The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation inClostridium perfringens

Abstract

Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G؉C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme ␤-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…CcpA of Lactobacillus pentosus represses the activity of xylulose 5-phosphate phosphoketolase, the central enzyme of the phosphoketolase pathway (674). In Clostridium perfringens, inactivation of ccpA diminishes the expression of the enterotoxinencoding cpe gene during entry into the stationary growth phase (913). S. mutans ccpA mutants exhibit diminished fructosyltransferase and glucosyltransferase gene expression (88) and biofilm formation (947).…”

Sporulation and Enterotoxin (CPE) Synthesis Are Controlled by the Sporulation-Specific Sigma Factors SigE and SigK inClostridium perfringens

“…OSM172 had a SacI restriction site, and OSM173 had a KpnI restriction site designed into the primer sequence. The resulting PCR product was then digested with SacI and KpnI and ligated into the C. perfringens suicide vector pSM300 (68), resulting in pNLDK.…”

“…Repeated attempts were made to mutagenize the sigE and sigK genes by using transformation with monomeric forms of the plasmids and also transforming with large quantities (ϳ20 g) of linearized DNA to enhance the frequency of allele replacement, but none of these experiments resulted in antibioticresistant colonies (data not shown). In the past, our group has constructed mutations in strain SM101 using allele replacement by homologous recombination methods (68), so the lack of transformants with anything except the multimeric form of the plasmids indicates that the sigE and sigK genes are relatively resistant to recombination events.…”

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

“…However, CcpA is known to positively regulate gene expression in some Clostridium spp. For example, CcpA positively regulates expression of the key solventogenic operon sol in C. acetobutylicum and is necessary for efficient sporulation of C. acetobutylicum and C. perfringens (30,46,48). It is also necessary for C. perfringens enterotoxin (CPE) production by C. perfringens sporulating cultures (30).…”

“…There is accumulating evidence that CcpA can also be involved in the control of virulence gene expression by several Gram-positive pathogens, including C. difficile and S. aureus (25)(26)(27)(28)(29). In C. perfringens, CcpA has been shown to control expression of the enterotoxin gene (cpe) and genes involved in type IV pilus formation and function (30,31). In addition to toxins, C. perfringens produces many enzymes, including three sialidases named NanJ, NanI, and NanH.…”