All Three TonB Systems Are Required for Vibrio vulnificus CMCP6 Tissue Invasiveness by Controlling Flagellum Expression

Abstract

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...

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

“…The transcriptional levels of the three structural flp genes, which encoded the major structural components of Flp pili, were measured by conventional and real-time RT-PCR. gyrA was chosen as the reference gene forn the qRT-PCR as previously reported (43). Forward and reverse primer pairs were designed and are provided in S2 Table.…”

“…To compare surface structure of the wild-type, Δ tad123 and Δ tad123 (pLAFR3:: tad1 ), we performed SEM observation. Bacteria were grown in vivo using a rat peritoneal infection model as previously described (41). To minimize shearing force during bacterial preparations, all procedures were carefully performed.…”

“…Bacterial cells that translocated from the intestine to the bloodstream were measured as previously described (43). Seven-week-old randomly bred SPF female ICR mice were starved for 16 h. The ileum was tied off in a 5-cm segment and log-phase V. vulnificus cells (4.0 × 10 6 CFU/400 µl) were inoculated into the ligated segment.…”

“…In vivo growth of V. vulnificus was measured using the dialysis tube implantation model as previously described (43). CelluSep ® H1 dialysis tubing (MWCO 12,000~14,000; Membrane Filtration Products, Inc. Texas) was incubated with PBS overnight.…”

Hepcidin-mediated iron sequestration protects against bacterial dissemination during pneumonia

“…in 100 μl PBS. To induce transient hyperferremia without iron overload, 900 μg of ferric ammonium citrate in 100 μl PBS was delivered i.p., using a previously characterized protocol (54,55). In some experiments, mice were given 1 mg anti-IL-6 antibody or isotype control i.p.…”