Suppressed Induction of Proinflammatory Cytokines by a Unique Metabolite Produced by Vibrio cholerae O1 El Tor Biotype in Cultured Host Cells

Abstract

ABSTRACT Vibrio cholerae O1 has two biotypes, El Tor and Classical, and the latter is now presumed to be extinct in nature. Under carbohydrate-rich growth conditions, El Tor biotype strains produce the neutral fermentation end product 2,3-butanediol (2,3-BD), which prevents accumulation of organic acids from mixed acid fermentation and thus avoids a lethal decrease in the medium pH, while the Classical biotype strains fail to do the same. In this study, we investigated the inhibitory effect of 2,3-BD on the production of two proinflammatory biomarkers, intreleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), in human intestinal epithelial HT29 and alveolar epithelial A549 cells. Cell-free culture supernatants of El Tor strain N16961 grown in LB supplemented with 1% glucose induced a negligible amount of IL-8 or TNF-α, while the Classical O395 strain induced much higher levels of these proinflammatory cytokines. On the other hand, three mutant strains constructed from the N16961 strain with defects in the constitutive 2,3-BD pathway were also able to induce high levels of cytokines. When HT29 and A549 cells were treated with bacterial flagella, known proinflammatory cytokine inducers, and chemically synthesized 2,3-BD at various concentrations, a dose-dependent decrease in IL-8 and TNF-α production was observed, demonstrating the suppressive effect of 2,3-BD on the production of proinflammatory cytokines in epithelial cells. Upon cotreatment with extraneous 2,3-BD, elevated levels of IκBα, the inhibitor of the NF-κB pathway, were detected in both HT29 and A549 cells. Furthermore, treatments containing 2,3-BD elicited lower levels of NF-κB-responsive luciferase activity, demonstrating that the reduced cytokine production is likely through the inhibition of the NF-κB pathway. These results reveal a novel and potential role of 2,3-BD as an immune modulator that might have conferred a superior pathogenic potential of the El Tor over the Classical biotype.