Expression of the large clostridial toxins is controlled by conserved regulatory mechanisms
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.
Section snippets
Bacterial strains and culture conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were cultured aerobically in 2YT broth with agitation, or on 2YT agar (16 g tryptone, 10 g yeast extract, 5 g NaCl and 10 g agar in 1 l of distilled H2O) at 37 °C. When selection was required, cultures were supplemented with chloramphenicol (25 μg/ml), tetracycline (10 μg/ml), or kanamycin (50 μg/ml). C. perfringens, C. sordellii, and C. difficile strains were cultured using supplemented heart infusion
LCT production in C. sordellii and C. perfringens is temporally regulated and inhibited by glucose
Previous studies have shown that TcdA and TcdB production in C. difficile is temporally regulated, occurring after the onset of stationary phase growth (Hundsberger et al., 1997), and is influenced by a number of environmental factors including the presence of glucose, which represses production of these toxins (Dupuy and Sonenshein, 1998). However, little is known about the regulatory pathways or signals that control the production of the other LCTs. Therefore, to gain a better understanding
Discussion and conclusions
This study has provided evidence which shows that the expression of TcsL and TcsH from C. sordellii and TpeL from C. perfringens is repressed by glucose and is subject to temporal regulation, with toxin production associated with later stages of growth. These observations mirror those previously reported for C. difficile (Dupuy and Sonenshein, 1998, Hundsberger et al., 1997), suggesting that conserved regulatory pathways control LCT production in each of these bacterial species. In C. difficile
Acknowledgements
This work was supported by funding from the Australian National Health and Medical Research Council (NHMRC) and the Australian Research Council (ARC). DL was supported by ARC Future Fellowship FT120100779 from the Australian Research Council. We thank David Aronoff and Teresa Erdman for providing the TcsL-specific Mab.
References (57)
- et al.
A complementation analysis of the restriction and modification of DNA in Escherichia coli
J. Mol. Biol.
(1969) - et al.
Definition of the single integration site of the pathogenicity locus in Clostridium difficile
Gene
(1996) - et al.
Lethal toxin is a critical determinant of rapid mortality in rodent models of Clostridium sordellii endometritis
Anaerobe
(2010) - et al.
Structure and mode of action of clostridial glucosylating toxins: the ABCD model
Trends Microbiol.
(2008) - et al.
Conjugative transfer of RP4-oriT shuttle vectors from Escherichia coli to Clostridium perfringens
Plasmid
(1998) - et al.
Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene
Microb. Pathog.
(2012) - et al.
Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1
Plasmid
(1989) - et al.
Topological and phylogenetic analyses of bacterial holin families and superfamilies
Biochim. Biophys. Acta
(2013) - et al.
Non-toxigenic Clostridium sordellii: clinical and microbiological features of a case of cholangitis-associated bacteremia
Anaerobe
(2011) - et al.
Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment
Clin. Infect. Dis.
(2006)
A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C
Microbiology
Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile
Nucleic Acids Res.
CcpA-mediated repression of Clostridium difficile toxin gene expression
Mol. Microbiol.
TcdC does not significantly repress toxin expression in Clostridium difficile 630DeltaErm
PLoS One
Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers
Nat. Commun.
Characterization of the catalytic domain of Clostridium novyi alpha-toxin
Infect. Immun.
TcsL is an essential virulence factor in Clostridium sordellii ATCC9714
Infect. Immun.
The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile
PLoS Pathog.
Binary toxin production in Clostridium difficile is regulated by CdtR, a LytTR family response regulator
J. Bacteriol.
Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production
Appl. Environ. Microbiol.
Role of the Agr-like quorum-sensing system in regulating toxin production by Clostridium perfringens type B strains CN1793 and CN1795
Infect. Immun.
Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system
mBio
The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens
Infect. Immun.
Regulation of neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic Clostridium botulinum
Appl. Environ. Microbiol.
Combination bacteriolytic therapy for the treatment of experimental tumors
Proc. Natl. Acad. Sci. U.S.A.
Clostridium difficile toxin synthesis is negatively regulated by TcdC
J. Med. Microbiol.
Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor
Mol. Microbiol.
Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors
Mol. Microbiol.
Cited by (27)
Evaluation of different antigenic preparations against necrotic enteritis in broiler birds using a novel Clostridium perfringens type G strain
2021, AnaerobeCitation Excerpt :However, available literature revealed a discrepancy in the release of immunogenic proteins to the influence of incubation period and extract preparation. Contrary to the present observation on immuno-detection of tpeL toxin in SS of 8h growth of C. perfringens type G, the release of TpeL toxin was recorded in a strain of C. perfringens during the late stationary phase [48]. Although clostridial vaccines in livestock animals, including cattle, sheep, and pigs, have been available for many decades, an effective vaccine for NE in chickens remains limited.
Clostridium botulinum type A-virulome-gut interactions: A systems biology insight
2018, Human Microbiome JournalCitation Excerpt :The genome of Hall A harbors a large number of genes encoding secreted proteases and enzymes involved in the uptake and metabolism of amino acids [50]. Hall A contains a variety of extracellular enzymes to degrade proteins and carbohydrates, particularly chitin in order to obtain nutrients for massive spore and toxin production [10,11,12,13,9]. Sebaihia et al.
The CpAL system regulates changes of the trans-epithelial resistance of human enterocytes during Clostridium perfringens type C infection
2016, AnaerobeCitation Excerpt :This hypothesis is supported by evidence showing that cultures of CN3685ΔagrB were able to significantly decrease the TEER of enterocytes in comparison to uninfected control T84 cells. For example, another type C toxin called TpeL [46], whose regulation is mediated by conserved regulatory mechanisms [47], has been recently purified from culture supernatants of C. perfringens type C strains. While cytotoxicity on cells cultures has been demonstrated for TpeL, its role in type C-induced enteric disease has yet to be determined.
Integration of metabolism and virulence in Clostridium difficile
2015, Research in MicrobiologyCitation Excerpt :Interestingly, all of these σ factors are encoded within genetic elements that might have been acquired by horizontal gene transfer. UviA and TetR are encoded on plasmids [28,29], BotR within bacteriophage genomes in some Clostridium botulinum strains [30], TpeR within an uncharacterized pathogenicity locus [27] and TcsR within a toxin locus that shows signatures of integrative and conjugative elements [26]. The organization of the sigma factor gene and its target gene promoters has also been largely conserved [26,31].