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

The pathogenic bacterium 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 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 biofilm, significant levels of FlaEF were detected. Mutants defective in both and 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. 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 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 species demonstrated both the presence of FHPs and their important role in biofilm formation. Flagellar filaments of the pathogenic species, including , , and , are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.