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

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.

Fig 1. 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.

Fig 2. 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).

Fig 3. PFGE analyses of plasmids from canine and equine C. perfringens strains.

Agarose plugs containing DNA from each specified isolate were digested with NotI and subjected to PFGE and staining with ethidium bromide. Line numbers indicate isolate numbers; M: Mid-Range II PFG molecular DNA ladder (Kb).

Fig 4. 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).

Fig 5. Dendogram of Clostridium perfringens isolates.

Dendogram of C.perfringens isolates typed by pulsed-field gel electrophoresis and analysed using BioNumerics software. The BioNumerics software used was version 7.1 from Applied Maths, Austin, TX.

Fig 6. 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 null 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.