Reactogenicity of live-attenuated Vibrio cholerae vaccines is dependent on flagellins

Abstract

Cholera is a severe diarrheal disease caused by the motile Gramnegative rod Vibrio cholerae. Live-attenuated V. cholerae vaccines harboring deletions of the genes encoding cholera toxin have great promise for reducing the global burden of cholera. However, development of live vaccines has been hampered by the tendency of such strains to induce noncholeric reactogenic diarrhea in human subjects. The molecular bases of reactogenicity are unknown, but it has been speculated that reactogenic diarrhea is a response to V. cholerae's flagellum and/or the motility that it enables. Here, we used an infant rabbit model of reactogenicity to determine what V. cholerae factors trigger this response. We found that V. cholerae ctx mutants that produced flagellins induced diarrhea, regardless of whether the proteins were assembled into a flagellum or whether the flagellum was functional. In contrast, ∼90% of rabbits infected with V. cholerae lacking all five flagellin-encoding genes did not develop diarrhea. Thus, flagellin production, independent of flagellum assembly or motility, is sufficient for reactogenicity. The intestinal colonization and intraintestinal localization of the nonreactogenic flagellin-deficient strain were indistinguishable from those of a flagellated motile strain; however, the flagellin-deficient strain stimulated fewer mRNA transcripts coding for proinflammatory cytokines in the intestine. Thus, reactogenic diarrhea may be a consequence of an innate host inflammatory response to V. cholerae flagellins. Our results suggest a simple genetic blueprint for engineering defined nonreactogenic live-attenuated V. cholerae vaccine strains.animal model | cholera | innate immunity | diarrhea

Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

“…2). In fact, these data are consistent with rabbit studies wherein both nontoxigenic bacteria that produce flagellin and those deleted of fla genes show equivalent colonization (47). This equivalent colonization is observed despite a reduction of proinflammatory markers in the absence of flagella, which should correlate to decreased recruitment of neutrophils.…”

“…Although the absence of flagella reduces the proinflammatory response and thus reduces diarrhea and inflammatory markers in an infant rabbit model, there is no concurrent reduction in colonization, suggesting that the proinflammatory response fails to clear bacterial infection even in the absence of diarrhea, a colonization known as asymptomatic carriage (47). We questioned whether this state was being controlled by the innate immune response even when disease is absent.…”

“…This equivalent colonization is observed despite a reduction of proinflammatory markers in the absence of flagella, which should correlate to decreased recruitment of neutrophils. Further, while infections in a mouse pneumonia model (13), rabbits (47), humans (43), and now adult mice (Fig. 3) show increased expression of regulatory and chemotactic markers IL-6, IL-10, and MIP-2 in the local tissue, there is no difference in these markers when neutrophils are depleted.…”

“…These studies correlate with clinical observations in which naturally occurring "nontoxigenic" V. cholerae O1 strains that have not acquired the CTX⌽ prophage induce diarrhea that is more inflammatory (5,33). In an infant rabbit model of infection, this diarrhea has been specifically linked to expression of flagellin that may be functioning by increasing inflammatory signaling through TLR5 (20,46,47). Studies using lung infection in mice have also linked proinflammatory signaling from CT-negative strains to LPS-dependent signaling through TLR4 (18), whereas lipoprotein has been shown in vitro to stimulate inflammation via TLR1/TLR2 (16,25).…”

In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

“…These mechanisms could account for the increased number of V. cholerae recovered after induction of T6SS-mediated inflammation. It is also worth noting that reactogenic diarrheas inducing by nontoxigenic V. cholerae vaccine prototypes apparently involves intestinal inflammation elicited by innate immune agonists such as flagellins (43).…”