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

Abstract

30The tad operons encode the machinery required for adhesive Flp (fimbrial low-31 molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors 32 three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing 33 bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three 34 tad loci during infection, we constructed single, double and triple tad loci deletion mutants. 35 Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in 36 mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the 37 Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to 38 Flp to visualize the pilin protein by using immunogold EM and immunofluorescence 39 microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, delayed RtxA1 40 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium 41 compared to wild type, which could be partially complemented with each wild type operon. The 42Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the 43 alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all three tad 44 loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from 45 host defense mechanisms, ultimately resulting in septicemia. 47To understand the roles of the three Tad operons in the pathogenesis of V. vulnificus 53 infection, we constructed mutant strain with single, double and triple Tad loci deletions. 54Employing a variety of mouse infection models coupled with molecular genetic analyses, we 55 demonstrate here that all three Tad operons are required for V. vulnificus pathogenicity as the 56 cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to 57 complement activation. 58 4 59 60Vibrio vulnificus is an opportunistic marine pathogen that causes fatal septicemia and 61 necrotizing wound infections in susceptible individuals with underlying hepatic diseases and 62 other immunocompromised conditions. In humans, this pathogen frequently causes rapidly 63 progressing fatal sepsis with a mortality rate of greater than 50% within a few days post-64 infection (1-4). During the infectious process, V. vulnificus must cope with dramatic 65 environmental changes by sensing changes in the host milieu (5). To establish successful 66 infections in vivo, V. vulnificus must manage spatiotemporally coordinated changes in the 67 expression levels of various virulence genes. 68 To understand the genome-wide gene expression changes in V. vulnificus after infection, 69 we recently performed a transcriptomic analysis of cells grown in vivo using a rat peritoneal 70 infection model. Notably, among the newly identified in vivo-expressed genes, a Flp/Tad pilus-71 encoding gene cluster (the tad1 locus) was found to be highly upregula...