A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

Abstract

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δ tad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δ tad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δ tad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δ tad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δ tad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

Author summary

Vibrio vulnificus is so called “flesh eating bacterium” causing fatal sepsis accompanying destruction (necrosis) of soft tissue. The fatal infection occurs after eating contaminated seafood such as oysters or exposure of pre-existing wounds to seawater. Here we show an important bacterial factor that should be used to adhere to human cells and avoid from host immune system. It is very thin thread-like projections from bacterial surface called Tad (tight adhesion) pili. V. vulnificus interestingly harbors three Tad gene genetic loci called operons. To understand the roles of the three Tad operons in the pathogenesis, we deleted each of those three gene loci. Employing mouse infection models coupled with molecular genetic analyses, we demonstrate here that all those three Tad operons are cooperatively required for V. vulnificus pathogenicity. More specifically, the thin pili threads, hardly observed even under electron microscope, contribute to host cell and tissue invasion, survival in the blood, and resistance to killing activities of serum. These findings explain why V. vulnificus has propensity for invading into blood stream from intestine and growing well in the blood resisting against protective immune responses.