title: What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.
abstract: Clostridium difficile is a major cause of antibiotic induced diarrhea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. The two main C. difficile virulence factors are toxins A and B. Isogenic toxin B mutants of 2 independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. Compared to 630, strain 630E and its progeny grow slower, achieve lower final cell densities, exhibit a reduced capacity for spore-formation, produce lower levels of toxin and are less virulent in the hamster infection model. By the same measures, strain 630Δerm and its derivatives more closely mirror the behavior of 630. Genome sequencing revealed that 630Δerm had acquired 7 unique Single Nucleotide Polymorphisms (SNPs) compared to 630 and 630E, while 630E had 9 SNPs and a DNA inversion not found in the other 2 strains. The relatively large number of mutations meant that the identification of those responsible for the altered properties of 630E was not possible, despite the restoration of 3 mutations to wildtype by allelic exchange and comparative RNAseq analysis of all 3 strains. The latter analysis revealed large differences in gene expression between the 3 strains, explaining in part why no single SNP could restore the phenotypic differences. Our findings suggest that strain 630Δerm should be favored over 630E as a surrogate for 630 in genetic-based studies. They also underline the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible.
authors.affiliation: a Clostridia Research Group , BBSRC/EPSRC Synthetic Biology Research Center (SBRC), School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Collery
authors.affiliation: a Clostridia Research Group , BBSRC/EPSRC Synthetic Biology Research Center (SBRC), School of Life Sciences, University of Nottingham , Nottingham , UK.|b NIHR Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust , University of Nottingham , Nottingham , UK.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kuehne
authors.affiliation: c Department of Microbiology and Immunology , Emory Antibiotic Resistance Center, Emory University , Atlanta , GA , USA.
authors.forename: Shonna M
authors.initials: SM
authors.lastname: McBride
authors.affiliation: a Clostridia Research Group , BBSRC/EPSRC Synthetic Biology Research Center (SBRC), School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Kelly
authors.affiliation: d Laboratoire Pathogenèse des Bactéries Anaérobies , Institut Pasteur , Paris , France.
authors.forename: Marc
authors.initials: M
authors.lastname: Monot
authors.affiliation: a Clostridia Research Group , BBSRC/EPSRC Synthetic Biology Research Center (SBRC), School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Cockayne
authors.affiliation: d Laboratoire Pathogenèse des Bactéries Anaérobies , Institut Pasteur , Paris , France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Dupuy
authors.affiliation: a Clostridia Research Group , BBSRC/EPSRC Synthetic Biology Research Center (SBRC), School of Life Sciences, University of Nottingham , Nottingham , UK.|b NIHR Biomedical Research Unit in Gastrointestinal and Liver Diseases at Nottingham University Hospitals NHS Trust , University of Nottingham , Nottingham , UK.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics*; D016360:Clostridioides difficile / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity*; D006224:Cricetinae; D003967:Diarrhea / Q000382:microbiology; D004768:Enterotoxins / Q000235:genetics*; D015870:Gene Expression; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile
keywords: ClosTron; motility; mutation; single nucleotide polymorphism; sporulation; toxin expression; virulence