Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences

Abstract

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C. perfringens isolates.

FIG. 1

Multiplex PCR for C. perfringens toxin genes. Representative results of multiplex PCR using primers designed to amplify genes for each “typing” toxin and CPE. PCR products derived from each gene are shown in lane 1 (standards), and their sizes are indicated on the left. Results from five C. perfringens type E veterinary isolates and the type E reference strain NCIB 10748 (positive for cpa, iap, and cpe), as well as the type A strain F4406 (positive for cpa and cpe), are shown.

FIG. 2

Western immunoblot analysis for CPE expression. The expression of CPE by type E and type A control isolates was evaluated by using a CPE-specific Western immunoblot procedure. Isolates were grown for 8 h at 37°C in DS-B. After sonication, an aliquot (100 μl) of each sonicated culture lysate, as well as the specified amounts of purified CPE, was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel), followed by immunoblotting with anti-CPE antibodies and ¹²⁵I-protein A. The blot was then autoradiographed to identify radioactive species. Molecular weight markers (in thousands) are shown at the right; arrow indicates migration of CPE.

FIG. 3

Gene arrangement in type E strains. Schematic representation of the arrangement of sequences in the cpe-iap region of the five type E veterinary enteritis isolates and NCIB 10748. Long arrows indicate the positions and orientations of gene sequences. Short arrows show the positions and orientations of primers used in the PCRs. The EcoRV sites used in inverse PCR are also indicated, and a scale (in base pairs) is shown below the map.

FIG. 4

Comparison between the cpe sequence of type E strains and the cpe ORF of NCTC 8239. The consensus cpe sequence present in type E isolates (upper line) was compared with the cpe ORF of the CPE-positive type A strain NCTC 8239 (lower line). Abbreviations: RBS, ribosome binding site; FS, frame shift; OPA, OCH, and AMB, opal, ochre, and amber termination codons, respectively. P1, P2, and P3 represent the three recently identified (31) promoters for the type A cpe of NCTC 8239.

FIG. 5

Analysis of clonal relationships among C. perfringens type E isolates. DNA, prepared in agarose plugs, from each of the specified C. perfringens isolates was digested with MluI and subjected to PFGE and ethidium bromide staining. The gel was calibrated with lambda ladder DNA. Molecular sizes of the DNA markers are shown at the right.

FIG. 6

Localization of cpe to episomal DNA in C. perfringens type E isolates. Southern hybridization analysis was performed for PFGE gels containing undigested (U) and I-CeuI-digested (C) DNA from selected C. perfringens type E isolates. (Left) Southern blot probed with a 639-bp DIG-labeled cpe probe (5). (Right) The cpe probe was stripped off, and the blot was reprobed with a 443-bp DIG-labeled iap-specific probe. Molecular size DNA markers are shown in the center.