Evolutionary history of the Clostridium difficile pathogenicity locus

The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19 kb pathogenicity locus (Pa Loc). Stable integration of the Pa Loc is suggested by its single chromosomal location and the clade specificity of its different genetic variants. However, the Pa Loc 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 Pa Loc. Phylogenetic analyses and annotation of the regions spanning the Pa Loc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (Pa Loc present) and non toxigenic (Pa Loc absent) isolates. Comparison of the core genome and Pa Loc phylogenies demonstrated aneventful evolutionary history, with distinct Pa Loc variants acquired clade specifically after divergence. In particular, our data suggest a relatively recent Pa Loc acquisitionin clade 4. Exchanges and losses of the Pa Loc 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 Pa Loc was unique in containing a stably integrated novel trans poson (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related but non conjugative 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 Pa Loc, mobilized rarely via homologous recombination, and Tn6218, mobilized frequently through transpositio