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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 630?erm) 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 630?erm and its derivatives more closely mirror the behavior of 630. Genome sequencing revealed that 630?erm 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 630?erm 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.

SUBMITTER: Collery MM

PROVIDER: S-EPMC5626343 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.

Collery Mark M MM Kuehne Sarah A SA McBride Shonna M SM Kelly Michelle L ML Monot Marc M Cockayne Alan A Dupuy Bruno B Minton Nigel P NP

Virulence 20160921 6

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 ...[more]

PMID: 27652799

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Project description:Isogenic, toxin B (tcdB) mutants of two independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of the Clostridium difficile strain CD630 have previously been shown to behave differently in the hamster model of infection. Those based on strain 630E failed to cause disease, whereas those based on 630Δerm did. Here we show that whilst 630Δerm and its derivatives behave in a comparable way to the parental strain CD630 in terms of growth rate, motility, sporulation, toxin production and virulence, strain 630E and its derivatives do not. They grow more slowly, achieve lower final cell densities, exhibit a reduced capacity for spore formation, express lower levels of toxin and are less virulent in the hamster model. Using Next Generation Sequencing, a total of three SNPs were found that were in both CD630 and 630Δerm. In addition to these, 630Δerm had eight unique SNPs compared to CD630 and 630E, while 630E had 11 SNPs not found in the other two strains. The strain-specific SNPs most likely arose during the repeated subculture experiments undertaken to isolate these ermB deletion strains. The relatively large number of mutations present meant that the identification of those SNPs responsible for the altered properties of 630E was not possible. Allelic exchange was used to correct three mutational changes in 630E, but with essentially no effect on phenotype. These were a 150 bp inversion in the promoter region of a flagella operon, the truncation of CD630_12740 (topoisomerase I), and the correction of a SNP that results in the fusion of two components of a glucose phosphor-transferase-system. To complete the analysis we performed RNAseq, comparing the transcriptome of the three strains at three different time points. The finding corroborated the many differences between the strains, but also highlighted that one SNP cannot necessarily be attributed to one particular expression pattern or even phenotype. In conclusion 630Δerm more accurately mirrors the behaviour of the parental strain CD630, hence if genetic-based studies with C. difficile strain CD630 are necessary, then the use of strain 630∆erm should be favoured over strain 630E. At a fundamental level, our data underlines the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible.

Project description:Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

Dataset Information

What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.

Publications

What's a SNP between friends: The influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives.

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