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RelA is a global regulator for stationary phase development in the model bacterium Bacillus subtilis. The relA gene forms a bicistronic operon with the downstream dtd gene. In this study, we evaluated the significance of RelA and DTD proteins in spore formation and toxin production by an important gastrointestinal pathogen Clostridium perfringens. Our β-glucuronidase assay showed that in C. perfringens strain SM101, relA forms a bicistronic operon with its downstream dtd gene, and the relA promoter is expressed during both vegetative and sporulation conditions. By constructing double relA dtd and single dtd mutants in C. perfringens SM101, we found that: (1) RelA is required for maintaining the efficient growth capacity of SM101 cells during vegetative conditions; (2) both RelA and DTD are required for spore formation and enterotoxin (CPE) production by SM101; (3) RelA/DTD activate CodY, which is known to activate spore formation and CPE production in SM101 by activating a key sporulation-specific σ factor F; (4) as expected, RelA/DTD activate sporulation-specific σ factors (σ E , σ F , σ G and σ K ) by positively regulating Spo0A production; and finally (5) RelA, but not DTD, negatively regulates phospholipase C (PLC) production by repressing plc gene expression. Collectively, our results demonstrate that RelA modulates cellular physiology such as growth, spore formation and toxin production by C. perfringens type A strain SM101, although DTD also plays a role in these pleiotropic functions in coordination with RelA during sporulation. These findings have implications for the understanding of the mechanisms involved in the infectious cycle of C. perfringens.

Sporulation is an important strategy for certain bacterial species within the phylum Firmicutes to survive longer periods of time in adverse conditions. All spore-forming bacteria have two phases in their life; the vegetative form, where they can maintain all metabolic activities and replicate to increase numbers, and the spore form, where no metabolic activities exist. Although many essential components of sporulation are conserved among the spore-forming bacteria, there are differences in the regulation and the pathways among different genera, even at the species level. While we have gained much information from the most studied spore-forming bacterial genus, Bacillus, we still lack an in-depth understanding of spore formation in the genus Clostridium. Clostridium and Bacillus share the master regulator of sporulation, Spo0A, and its downstream pathways, but there are differences in the activation of the Spo0A pathway. While Bacillus species use a multi-component phosphorylation pathway for phosphorylation of Spo0A, termed phosphorelay, such a phosphorelay system is absent in Clostridium. On the other hand, a number of genes regulated by the different sporulation-specific transcription factors are conserved between different Clostridium and Bacillus species. In this review, we discuss the recent findings on Clostridium sporulation and compare the sporulation mechanism in Clostridium and Bacillus., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Members of Clostridium genus are a diverse group of anaerobic spore-formers that includes several pathogenic species. Their anaerobic requirement enhances the importance of the dormant spore morphotype during infection, persistence and transmission. Bacterial spores are metabolically inactive and may survive for long times in the environment and germinate in presence of nutrients termed germinants. Recent progress with spores of several Clostridium species has identified the germinant receptors (GRs) involved in nutrient germinant recognition and initiation of spore germination. Signal transduction from GRs to the downstream effectors remains poorly understood but involves the release of dipicolinic acid. Two mechanistically different cortex hydrolytic machineries are present in Clostridium spores. Recent studies have also shed light into novel biological events that occur during spore formation (accumulation of transcriptional units) and transcription during early spore outgrowth. In summary, this review will cover all of the recent advances in Clostridium spore germination., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Unlabelled: CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection., Importance: Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).

Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe+ C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe+ type A, and 12 of the 14 cpe+ isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe+ SD isolates. However, only 10 of the 12 cpe+/cpb2+ SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD.

Recent epidemiological studies suggested that cpb2-positive Clostridium perfringens isolates are associated with gastrointestinal (GI) diseases in horses. These putative relationships, indicated by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 23 cpb2-positive C. perfringens isolates from horses with GI disease (referred to hereafter as horse GI disease isolates). Our beta2-toxin (CPB2) Western blot analyses demonstrated that all of the tested isolates were unable to produce detectable levels of CPB2. However, Southern blot and nucleotide sequencing analyses identified intact cpb2 open reading frames in all of our surveyed horse GI disease isolates. Furthermore, reverse transcriptase PCR and Northern blot analyses showed that cpb2 genes in all of our surveyed horse GI disease isolates were transcriptionally active, i.e., an approximately 1.2-kb cpb2-specific mRNA was identified in total RNA from our surveyed isolates. The levels of cpb2 mRNA in CWC245 (a high-CPB2-producing pig strain) and our surveyed horse GI disease isolates differed to such an extent (35-fold) that this difference could be considered as a major cause of the difference in levels of CPB2 production by CWC245 and horse GI disease isolates. This finding received further support from our observation that the complementing strain 106902(pMRS140), which produced significantly higher levels of mRNA than strain 106902, produced high levels of CPB2. Collectively, our results indicated that there is a positive correlation between cpb2 transcription levels and the amount of CPB2 produced by a C. perfringens cell and that decreased transcription and/or message instability may be involved, at least in part, in the low CPB2 production noted for horse GI disease isolates in comparison to that noted for pig GI disease isolate CWC245.