Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens

The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.     

Identification of a two-component VirR/VirS regulon in Clostridium perfringens

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.     

The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens

The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.     

Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens

Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR_D57N onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.     

Identification and proteome analysis of the two-component VirR/VirS system in epidemic Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects pigs and sporadically causes serious infections in humans. Two recent large-scale outbreaks of human streptococcal toxic-shock-like syndrome with high mortality occurred in China, posing new challenges for global public health. However, the global regulation of the virulence of epidemic SS2 isolates lacks a systematic understanding. In this study, we performed a mutational and functional analysis of an SS2 two-component system that is orthologous to the VirR/VirS regulatory system of Clostridium perfringens. An isogenic knockout mutant of VirR/VirS (ΔvirRS) was found to exhibit marked phenotypic changes, including the formation of shorter chains and thinner capsular walls, more easily cleared in whole blood, and decreased oxidative stress tolerance. Furthermore, the ΔvirRS mutant was greatly attenuated in a mouse model. Comparative proteome analysis of the expression profiles of the wild-type strain with the ΔvirRS mutant allowed us to identify 72 proteins that are differentially expressed in the absence of the VirR/VirS system and that are directly responsible for the pleiotropic phenotype of the ΔvirRS mutant.     

Genome sequencing and analysis of a type A Clostridium perfringens isolate from a case of bovine clostridial abomasitis

Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ～3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (～18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified.     

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.     

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.