Abstract
Vibrio fluvialis causes human diarrhea, but the pathogenesis is not well-studied. We hypothesized that V. fluvialis-secreted hemolysin (VFH) may induce IL-1β secretion through the activation of the NLRP3 inflammasome and contribute to the pathogenicity of V. fluvialis. To examine this possibility, we constructed VFH mutant and complement strains and demonstrated that V. fluvialis-induced IL-1β production and cytotoxicity in human monocytic THP-1 cells and mouse macrophages is attributed to VFH. To evaluate the role of VFH in vivo, we infected adult C57BL/6 mice intraperitoneally and suckling C57/B6 mice orally with various strains. The mice treated with 108 CFU wild-type V. fluvialis or cell-free supernatant containing VFH induced significantly higher IL-1β production in peritoneal lavage fluid or in colon compared with those infected with the mutant strain, while no effect on TNF and IL-6 production was observed at day 5 or 24 h post-infection. VFH contributed to pathological changes and IL-1β release independent of colonization of V. fluvialis in the colon. VFH has no effect on the synthesis of pro-IL-1β, but rather it triggers the processing of pro-IL-1β into IL-1β. Furthermore, using deficient mouse strains, we verified that V. fluvialis-induced IL-1β is mediated through activation of Caspase-1 and the NLRP3 inflammasome ex vivo. Confocal microscopy suggests that VFH contributes to cathepsin B release. Furthermore, V. fluvialis-induced IL-1β secretion requires potassium (K+) efflux and reactive oxygen species production. Our results provide new evidence for the role of VFH in the activation of the NLRP3 inflammasome and pathogenesis in response to V. fluvialis infection.Summary Sentence: Vibrio fluvialis-secreted hemolysin induces IL-1β secretion through the activation of the NLRP3 inflammasome and contributes to the pathogenicity of V. fluvialis.
Highlights
Vibrio fluvialis causes mild to moderate dehydration, vomiting, fever, abdominal pain, diarrhea and it can be isolated from human diarrheal feces and aquatic environments (Igbinosa and Okoh, 2010; Ramamurthy et al, 2014)
V. fluvialis induced IL-1β secretion and cytotoxicity in a dose-dependent manner, with peak levels of IL-1β production at 50 multiplicity of infection (MOI), which caused 20% cytotoxicity (Figures 1B,C). At this MOI, V. fluvialis promoted a time-dependent increase in IL-1β production, which peaked at 3 h p.i., as well as an increase in cytotoxicity (Figures 1D,E)
To assess the proinflammatory response induced by V. fluvialis in vivo, 4-weeks-old C57BL/6 mice were injected i.p. with 107 or 108 CFU of V. fluvialis and the cytokines levels in peritoneal lavage fluid (PLF) were measured by ELISA at day 5 p.i
Summary
Vibrio fluvialis causes mild to moderate dehydration, vomiting, fever, abdominal pain, diarrhea and it can be isolated from human diarrheal feces and aquatic environments (Igbinosa and Okoh, 2010; Ramamurthy et al, 2014). V. fluvialis infection has become an increasing public health hazard worldwide and frequently occurs in countries where the raw seafood is largely consumed (Ramamurthy et al, 2014). Cell-free culture filtrates of V. fluvialis strains are able to evoke distinct cytotoxic and vacuolization effects on HeLa cells (Chakraborty et al, 2005). Quorum sensing in V. fluvialis positively regulates production of an extracellular protease and hemolysin and affects cytotoxic activity against epithelial tissue cultures (Wang et al, 2013). The V. fluvialis-caused diarrhea with presence of blood is different from cholera. The underlying mechanisms of inflammatory bloody diarrhea caused by V. fluvialis have not been fully defined
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