in human monocytes. Lipidation of the rIlpA was observed by in vivo labeling in Escherichia coli. Experiments using the mutant IlpA, which is unable to be modified by lipidation, indicate that the lipid moiety of this protein has an essential property for cytokine production in human cells. Pretreatment of monocytes with antibodies against Toll-like receptor 2 (TLR2) inhibited production of both tumor necrosis factor-␣ and interleukin-6. The role of TLR2 in IlpA-induced cytokine production was confirmed by an in vitro assay, in which only the TLR2-expressing cells showed a dramatic induction of nuclear factor-B activity by rIlpA. In addition, rIlpA treatment resulted in induction of TLR2 transcription in human cells. In comparison with the wild type V. vulnificus, the ilpA mutant showed a reduced mortality in mice. These results demonstrate that IlpA of V. vulnificus functions as an immunostimulant to human cells via TLR2.Vibrio vulnificus, a Gram-negative bacterium found commonly in the estuarine environment, has been frequently associated with primary septicemia following the consumption of contaminated shellfish. Over 50% of the primary septicemia patients caused by V. vulnificus died of multiorgan failure as a result of a rapidly progressive shock syndrome (1, 2). Extracellular substances produced by V. vulnificus, such as hemolytic cytolysin (3, 4) and elastase (5), had been extensively studied as candidate virulence factors responsible for its pathogenesis. Surface structures such as lipopolysaccharide (LPS) 2 (6, 7) and outer membrane proteins (8, 9) were also studied as candidates for V. vulnificus virulence factors. Based on the attenuated mouse lethality by a noncapsulated mutant V. vulnificus, capsular polysaccharide was also proven to be important in the pathogenesis of V. vulnificus (10). Type IV pilin was confirmed to be involved in the virulence of V. vulnificus via genetic deletion of the pilD or pilA genes (11, 12). In addition, motility was discovered as a virulence determinant of V. vulnificus (13,14).

Vibrio vulnificus is a Gram-negative bacterium that multiplies rapidly in host tissue and causes extensive tissue damage. Human peripheral blood mononuclear cells (PBMC) were shown to be readily killed by exposure to live V. vulnificus. V. vulnificus induced production of intracellular reactive oxygen species (ROS) and nitric oxide (NO) in PBMC. Pretreatment of PBMC with diphenyleneiodonium chloride (DPI) abolished ROS generation upon exposure to V. vulnificus and decreased the bacterial ability to cause cell death. In contrast, pretreatment of these cells with inhibitors of inducible nitric oxide synthase (iNOS) blocked V. vunificus-induced NO production, but did not significantly alter cell death by V. vulnificus. V. vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 and ERK1/2 in PBMC. Inactivation of these MAPKs by selective inhibitors caused a reduction both in ROS generation and cell death induced by V. vulnificus. It was further shown that an inhibitor of ROS generation (DPI) blocked V. vulnificus-induced phosphorylation of p38 and ERK1/2 MAPK. This study demonstrates that V. vulnificus induces death of PBMC via ROS-dependent activation of p38 MAPK and ERK1/2 MAPK.