Clostridium chauvoei, an Evolutionary Dead-End Pathogen

Abstract

Full genome sequences of 20 strains of Clostridium chauvoei, the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951-2015) were determined and analyzed. The study reveals that the genome of the species C. chauvoei is highly homogeneous compared to the closely related species C. perfringens, a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most C. chauvoei strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 - 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of fliC1, fliC2 and fliC3 genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic C.chauvoei toxin A, the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of C. chauvoei. These data indicate that C. chauvoei is a strict ruminant-associated pathogen that has reached a dead end in its evolution.

Keywords: CRISPR; Clostridium chauvoei; blackleg; dead-end evolution; flagellin genes; virulence genes.

FIGURE 1

Phylogenetic Trees of the 20 C. chauvoei strains; tree generated with the alignment of concatenated sequence of 37 marker genes searched from the genome of C. chauvoei and C. perfringens strains whose full genome sequences are publicly accessible, using the PhyloSift software. The bar in the figure indicates substitutions per site. Confidence values are given at the major branching points.

FIGURE 2

Consensus sequences of all strains aligned against the reference Pacific Biosciences sequence of JF4335 using BLAST Ring Image Generator. Most prevalent gaps (labeled as island 1–9) were inspected for genes potentially involved in virulence or pathogenicity. None of these genes were regarded as such. The gaps mostly represent fragments of transposases and fragments of phage DNA. In red the phages of JF4335, identified with PHASTER (Arndt et al., 2016), are indicated.

FIGURE 3

Multi-sequence alignment of the genes (A) Clostridium chauvoei toxin A (cctA), (B) Hyaluronidase (nagH), (C) Hyaluronidase (nagI), and (D) Sialidase (nanA) created with MAFFT v7.222 and visualized in Geneious v10.1.2. The number of SNV differences for each of these genes are given in the results section. Subgroups (A,B) of the Australian/New Zealand/United Kingdom cluster are differentiated by letters in dark and bright purple color, respectively.

FIGURE 4

(A) Phylogenetic tree of the SNVs of all 20 C. chauvoei strains with a C. septicum strain as outgroup. (B) Cladogram of the same tree. Note that the bootstrap values of several branches are below 50% indicating that these branches are not well supported as this can be anticipated from the high genetic similarity between the individual strains of C. chauvoei.

FIGURE 5

Matrix representation of CRISPR spacers for each individual C. chauvoei strain. Identical spacers are assigned the same numerical value and color. The IS256 transposase is indicated with in pink and divides the CRISPR sequences into two arrays. White spaces are artificial due to the visual representation lining up identical CRISPR spacer sequences and do not represent DNA sequences of the individual genomes. The order of the strains, from top to bottom was arranged to follow clusters and subgroups of the phylogenetic tree of the SNV analysis (Figure 3).

FIGURE 6

Multi-sequence alignment (MSA), visualized in Geneious v10.1.2, of the ORFs, coding for Flagellin genes FliC, of strains JF4335, JF4252, JF4492, JF4494, and JF4495 as representatives of the detected difference in copy numbers in the strains. The MSA reveals that the N- and C- termini are mostly conserved, while the variable domain is in the middle of the FliC.