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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

Infection and cytotoxic effects on equine ovarian (EO) cells by supernatant from JFP838 and its isogenic derivatives.Confluent EO cell cultures were infected for 8 h at 37°C/5%CO2. Filter-sterile broth culture supernatants were used for these infections: (A) Typical morphology of EO cells, (B) JFP838-F05 (netF  mutant), (C) Wild-type JFP838, (D) Complementing strain VN-22C. Cytotoxic effects to EO cells in these conditions were cell rounding, detachment and death of cell in the cell plate, seen in Fig 6C and 6D. Magnification×100.
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pone.0122684.g006: Infection and cytotoxic effects on equine ovarian (EO) cells by supernatant from JFP838 and its isogenic derivatives.Confluent EO cell cultures were infected for 8 h at 37°C/5%CO2. Filter-sterile broth culture supernatants were used for these infections: (A) Typical morphology of EO cells, (B) JFP838-F05 (netF mutant), (C) Wild-type JFP838, (D) Complementing strain VN-22C. Cytotoxic effects to EO cells in these conditions were cell rounding, detachment and death of cell in the cell plate, seen in Fig 6C and 6D. Magnification×100.

Mentions: Secondly, the non-cytotoxic mutant strain JFP838F-05 was complemented in trans with pNetF-07, which contains the entire netF gene. The netF gene in pNetF-07 restored the cytotoxicity of the JFP838::netF mutant (JFP838F-05) similar to that of the wild-type JFP838 (Fig 6). Western blot attested to the expression of NetF in the complemented but not the mutant strain (S4 Fig).


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)

Infection and cytotoxic effects on equine ovarian (EO) cells by supernatant from JFP838 and its isogenic derivatives.Confluent EO cell cultures were infected for 8 h at 37°C/5%CO2. Filter-sterile broth culture supernatants were used for these infections: (A) Typical morphology of EO cells, (B) JFP838-F05 (netF  mutant), (C) Wild-type JFP838, (D) Complementing strain VN-22C. Cytotoxic effects to EO cells in these conditions were cell rounding, detachment and death of cell in the cell plate, seen in Fig 6C and 6D. Magnification×100.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122684.g006: Infection and cytotoxic effects on equine ovarian (EO) cells by supernatant from JFP838 and its isogenic derivatives.Confluent EO cell cultures were infected for 8 h at 37°C/5%CO2. Filter-sterile broth culture supernatants were used for these infections: (A) Typical morphology of EO cells, (B) JFP838-F05 (netF mutant), (C) Wild-type JFP838, (D) Complementing strain VN-22C. Cytotoxic effects to EO cells in these conditions were cell rounding, detachment and death of cell in the cell plate, seen in Fig 6C and 6D. Magnification×100.
Mentions: Secondly, the non-cytotoxic mutant strain JFP838F-05 was complemented in trans with pNetF-07, which contains the entire netF gene. The netF gene in pNetF-07 restored the cytotoxicity of the JFP838::netF mutant (JFP838F-05) similar to that of the wild-type JFP838 (Fig 6). Western blot attested to the expression of NetF in the complemented but not the mutant strain (S4 Fig).

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