Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Abstract

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

FIG. 1.

Confirmation of Δetx::catP mutants. (A and B) PCR analysis using etx- and catP-specific primers, respectively. Lanes 2 to 6 contained strains CN1020, JIR4891, CN3718, and JIR4892 and recombination plasmid pJIR3077, respectively. Lanes 1 and 7 contained size markers (Promega PCR marker and HindIII-digested λcI857, respectively). (C and D) Southern blots of HindIII-digested genomic DNA obtained using etx- and catP-specific probes, respectively. The contents of lanes 2 to 6 were the same as those described above. Lane 1 contained HindIII-digested λcI857 molecular size markers. (E) Representation of the etx region (based on previous studies [30]) in the wild-type ɛ-toxin plasmids pJIR3118 and pJIR3119, compared to the same region after insertional inactivation of the etx gene by allelic exchange with the recombination vector pJIR3077. The positions of HindIII sites (H) are indicated, as are the sizes of the predicted HindIII fragments (in kb). The etx and catP probes used for Southern blotting are indicated by the solid lines under the genes.

FIG. 2.

Multiplex PCR analysis of the wild type and transconjugants. (A) Lanes 2 to 8 contained type D strain CN1020, CN1020 etx mutant JIR4981, JIR4981 × JIR325 transconjugant JIR4983, type D strain CN3718, CN3178 etx mutant JIR4982, JIR4982 × JIR325 transconjugant JIR4985, and JIR325, respectively. Lane 1 contained Promega PCR molecular size markers. (B) Lanes 2 to 5 contained strains JIR325, JIR12012, JIR12013, and CN1020, respectively. Lane 1 contained Promega PCR molecular size markers.

FIG. 3.

Toxin production by pJIR3118-derived transconjugants. (A) Western blotting of culture supernatants with anti-ɛ-toxin MAb 5B7. Lanes 1 to 4 contained JIR325, CN1020, JIR12012, and JIR12013, respectively. (B) Quantification of ɛ-toxin in culture supernatants of transconjugants JIR12012 and JIR12013 (lanes 1 and 2, respectively) and CN1020 (lane 3). The other lanes contained different concentrations of the purified ɛ-toxin standard (50 to 500 ng). (C and D) Phospholipase C (Plc) and perfringolysin O (PfoA) activities of the strains indicated. The data are the averages for three independent biological samples.

FIG. 4.

ɛ-Toxin-mediated cytopathic effects of culture supernatants. Monolayers of MDCK cells were incubated for 24 h in the presence of trypsin-treated culture supernatants from different strains before examination with an Olympus 1X71 inverted microscope. ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html) was used to view the images. (A) JIR325. (B) CN1020 Δetx::catP. (C) Medium alone. (D) CN1020. (E) CN1020 pretreated with MAb 5B7. (F) CN1020 pretreated with ErmB-specific antibody. (G) JIR12012 [JIR325(pJIR3118)]. (H) JIR12012 pretreated with MAb 5B7. (I) JIR12012 pretreated with ErmB-specific antibody. (J) JIR4983 [JIR325(pJIR3118Δetx::catP)]. (K) JIR4983 pretreated with MAb 5B7. (L) JIR4983 pretreated with ErmB-specific antibody. The culture supernatant from CN1020 was diluted 50-fold prior to use. Bar, 100 μm.