Contribution of Six Flagellin Genes to the Flagellum Biogenesis of Vibrio vulnificus and In Vivo Invasion

Abstract

dVibrio vulnificus is a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum. V. vulnificus possesses a total of six flagellin genes organized into two loci (flaFBA and flaCDE). We proved that all six of the flagellin genes were transcribed, whereas only five (FlaA, -B, -C, -D, and -F) of the six flagellin proteins were detected. To understand roles of the six V. vulnificus flagellins in motility and virulence, mutants with single and multiple flagellin deletions were constructed. Mutations in flaB or flaC or the flaCDE locus resulted in a significant decrease in motility, adhesion, and cytotoxicity, whereas single mutations in the other flagellin genes or the flaFBA locus showed little or no effect. The motility was completely abolished only in the mutant lacking all six flagellin genes (flaFBA flaCDE). Surprisingly, a double mutation of flaB and flaD, a gene sharing 99% identity with the flaB at the amino acid level, resulted in the largest decrease in motility, adhesion, and cytotoxicity except for the mutant in which all six genes were deleted (the hexa mutant). Additionally, the 50% lethal doses (LD 50 s) of the flaB flaD and the flaFBA flaCDE mutants increased 23-and 91-fold in a mouse model, respectively, and the in vitro and in vivo invasiveness of the mutants was significantly decreased compared to that of the wild type. Taken together, the multiple flagellin subunits differentially contribute to the flagellum biogenesis and the pathogenesis of V. vulnificus, and among the six flagellin genes, flaB, flaD, and flaC were the most influential components.

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

“…To corroborate whether the three TonB systems contribute to the invasiveness of V. vulnificus CMCP6, we performed an in vivo invasion assay using the mouse ligated-ilealloop infection model (32). Viable V. vulnificus cells in the blood were counted at 3, 6, and 8 h to evaluate the tissue invasiveness of the ⌬tonB123 mutant.…”

“…Previously, Alice et al reported that the genes coding for flagellar proteins are induced under iron-rich conditions (20). It has been reported that flagellar biogenesis is a requisite for host cell adhesion and cytotoxicity (32,45,46). Hence, we speculated that the three TonB systems would play important roles in the flagellar biogenesis by supplying sufficient level of iron.…”

“…Polarized HCA-7 cells grown on Transwell filter chambers (8-m pore size; Costar, Cambridge, MA, USA) were apically exposed to log-phase V. vulnificus cells at a multiplicity of infection (MOI) of 5 under serum-free conditions. The invasiveness was determined by measuring the change in transepithelial electrical resistance (33) and the number of bacterial cells that translocated from the upper chamber to the lower chamber of the Transwell (32). Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates.…”

“…The challenged mice were monitored for 48 h. Seven mice per group were used In vivo invasion assay. The bacterial cells that translocated from the intestine to the bloodstream were measured as previously described (31,32). Seven-week-old SPF female CD-1 mice were starved for 16 h and anesthetized with a mixture of 10% zolazepam-tiletamine (Zoletil; Virbac Laboratories, France) and 5% xylazine (Rumpun; Byer Korea, South Korea) dissolved in PBS.…”

“…The RtxA1 proteins were detected using an anti-Cterminal GD-rich domain (anti-GD domain) antibody (RtxA1-C, a band of approximately 130 kDa) (36). For the Western blot analysis of FlaB, bacteria were cultured in 2.5% NaCl HI to log-phase growth, and then FlaB proteins in the bacterial cell pellet or the culture supernatant were detected using an anti-FlaB antibody (a band of approximately 42 kDa) as described previously (32).…”