The complexity and diversity of the Pathogenicity Locus in Clostridium difficile clade 5

Abstract

The symptoms of Clostridium difficile infection are caused by two closely related toxins, TcdA and TcdB, which are encoded by the 19.6 kb Pathogenicity Locus (PaLoc). The PaLoc is variably present among strains, and in this respect it resembles a mobile genetic element. The C. difficile population structure consists mainly of five phylogenetic clades designated 1-5. Certain genotypes of clade 5 are associated with recently emergent highly pathogenic strains causing human disease and animal infections. The aim of this study was to explore the evolutionary history of the PaLoc in C. difficile clade 5. Phylogenetic analyses and annotation of clade 5 PaLoc variants and adjoining genomic regions were undertaken using a representative collection of toxigenic and nontoxigenic strains. Comparison of the core genome and PaLoc phylogenies obtained for clade 5 and representatives of the other clades identified two distinct PaLoc acquisition events, one involving a toxin A(+)B(+) PaLoc variant and the other an A(-)B(+) variant. Although the exact mechanism of each PaLoc acquisition is unclear, evidence of possible homologous recombination with other clades and between clade 5 lineages was found within the PaLoc and adjacent regions. The generation of nontoxigenic variants by PaLoc loss via homologous recombination with PaLoc-negative members of other clades was suggested by analysis of cdu2, although none is likely to have occurred recently. A variant of the putative holin gene present in the clade 5 A(-)B(+) PaLoc was likely acquired via allelic exchange with an unknown element. Fine-scale phylogenetic analysis of C. difficile clade 5 revealed the extent of its genetic diversity, consistent with ancient evolutionary origins and a complex evolutionary history for the PaLoc.

Keywords: evolution; phylogeny; toxin locus.

Fig. 1.—

Distinct variants of the PaLoc found in clade 5. The genetic organization of the clade 5 PaLoc and flanking genes: (A) A⁺B⁺, toxinotypes XXX and XXXI, (B) A⁻B⁺, (C) PaLoc-negative strains, (D) A⁻B⁻ toxinotype XI strains, and (E) WA12 containing nontoxigenic clade C-1 related (non-PaLoc) sequence at the PaLoc integration site and consequently A⁻B⁻.

Fig. 2.—

Independent clade 5 PaLoc acquisitions are supported by the crosspopulation phylogeny of tcdB functional domains. Phylogenies constructed from the four domains of tcdB: (A) The glucosyltransferase domain, (B) the cysteine protease domain, (C) the translocation domain, and (D) the receptor-binding domain. Colored labels indicate clade (see Key). Strain labels are shown in supplementary figure S1, Supplementary Material online.

Fig. 3.—

Phylogenetic relationship of PaLoc accessory genes. Phylogenies of the PaLoc accessory genes (A) tcdR and (B) tcdE, which encode a positive regulator and a putative holin gene, respectively. The A⁻B⁺ tcdR gene (from strain AI27, marked with asterisk) clustered closest to the A⁺B⁺ lineage (A) was a hybrid of the two sequences suggesting it derived from a recombination event (data not shown). The putative holin gene present in the PaLoc of clade 5 A⁻B⁺ strains is distantly related to that found in the other clades (B), suggesting it was gained via allelic exchange with another source. Strain labels are shown in supplementary figure S2, Supplementary Material online.

Fig. 4.—

The two PaLoc variants were each acquired independently approximately 1,300 years ago. A time-scaled ClonalFrame tree of the core genome dating (approximately) the events of acquisition and loss of the PaLoc within clade 5. The different lineages are color coded according to toxin profiles: A⁺B⁺ (red), A⁻B⁺ (orange), and A⁻B⁻ (blue). The A⁻B⁺ isolates within clade 5 can be divided into two separate branches based on toxinotype (shown in black text on the right).

Fig. 5.—

Acquisition and loss of the clade 5 PaLoc via homologous recombination. In A⁻B⁻ clade 5 strains, the phylogenies of cdu2 (A) and cdu1 (B) genes upstream of the PaLoc are distinct from other clade 5 strains, with the exception of WA12. The genes seen in these strains are more closely related to those of clades 1–4, suggesting that the PaLoc was lost via homologous recombination with a nontoxigenic strain from one of these clades. In the A⁺B⁺ clade 5 strains, the same is seen with the cdd1 gene (C), suggesting these strains gained cdd1 along with the PaLoc via homologous recombination with a toxigenic strain from another clade, whereas the phylogeny of cdd2 (D) remains relatively consistent throughout clade 5. L033 is not included in the trees for cdu2 and cdu1 as these genes are not present in this strain. Strain labels are shown in supplementary figure S3, Supplementary Material online.

Fig. 6.—

Investigation of mechanisms of PaLoc acquisition and modification post acquisition using SNP plots. The distribution of polymorphisms between five different pairs of strains is shown for the genomic region containing the PaLoc relative to the reference strain M120. The PaLoc region is indicated by the blue dashed box. Each row represents a pairwise comparison, and individual polymorphisms are shown in red.

Fig. 7.—

Modification of clade 5 PaLoc postacquisition by large-scale deletions. A comparison between the PaLoc regions of strains L033 (top) and M120 (bottom). The insertion of Tn6218 (indicated in black box) has resulted in a deletion relative to M120 of 51,379 bp comprising most of the PaLoc and a large region upstream. Only tcdC and the 3′-terminal 2,456 bp of tcdA remain of the PaLoc in L033.

Fig. 8.—

Genetic organization of the toxin-encoding loci of C. difficile and C. sordellii. The typical C. difficile PaLoc genetic organization (A) compared with the organization in C. sordellii strains VPI 9048 (B) and the toxin variant (tcsH⁻tcsL⁺) ATCC 9714 (C).