Expression of the large clostridial toxins is controlled by conserved regulatory mechanisms

Abstract

The clostridia cause many human and animal diseases, resulting in significant morbidity and mortality. Host damage results from the action of potent exotoxins, an important group of which is the large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens and Clostridium novyi. Knowledge of the structure and function of these toxins has been attained, however, apart from C. difficile, the regulatory pathways that control LCT production remain largely unknown. Here we show that LCT production in C. sordellii and C. perfringens is temporally regulated and repressed by glucose in a similar manner to C. difficile. Furthermore, we show that the TpeL-encoding gene of C. perfringens is located in an uncharacterized Pathogenicity Locus (PaLoc), along with accessory genes predicted to encode a bacteriophage holin-type protein and a TcdR-family alternative sigma factor, TpeR. Inactivation of tpeR demonstrated that TpeR is critical for C. perfringens TpeL production, in a similar manner to C. difficile TcdR and C. sordellii TcsR, but cross-complementation showed that TpeR is not functionally interchangeable with TcdR or TcsR. Although conserved mechanisms are employed by the clostridia to control LCT production there are important functional differences that distinguish members of the TcdR-family of clostridial alternative sigma factors.

Introduction

The large clostridial toxin (LCT) family includes toxin A (TcdA) and toxin B (TcdB) from Clostridium difficile (Jank and Aktories, 2008), lethal toxin (TcsL) and haemorrhagic toxin (TcsH) from Clostridium sordellii (Voth et al., 2006), alpha toxin (TcnA) from Clostridium novyi (Busch et al., 2000) and TpeL from Clostridium perfringens (Amimoto et al., 2007). These toxins are all monoglycosyltransferases that inactivate Rho family GTPases through the covalent transfer of a glucose or N-acetylglucosamine moiety. Cellular intoxication with a LCT results in disruption of the actin cytoskeleton, cell rounding and, eventually, apoptosis and cell death (Jank and Aktories, 2008, Voth et al., 2006). The importance of the LCTs in disease is becoming increasingly clear, and there is now mounting evidence to suggest that some of these toxins are essential virulence factors (Carter et al., 2011a; Dang et al., 2001, Lyras et al., 2009). Furthermore, the LCT producing clostridia are important human and animal pathogens that cause severe disease and are increasingly being associated with high rates of morbidity and mortality (Aldape et al., 2006, Majumdar et al., 2004, Redelings et al., 2007).

With the exception of TcdA and TcdB from C. difficile, very little is known about how the clostridia regulate the expression of the LCTs. In C. difficile, TcdA and TcdB are encoded by tcdA and tcdB, respectively, within a chromosomal region known as the Pathogenicity Locus or PaLoc. In addition to the toxin genes, three accessory genes are encoded within PaLoc, designated tcdR, tcdE and tcdC. Substantial experimental evidence suggests that tcdR encodes an alternative sigma factor, TcdR, which is critical for the expression of both toxins A and B (Mani and Dupuy, 2001, Mani et al., 2002). The TcdC protein is thought to encode an anti-sigma factor which sequesters the TcdR protein and prevents its association with the core RNA polymerase (Dupuy et al., 2008, Matamouros et al., 2007), although the role of TcdC in controlling toxin production is controversial (Bakker et al., 2012, Carter et al., 2011b, Cartman et al., 2012). The tcdE gene encodes a protein similar to a class of bacteriophage proteins known as holins (Tan et al., 2001). This suggests that TcdE may be a component of a novel holin-based mechanism responsible for toxin export in C. difficile (Tan et al., 2001). Although this hypothesis is supported by a recent study involving the analysis of a tcdE mutant in C. difficile strain JIR8094, which showed that toxin secretion from this strain was reduced in comparison to the wild-type strain (Govind and Dupuy, 2012), a similar independent study suggested that a tcdE mutation in strain 630Δerm did not reduce toxin secretion (Olling et al., 2012).

More recently, the LCT-encoding tcsL and tcsH genes of C. sordellii were shown to reside within a region similar to the C. difficile PaLoc (Sirigi Reddy et al., 2013). In addition to tcsL and tcsH, a gene encoding a bacteriophage holin-like protein named TcsE was identified, as was a gene that encodes a TcdR-family alternative sigma factor. Like TcdR in C. difficile, this protein, TcsR, was shown to be critical for LCT production in C. sordellii (Sirigi Reddy et al., 2013).

In addition to TcdR and TcsR from C. difficile and C. sordellii, respectively, TcdR-family proteins have also been identified in TpeL-negative strains of C. perfringens, where the UviA protein has been shown to control the production of a UV inducible bacteriocin (Dupuy et al., 2005). Similarly, BotR (Marvaud et al., 1998b) and TetR (Marvaud et al., 1998a) from Clostridium botulinum and Clostridium tetani, respectively, control the production of neurotoxins. Although these TcdR-family proteins are involved in expression of toxins, these toxins are not LCTs and the genes encoding these proteins do not appear to be associated with PaLoc regions.

The identification of a PaLoc-like region in C. sordellii raises the possibility that each of the LCT genes may reside within similar PaLoc-like regions and that these regions may encode the proteins needed to control the expression and export of these toxins. However, little is known about the genomic regions within which the tpeL and tcnA genes reside. In this study, we show that LCT production in C. sordellii and C. perfringens is repressed by glucose and is temporally regulated in a similar manner to TcdA and TcdB in C. difficile. We have also identified a previously uncharacterized PaLoc-like region in a TpeL-positive strain of C. perfringens and shown that the tpeR gene, which is located within this region, encodes a TcdR-family protein which is critical for TpeL production. Finally, we have shown that TpeR is functionally distinct from TcsR and TcdR from C. sordellii and C. difficile, respectively, despite belonging to the same protein family.