title: Increased toxin expression in a Clostridium difficile mfd mutant.
abstract: BACKGROUND
  The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY.
  
  
  RESULTS
  During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations.
  
  
  CONCLUSIONS
  This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.
authors.affiliation: Department of Life Sciences, Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK. Stephanie.Willing@rhul.ac.uk.|School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK. Stephanie.Willing@rhul.ac.uk.
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Willing
authors.affiliation: Department of Life Sciences, Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK. e.richards12@imperial.ac.uk.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Richards
authors.affiliation: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK. aaron.dale@medschool.ac.uk.
authors.forename: Lluis
authors.initials: L
authors.lastname: Sempere
authors.affiliation: Department of Life Sciences, Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK. luissempere@yahoo.es.
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Dale
authors.affiliation: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK. s.cutting@rhul.ac.uk.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cutting
authors.affiliation: Department of Life Sciences, Centre for Molecular Bacteriology and Infection, Imperial College London, London, SW7 2AZ, UK. n.fairweather@imperial.ac.uk.
authors.forename: Neil F
authors.initials: NF
authors.lastname: Fairweather
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D002473:Cell Wall / Q000235:genetics; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D015964:Gene Expression Regulation, Bacterial; D019073:HT29 Cells; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000276:immunology; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D015854:Up-Regulation; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D014157:Transcription Factors; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; C072103:transcription repair coupling factor protein, Bacteria