Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing

Abstract

Background: Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30%. The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster. Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens.

Methods: Whole-genome sequences were determined for 57 C. difficile isolates representative of the population structure and different clinical phenotypes. Phylogenetic analyses were performed on their genomic region (>63 kb) spanning the cwp cluster.

Results: Genetic diversity across the cwp cluster peaked within slpA, cwp66 (adhesin), and secA2 (secretory translocase). These genes formed a 10-kb cassette, of which 12 divergent variants were found. Homologous recombination involving this cassette caused it to associate randomly with genotype. One cassette contained a novel insertion (length, approximately 24 kb) that resembled S-layer glycosylation gene clusters.

Conclusions: Genetic exchange of S-layer cassettes parallels polysaccharide capsular switching in other species. Both cause major antigenic shifts, while the remainder of the genome is unchanged. C. difficile genotype is therefore not predictive of antigenic type. S-layer switching and immune escape could help explain temporal and geographic variation in C. difficile epidemiology and may inform genotyping and vaccination strategies.

Figure 1.

Density of polymorphism in the cwp cluster and phylogeny of whole genomes and S-layer. A, Genetic organization in and around the cwp cluster. B, Density of polymorphism across the cwp cluster. C, Phylogeny constructed from the whole genomes. Solid black shapes indicate the clades. D, Phylogeny constructed from the slpA gene. Shapes indicate the clade, and colors indicate the S-layer variant with the exception of 2 hybrids, designated H and indicated by a star. Numbers indicate bootstrap support. Abbreviation: ORF, open reading frame.

Figure 2.

Phylogeny of cwp66 and secA2. A and B, Phylogeny constructed from cwp66 (A) and secA2 (B). Shapes indicate the clade of each genome (Figure 1C). S-layer cassette variants are shown using colors, with the exception of 2 hybrids, indicated by a star. Numbers indicate bootstrap support.

Figure 3.

Annotation of the putative S-layer glycosylation cluster within ST5 relative to the reference genome CD630 [14]. The annotation of CD630 is shown at the top, and the location of each CD630 gene in the ST5 genome is shown using grey blocks. Color has been used to highlight the glycosylation cluster insertion, the rearrangement of the 2 flanking open reading frames (ORFs), and the deletion of cwp2. Annotation of the genes in the glycosylation cluster is given at the bottom. Numbers 1–19 indicate the ORFs of the glycosylation cluster as in Table 2.

Figure 4

S-layer switching at the chromosomal scale and at the scale of the cwp cluster. A, Whole-genome distributions of polymorphism between pairs of isolates very closely related in terms of whole-genome phylogeny (Figure 1C) but with distinct S-layer cassettes (Figure 1D and Figure 2A and 2B). The 2 outer rings composed of small red lines indicate the open reading frames (ORFs) annotated on the forward and reverse strands of reference genome CD630 [14]. The left-hand plot shows, from inside out, pairs ST10[4]/44[5], ST54[8]/54[1], and ST14[6]/49[10]; the middle plot shows, from inside out, ST41[5]/41[10], ST11(078)[H]/ST11(033)[3], and ST11(078)[H]/ST11(066)[8]; and the right-hand plot shows, from inside out, ST2[10]/ST2[12],ST36[4]/ST36[8], and ST160 [11]/ST44[5]. Blue highlighting indicates the location of the cwp cluster within the chromosome. B, Distribution of polymorphism between the pairs of genomes shown in (A) within the region of the genome containing the cwp cluster. By enlarging and comparing only the region of the cwp cluster involved in S-layer switching (shown in blue in panel A), the distribution of polymorphisms can be used to estimate the size of recombination events. Each row represents a pairwise comparison of 2 isolates, and polymorphisms are shown in red. Abbreviation: SNV, single nucleotide variant.