Molecular characterization of fliD gene encoding flagellar cap and its expression among Clostridium difficile isolates from different serogroups

The FliD gene encoding the flagellar cap protein (FliD) of Clostridium diff icile was studied in 46 isolates belonging to serogroups A, B, C, D, F, G,H , I, K, X, and S3, including 30 flagellated strains and 16 nonflagellated s trains. In ail but three isolates, amplification by PCR and reverse transcr iption-PCR demonstrated that the fliD gene is present and transcribed in bo th flagellated and nonflagellated strains. PCR-restriction fragment length polymorphism (RFLP) analysis of amplified fliD gene products revealed inter strain homogeneity, with one of two major patterns (a and b) found in all b ut one of the strains, which had pattern c. A polyclonal monospecific antis erum raised to the recombinant FliD protein reacted in immunoblots with cru de flagellar preparations from 28 of 30 flagellated strains but did not rec ognize FliD from nonflagellated strains. the fliD genes from five strains r epresentative of the three different RFLP groups,were sequenced, and sequen cing revealed 100% identity between the strains with the same pattern and 8 8% identity among strains with different patterns. Our results show that ev en though FliD is a structure exposed to the outer environment, the flagell ar cap protein is very well conserved, and this high degree of conservation suggests that It has a very specific function in attachment to cell or muc us receptors.