Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors

Abstract

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

Keywords: Clostridium perfringens; avian; hemorrhagic bowel syndrome; necrotic enteritis; ruminant.

Figure 1

Maximum likelihood phylogenetic tree of C. perfringens genome assemblies determined by single nucleotide polymorphisms compared to the reference strain ATCC 13124. The reference strain and Clade I are shown in red. Clade II is the food poisoning associated clade and Clade V is the alveolysin clade. Host associations are shown on the inner ring, followed by specific virulence factors, the outer three rings indicate the beta2 variant, toxinotype, and health or disease association (if known) of each strain. The tree is rooted at the midpoint.

Figure 2

Beta2 (cpb2) toxin clustal alignment of each representative beta2 protein type. Amino acids are colored based on their physical properties: blue (acidic), red [small hydrophobic (includes aromatic except Y)], magenta (basic), and green [other (polar, Y)]. The signal peptide sequences are shown in the black box.

Figure 3

The percentage of isolates within each category for each of the individual toxins and virulence factors and whether they are significantly associated with the category are marked with a red circle. The number of strains per category is as follows: avian (61); ruminant (34); human (29); canine (17); equine (16) food (15), and environmental strains (5). Unknown (4), porcine strains (3), and mouse (1) are shown, but were not evaluated for associations. Cells are colored by lift: values greater than 1 indicate a higher presence in the category compared to the presence in all strains and, conversely, lift values less than indicate lower prevalence in the category.