Vibrio vulnificus IlpA-induced Cytokine Production Is Mediated by Toll-like Receptor 2

Abstract

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).

MARTX Toxin in the Zoonotic Serovar of Vibrio vulnificus Triggers an Early Cytokine Storm in Mice

“…In agreement with the pattern of DUSP1 activation, the Mapk14 gene (or p38α) was up-regulated by the Bt2-SerE strain, but down-regulated by the selected Bt1-RtxA1 1 strain. In contrast, mice infected with the Bt1-RtxA1 1 -strain developed a typical immune response against an extracellular pathogen: most of the CKS genes were down-regulated in these mice while genes known to have a beneficial role during extracellular bacterial infections ( Il6, Tnf or Lcn2 ) were clearly up-regulated (Shin et al, 2002; Berger et al, 2006; Goo et al, 2007; Chuang et al, 2010). Finally, we also found that the Bt1-RtxA1 1 strain induced high levels of the CCs Ccl3 and Cxcl1 .…”

Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes

“…Cells were pre-incubated with anti-mouse TLR2 monoclonal antibody (Clone: T2.5, BioLegend), anti-human TLR2 monoclonal antibody (Clone: TL2.1, BioLegend) or its isotype control IgG (BioLegend). These antibodies are useful for blocking studies and have been reported to block TLR2-specific cell activation in vitro and in vivo 39–41 .…”

Vibrio vulnificus: An Environmental and Clinical Burden

“…It is able to accomplish this due to its innate virulence factors/determinants which enhance its pathogenicity, conferring the ability to survive in the human body (Jones and Oliver, 2009). Severe mechanisms contribute in attachment and invasion of a host, which including pili, OmpU , and IlpA membrane proteins and flagella (Jung et al, 2005; Goo et al, 2006, 2007; Jones and Oliver, 2009). V. vulnificus are also able to utilize Neu5Ac as a nutrient during infection in humans and survive in highly acidic environments by breaking down amino acids to amines and CO 2 (Jeong et al, 2009; Jones and Oliver, 2009).…”