A Clostridium difficile gene encoding flagellin

Six strains of Clostridium difficile examined by electron microscopy were f ound to carry flagella, The flagella of these strains were extracted and th e N-terminal sequences of the flagellin proteins were determined. Four of t he strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-te rminal sequence and the conserved C-terminal sequence of several flagellins , the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-stra in identity. The gene consists of 870 nt encoding a protein of 290 amino ac ids with an estimated molecular mass of 31 kDa, while the extracted flagell in has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein d isplays a high degree of identity in the N- and C-terminal amino acids wher eas the central region is variable. A second ORF is present downstream of f liC displaying homology to glycosyltransferases. The fliC gene was expresse d in fusion with glutathione S-transferase, purified and a polyclonal monos pecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified fla gellin gene products and Southern analysis revealed inter-strain heterogene ity; this could be useful for epidemiological and phylogenetic studies of t his organism.