Contribution of Six Flagellin Genes to the Flagellum Biogenesis of Vibrio vulnificus andIn VivoInvasion

Abstract

ABSTRACTVibrio vulnificusis a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum.V. vulnificuspossesses a total of six flagellin genes organized into two loci (flaFBAandflaCDE). 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 sixV. vulnificusflagellins in motility and virulence, mutants with single and multiple flagellin deletions were constructed. Mutations inflaBorflaCor theflaCDElocus resulted in a significant decrease in motility, adhesion, and cytotoxicity, whereas single mutations in the other flagellin genes or theflaFBAlocus 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 offlaBandflaD, a gene sharing 99% identity with theflaBat 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 (LD50s) of theflaB flaDand theflaFBA flaCDEmutants increased 23- and 91-fold in a mouse model, respectively, and thein vitroandin vivoinvasiveness 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 ofV. vulnificus, and among the six flagellin genes,flaB,flaD, andflaCwere the most influential components.