Reevaluation of whether a Functional Agr-like Quorum-Sensing System Is Necessary for Production of Wild-Type Levels of Epsilon-Toxin by Clostridium perfringens Type D Strains

Abstract

Clostridium perfringens type B and D strains produce epsilon-toxin (ETX). Our 2011 mBio study (mBio 2:e00275-11, 2011, https://doi.org/10.1128/mBio.00275-11) reported that the Agr quorum-sensing (QS) system regulates ETX production by type D strain CN3718. However, subsequent studies have brought that conclusion into question. For example, we reported in 2012 (Infect Immun 80:3008-3017, 2012, https://doi.org/10.1128/IAI.00438-12) that the Agr-like QS system is not required for wild-type ETX production levels by two type B strains. Consequently, we reexamined whether the Agr-like QS system regulates ETX production in type D strains by using Targetron insertional mutagenesis to construct new agrB null mutants of two type D strains, CN3718 and CN2068. Western blotting showed that both agrB mutants still produce wild-type ETX levels. However, the newly constructed agrB mutants of both type D strains produced reduced amounts of alpha-toxin, and this effect was reversible by complementation, which confirms loss of functional AgrB production by these mutants since alpha-toxin production is known to be regulated by AgrB. Coupled with the previously published results for type B strains, these new findings indicate the Agr-like QS system is not usually necessary for C. perfringens to produce wild-type ETX levels. IMPORTANCE Since epsilon-toxin (ETX) is necessary for the virulence of C. perfringens type D and, likely, type B strains, understanding the regulation of ETX production is important. In 2011, we reported that an agrB null mutant of type D strain CN3718 produces less ETX than its wild-type parent. However, when new agrB mutants were constructed in type D strains CN3718 and C2068, ETX production was unaffected. Those newly constructed agrB mutants produced less alpha-toxin, and this phenotype was reversible by complementation, confirming construction of agrB null mutants since alpha-toxin production is regulated by AgrB. Coupled with previous results for type B strains, these new type D results support the conclusion that the Agr QS is not usually necessary for wild-type ETX production levels.

FIG 1

Construction and characterization of newly constructed agrB mutants of type D strains CN3718 and CN2068. (A) PCR confirmation of CN3718 and CN2068 agrB mutant strains. DNA purified from wild-type CN3718 or CN2068 supported amplification of a 366-bp product using internal agrB primers, while the same PCR assays amplified an ∼1.3-kb product using DNA purified from the mutant strains due to insertion of an ∼900-bp product into their agrB gene. (B) Southern blot hybridization of an intron-specific probe to DNA from CN3718 or CN2068 or their agrB mutants. DNA from each strain was digested with EcoRI and electrophoresed on a 1% agarose gel prior to blotting and hybridization with the intron-specific probe. (C) RT-PCR evaluation of agrB expression shows that the agrB mutants (left, CN3718 agrB KO; right, CN2068 agrB KO) expressed an intron::agrB fusion transcript, while the complementing strains (CN3718 agrB Comp and CN2068 agrB Comp) expressed the wild-type agrB transcript. These PCR assays were repeated three times, and a representative result is shown. For size reference, a 1-kb marker is shown (Fisher Scientific).

FIG 2

Phenotypic comparisons of Fig. 1 type D strains. Western blots showing (A) timeline of ETX production by wild-type CN3718 and CN2068, (B) a time point comparison of ETX production by wild-type CN3718 or CN2068 versus their agrB mutants (CN3718 agrB KO or CN2068 agrB KO) or complementing strains (CN3718 agrB Comp or CN2068 agrB Comp), and (C) CPA production by CN3718 and its derivatives (left) or CN2068 and its derivatives (right). All Western blot results shown in panels A to C are representative of three repetitions.