A Clostridium difficile gene encoding flagellin.
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.
Tasteyre
Albert
A
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1.
Barc
Marie-Claude
MC
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1.
Karjalainen
Tuomo
T
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1.
Dodson
Paul
P
Institute of Infection and Immunity, Queen's Medical Centre, University of Nottingham, University Park, Nottingham NG7 2RD, UK2.
Hyde
Susan
S
Institute of Infection and Immunity, Queen's Medical Centre, University of Nottingham, University Park, Nottingham NG7 2RD, UK2.
Bourlioux
Pierre
P
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, 92296 Châtenay-Malabry cedex, France1.
Borriello
Peter
P
PHLS Central Public Health Laboratory, 61 Colindale Ave, London NW9 5HT, UK3.|Institute of Infection and Immunity, Queen's Medical Centre, University of Nottingham, University Park, Nottingham NG7 2RD, UK2.
D000595:Amino Acid Sequence; D016360:Clostridioides difficile / Q000235:genetics*; D005408:Flagellin / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D005408:Flagellin