e TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis. Vibrio vulnificus is a halophilic estuarine pathogen that causes fatal septicemia and necrotizing wound infections in patients suffering from hepatic diseases with high levels of circulating iron or who are immunocompromised (1-5). Infection with V. vulnificus typically shows rapid progression and mortality rates greater than 50% (6, 7). In Gram-negative bacteria, the inner membrane protein complex TonB plays a crucial role in the uptake of iron (8, 9), which is an important micronutrient for numerous biological processes (10-12). TonB complexes transduce the proton motive force (PMF) of the cytoplasmic membrane to energize iron-siderophore complex transport through a specific TonB-dependent transporter (TBDT) across the outer membrane (OM) (9,13,14). This system's known biological roles had been restricted to iron complexes (15, 16) and vitamin B 12 (14), but recent experimental evidence of the TonB-energized transport of nickel and various carbohydrates suggests that the number and variety of TonB-dependent substrates have been underestimated (17)(18)(19). Unlike the single TonB system in Escherichia coli (8), the genomes of Vibrio species carry multiple TonB systems. Interestingly, three TonB systems were first report...
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 Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 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 Δtad123 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 Δtad123 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 Δtad123 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.
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...
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