title: Modulation of toxin production by the flagellar regulon in Clostridium difficile.
abstract: We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: Annie
authors.initials: A
authors.lastname: Aubry
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.forename: Wangxue
authors.initials: W
authors.lastname: Chen
authors.forename: Rhonda
authors.initials: R
authors.lastname: KuoLee
authors.forename: Susan M
authors.initials: SM
authors.lastname: Twine
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Fulton
authors.forename: Simon
authors.initials: S
authors.lastname: Foote
authors.forename: Catherine D
authors.initials: CD
authors.lastname: Carrillo
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D016360:Clostridioides difficile / Q000378:metabolism*; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D009154:Mutation; D040901:Proteomics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C064971:FliA protein, Bacteria; D012329:RNA, Bacterial; D012808:Sigma Factor