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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

Phylogenetic analysis of representative members of the Leukocidin/Hemolysin superfamily.The phylogenetic tree was built by the Neighbor-joining algorithm using (1000 interactions) MEGA5 software (35). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Toxins that were used included: alpha-hemolysin of C. botulinum (YP_004394739.1), hemolysin II of B. cereus (YP_002447023.1), alpha-hemolysin of S. aureus (WP_001788633), putative CctA of C. chauvoei (WP_021874975) and beta-toxin of C. perfringens (CAA58246.1).
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pone.0122684.g002: Phylogenetic analysis of representative members of the Leukocidin/Hemolysin superfamily.The phylogenetic tree was built by the Neighbor-joining algorithm using (1000 interactions) MEGA5 software (35). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Toxins that were used included: alpha-hemolysin of C. botulinum (YP_004394739.1), hemolysin II of B. cereus (YP_002447023.1), alpha-hemolysin of S. aureus (WP_001788633), putative CctA of C. chauvoei (WP_021874975) and beta-toxin of C. perfringens (CAA58246.1).

Mentions: Sequence analyses of these ORFs were performed using BLAST, BLASTP, the Conserved Domains Database (CDD) [31], SignalP [32], pSortB [33], and InterProScan [34] (Table 1). Analysis against the CDD conserved domain database showed that these newly described net genes encode proteins belonging to the Leukocidin/Hemolysin superfamily. InterProScan analysis and classification also confirmed the presence of the Leukocidin/Hemolysin domain (IPR001340) and classified them as members of the alpha-hemolysin branch of the beta-pore-forming toxin family. The new genes are predicted by pSortB and SignalP to encode extracellular proteins. netE encodes a putative 322 amino acid (aa) protein with a signal peptide region of 30 residues, netF encodes a 305 aa protein, and netG encodes a 306 aa protein, both with a signal peptide region of 24 residues. The predicted respective molecular size of the mature NetE toxin is 32.9 kDa, and of NetF and NetG are 31.7 kDa. The close relationship of the novel Net toxins to other members of the Leukocidin/Hemolysin pore-forming toxin family is shown through the Clustal W alignment (S1 Fig) and phylogenetic tree (Fig 2) [35]. For NetE, the closest ortholog (79% identity) is NetB from C. perfringens, whereas NetF and NetG were placed in their own branch (Fig 2).


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)

Phylogenetic analysis of representative members of the Leukocidin/Hemolysin superfamily.The phylogenetic tree was built by the Neighbor-joining algorithm using (1000 interactions) MEGA5 software (35). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Toxins that were used included: alpha-hemolysin of C. botulinum (YP_004394739.1), hemolysin II of B. cereus (YP_002447023.1), alpha-hemolysin of S. aureus (WP_001788633), putative CctA of C. chauvoei (WP_021874975) and beta-toxin of C. perfringens (CAA58246.1).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122684.g002: Phylogenetic analysis of representative members of the Leukocidin/Hemolysin superfamily.The phylogenetic tree was built by the Neighbor-joining algorithm using (1000 interactions) MEGA5 software (35). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Toxins that were used included: alpha-hemolysin of C. botulinum (YP_004394739.1), hemolysin II of B. cereus (YP_002447023.1), alpha-hemolysin of S. aureus (WP_001788633), putative CctA of C. chauvoei (WP_021874975) and beta-toxin of C. perfringens (CAA58246.1).
Mentions: Sequence analyses of these ORFs were performed using BLAST, BLASTP, the Conserved Domains Database (CDD) [31], SignalP [32], pSortB [33], and InterProScan [34] (Table 1). Analysis against the CDD conserved domain database showed that these newly described net genes encode proteins belonging to the Leukocidin/Hemolysin superfamily. InterProScan analysis and classification also confirmed the presence of the Leukocidin/Hemolysin domain (IPR001340) and classified them as members of the alpha-hemolysin branch of the beta-pore-forming toxin family. The new genes are predicted by pSortB and SignalP to encode extracellular proteins. netE encodes a putative 322 amino acid (aa) protein with a signal peptide region of 30 residues, netF encodes a 305 aa protein, and netG encodes a 306 aa protein, both with a signal peptide region of 24 residues. The predicted respective molecular size of the mature NetE toxin is 32.9 kDa, and of NetF and NetG are 31.7 kDa. The close relationship of the novel Net toxins to other members of the Leukocidin/Hemolysin pore-forming toxin family is shown through the Clustal W alignment (S1 Fig) and phylogenetic tree (Fig 2) [35]. For NetE, the closest ortholog (79% identity) is NetB from C. perfringens, whereas NetF and NetG were placed in their own branch (Fig 2).

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