Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis

• Published in: PloS one Vol. 5; no. 5; p. e10795
• Main Authors: Lepp, Dion, Roxas, Bryan, Parreira, Valeria R, Marri, Pradeep R, Rosey, Everett L, Gong, Joshua, Songer, J Glenn, Vedantam, Gayatri, Prescott, John F
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.05.2010; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Bacillus thuringiensis; Bacteria; Base Sequence; Blotting, Southern; Chickens; Clostridium Infections - genetics; Clostridium Infections - microbiology; Clostridium perfringens; Clostridium perfringens - genetics; Clostridium perfringens - pathogenicity; Economic importance; Electrophoresis, Gel, Pulsed-Field; Enteritis; Enteritis - microbiology; Enteritis - veterinary; Gel electrophoresis; Gene sequencing; Genes; Genes, Bacterial - genetics; Genetic Loci - genetics; Genetic Variation; Genetics and Genomics/Bioinformatics; Genetics and Genomics/Genetics of Disease; Genetics and Genomics/Genome Projects; Genetics and Genomics/Microbial Evolution and Genomics; Genome, Bacterial - genetics; Genomes; Genomic Islands - genetics; Genomics; Gram-positive bacteria; Infectious Diseases/Antimicrobials and Drug Resistance; Infectious Diseases/Bacterial Infections; Infectious Diseases/Gastrointestinal Infections; Internalin; Intestinal microflora; Intestine; Loci; Microbiology/Cellular Microbiology and Pathogenesis; Microbiology/Microbial Evolution and Genomics; Microbiota; Molecular Sequence Data; Next-generation sequencing; Open reading frames; Pathogenesis; Pathogenicity; Pathogens; Plasmids; Plasmids - genetics; Pore formation; Poultry; Poultry Diseases - genetics; Poultry Diseases - microbiology; Pulsed-field gel electrophoresis; Ricin; Sequence Analysis, DNA; Southern blotting; Staphylococcus aureus; Virulence; Virulence (Microbiology); Virulence factors; Canada; United States > US; Arizona; Guelph Ontario Canada
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0010795

Type A Clostridium perfringens causes poultry necrotic enteritis (NE), an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s) not present in commensal type A isolates. We used high-throughput sequencing (HTS) technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs) were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb), NELoc-2 (11.2 kb) and NELoc-3 (5.6 kb). The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes approximately 85 and approximately 70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.