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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

PFGE-Southern blot of plasmids from canine and equine C. perfringens strains.Southern blotting of PFGE was performed with DIG-labelled probes for netE and cpe genes. Results from both netE and cpe probes are shown overlayed. In all lanes with two bands, the upper band represents netE and the lower band cpe. M: Mid-Range IIPFG molecular DNA ladder (Kb).
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pone.0122684.g004: PFGE-Southern blot of plasmids from canine and equine C. perfringens strains.Southern blotting of PFGE was performed with DIG-labelled probes for netE and cpe genes. Results from both netE and cpe probes are shown overlayed. In all lanes with two bands, the upper band represents netE and the lower band cpe. M: Mid-Range IIPFG molecular DNA ladder (Kb).

Mentions: To determine the presence of large plasmids in equine and canine type A C. perfringens isolates, genomic DNA from six canine and six equine isolates (Table 4) were subjected to PFGE. The PFGE profiles of the canine and equine C. perfringens strains digested with NotI revealed the presence of two to three large plasmids ranging in size from 45–97 kb in all strains (Fig 3 and Table 5). PFGE analysis (Fig 3) shows the diversity of plasmids among these type A isolates and their marked size variations, which were confirmed by PFGE/SB studies (Fig 4). Specifically, the partially sequenced canine type A C. perfringens JFP718 strain carried three large plasmids (Fig 3) in which the netE and cpe genes were on a distinct plasmid (Fig 4). The SBs showed the presence of tcpF in all the large plasmids (data not shown). In addition, cpb2 was identified by SB in the netG plasmid as described in the scaffold 00012 (data not shown) confirming the hypothesis that these two scaffolds represent two distinct plasmids. Interestingly, the non-cytotoxic strains JFP134 (canine) and JFP738 (equine) which showed one and no large plasmids, respectively, were also SB negative for netE (Fig 4).


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)

PFGE-Southern blot of plasmids from canine and equine C. perfringens strains.Southern blotting of PFGE was performed with DIG-labelled probes for netE and cpe genes. Results from both netE and cpe probes are shown overlayed. In all lanes with two bands, the upper band represents netE and the lower band cpe. M: Mid-Range IIPFG molecular DNA ladder (Kb).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122684.g004: PFGE-Southern blot of plasmids from canine and equine C. perfringens strains.Southern blotting of PFGE was performed with DIG-labelled probes for netE and cpe genes. Results from both netE and cpe probes are shown overlayed. In all lanes with two bands, the upper band represents netE and the lower band cpe. M: Mid-Range IIPFG molecular DNA ladder (Kb).
Mentions: To determine the presence of large plasmids in equine and canine type A C. perfringens isolates, genomic DNA from six canine and six equine isolates (Table 4) were subjected to PFGE. The PFGE profiles of the canine and equine C. perfringens strains digested with NotI revealed the presence of two to three large plasmids ranging in size from 45–97 kb in all strains (Fig 3 and Table 5). PFGE analysis (Fig 3) shows the diversity of plasmids among these type A isolates and their marked size variations, which were confirmed by PFGE/SB studies (Fig 4). Specifically, the partially sequenced canine type A C. perfringens JFP718 strain carried three large plasmids (Fig 3) in which the netE and cpe genes were on a distinct plasmid (Fig 4). The SBs showed the presence of tcpF in all the large plasmids (data not shown). In addition, cpb2 was identified by SB in the netG plasmid as described in the scaffold 00012 (data not shown) confirming the hypothesis that these two scaffolds represent two distinct plasmids. Interestingly, the non-cytotoxic strains JFP134 (canine) and JFP738 (equine) which showed one and no large plasmids, respectively, were also SB negative for netE (Fig 4).

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