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A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis.

Mehdizadeh Gohari I, Parreira VR, Nowell VJ, Nicholson VM, Oliphant K, Prescott JF - PLoS ONE (2015)

Bottom Line: Mutation and complementation showed that only netF was associated with the cytotoxicity.Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro.The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada.

ABSTRACT
A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

No MeSH data available.


Related in: MedlinePlus

Genetic organization of JFP718 scaffolds.The genetic organization of (A) partial view of scaffold00006 (GenBank KP739975), (B) partial view of scaffold00012 (GenBank KP739976) is shown, each arrow representing a predicted gene and its orientation. Predicted functional annotations and respective positions are shown above each gene, respectively. Genes are color-coded by their putative role based upon sequence analyses.
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pone.0122684.g001: Genetic organization of JFP718 scaffolds.The genetic organization of (A) partial view of scaffold00006 (GenBank KP739975), (B) partial view of scaffold00012 (GenBank KP739976) is shown, each arrow representing a predicted gene and its orientation. Predicted functional annotations and respective positions are shown above each gene, respectively. Genes are color-coded by their putative role based upon sequence analyses.

Mentions: C. perfringens strain JFP718 was isolated from a case of fatal canine hemorrhagic gastroenteritis diagnosed in a 2-year-old female Pomeranian dog that was found dead the day after attending a dog show [13]. The strain was sequenced because of both the classic appearance of the disease in this dog and the highly cytotoxic effect of its supernatant. The automated annotation of the draft genome sequences of C. perfringens canine strain JFP718 were essential to the discovery of the three putative toxin genes, initially denominated as Panton-Valentine leukocidins (PVLs). Two PVL open reading frames (ORF) were located in scaffold00006 (nucleotides 139809–140777 [–] and 154804–155721[+]) (GenBank KP739975), while the third was in scaffold00012 (nucleotides 9399–10319 [+]) (GenBank KP739976), both surrounded by transposon-related sequences. Interestingly, both scaffolds (00006 and 00012) contained mainly plasmid genes, suggesting that these sequences were associated with a transposable element and/or plasmid. The newly identified toxin genes were named netE, netF and netG. The predicted proteins (NetE, NetF and NetG) encoded by these three ORFs showed 79%, 48% and 52% identity, respectively, with the pore-forming toxin NetB from C. perfringens (EU143239) (Table 1 and S1 Fig). Scaffold 00006 harbors netE and netF toxin genes among 140 ORFs. Immediately upstream of the netE and netF genes, which are 14,027 nt apart, are two mobile element proteins classified as an integrase (C. botulinum BKT015925) and a transposase (C. perfringens), whereas downstream of the netF gene there is another integrase (C. botulinum BKT015925) (Fig 1A). Scaffold 00012 harbors the netG gene as well as the cpb2 (CPB2 toxin) (54351–55052) and cpe (67515–68474) genes (Fig 1B).


A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis.

Mehdizadeh Gohari I, Parreira VR, Nowell VJ, Nicholson VM, Oliphant K, Prescott JF - PLoS ONE (2015)

Genetic organization of JFP718 scaffolds.The genetic organization of (A) partial view of scaffold00006 (GenBank KP739975), (B) partial view of scaffold00012 (GenBank KP739976) is shown, each arrow representing a predicted gene and its orientation. Predicted functional annotations and respective positions are shown above each gene, respectively. Genes are color-coded by their putative role based upon sequence analyses.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390311&req=5

pone.0122684.g001: Genetic organization of JFP718 scaffolds.The genetic organization of (A) partial view of scaffold00006 (GenBank KP739975), (B) partial view of scaffold00012 (GenBank KP739976) is shown, each arrow representing a predicted gene and its orientation. Predicted functional annotations and respective positions are shown above each gene, respectively. Genes are color-coded by their putative role based upon sequence analyses.
Mentions: C. perfringens strain JFP718 was isolated from a case of fatal canine hemorrhagic gastroenteritis diagnosed in a 2-year-old female Pomeranian dog that was found dead the day after attending a dog show [13]. The strain was sequenced because of both the classic appearance of the disease in this dog and the highly cytotoxic effect of its supernatant. The automated annotation of the draft genome sequences of C. perfringens canine strain JFP718 were essential to the discovery of the three putative toxin genes, initially denominated as Panton-Valentine leukocidins (PVLs). Two PVL open reading frames (ORF) were located in scaffold00006 (nucleotides 139809–140777 [–] and 154804–155721[+]) (GenBank KP739975), while the third was in scaffold00012 (nucleotides 9399–10319 [+]) (GenBank KP739976), both surrounded by transposon-related sequences. Interestingly, both scaffolds (00006 and 00012) contained mainly plasmid genes, suggesting that these sequences were associated with a transposable element and/or plasmid. The newly identified toxin genes were named netE, netF and netG. The predicted proteins (NetE, NetF and NetG) encoded by these three ORFs showed 79%, 48% and 52% identity, respectively, with the pore-forming toxin NetB from C. perfringens (EU143239) (Table 1 and S1 Fig). Scaffold 00006 harbors netE and netF toxin genes among 140 ORFs. Immediately upstream of the netE and netF genes, which are 14,027 nt apart, are two mobile element proteins classified as an integrase (C. botulinum BKT015925) and a transposase (C. perfringens), whereas downstream of the netF gene there is another integrase (C. botulinum BKT015925) (Fig 1A). Scaffold 00012 harbors the netG gene as well as the cpb2 (CPB2 toxin) (54351–55052) and cpe (67515–68474) genes (Fig 1B).

Bottom Line: Mutation and complementation showed that only netF was associated with the cytotoxicity.Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro.The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada.

ABSTRACT
A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

No MeSH data available.


Related in: MedlinePlus