Evolutionary History of the Clostridium difficile Pathogenicity Locus

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Abstract
The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19 kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and nontoxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ∼30 years ago within a clade 1 genotype. The genetic organization of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related but nonconjugative and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting but clinically important genetic elements were thus characterized: the PaLoc, mobilized rarely via homologous recombination, and Tn6218, mobilized frequently through transposition.