Abstract
ABSTRACTClostridium 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.
Highlights
Clostridium difficile is a Gram-positive, anaerobic sporeforming bacterium capable of causing a range of diseases from mild diarrhea to potentially fatal toxic pseudomembranous colitis
The ClosTron plasmid pMTL007C-E2::CditcdB-1511a that had previously been used to generate strain 630Derm a tcdB mutant producing only TcdA (ACB-)9 was used to create an equivalent mutant in strain 630E as described.[9]
Our study has established that the parental strains (630E and 630Derm) used in the 2 previous studies, that explored the relative roles of toxins are 308kDa (toxin A) and toxin B in disease,[8,9] are phenotypically and genetically distinct
Summary
Clostridium difficile is a Gram-positive, anaerobic sporeforming bacterium capable of causing a range of diseases from mild diarrhea to potentially fatal toxic pseudomembranous colitis. The two toxins are 308kDa (toxin A) and 270kDa (toxin B) in size[1,2,3] and are encoded by the chromosomally located genes tcdA and tcdB, respectively Both are cytopathic to cultured cells due to disruption of the cytoskeleton, TcdB is thought to be up to 1000-times more potent.[1] Historically, toxin A was regarded as the main causative agent of the symptoms of C. difficile infection (CDI). Pivotal data was provided by Lyerly et al.[4] who were only able to detect disease when hamsters were subject to intragastric challenge with purified TcdA alone and not with TcdB The latter could, cause disease symptoms if prior damage to the mucosa had been inflicted by co-administration of sublethal concentrations of toxin A. It was generally accepted that both toxins acted in concert to bring about disease symptoms, with toxin A leading to the initial damage to the colon allowing the subsequent access of the more potent toxin B
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