Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species

The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation.