Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

Abstract

ABSTRACTWe show in this study that toxin production inClostridium difficileis altered in cells which can no longer form flagellar filaments. The impact of inactivation offliC,CD0240,fliF,fliG,fliM, andflhB-fliRflagellar 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 afliCmutant compared to that in theC. difficile630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parentalC. difficile630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR,tcdB,tcdE, andtcdA) revealed a significant increase of all four genes in thefliCmutant strain, while transcription of all four genes was significantly reduced infliM,fliF,fliG, andflhB-fliRmutants. These results demonstrate that toxin transcription inC. difficileis 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 ofC. difficileinfection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production inC. difficileand provides evidence for elaborate regulatory networks for virulence genes inC. difficile.