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Pathogenicity and Medical Microbiology |
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1
Institute of Infection and Immunity, Queen’s Medical Centre, University of Nottingham, University Park, Nottingham NG7 2RD, UK2
PHLS Central Public Health Laboratory, 61 Colindale Ave, London NW9 5HT, UK3
Author for correspondence: Marie-Claude Barc. Tel: +33 1 46 83 55 49. Fax: +33 1 46 83 58 83. e-mail: marie-claude.barc{at}cep.u-psud.fr
Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal 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-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific 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 flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.
Keywords: Clostridium difficile, flagella, flagellin, PCR, cloning
Abbreviations: GST, glutathione S-transferase
The GenBank accession numbers for the sequences reported in this paper are AF065259 (strain 79-685) and AF077341 (strain VPI 10463).
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