Analysis of the Phospholipase C Gene of Clostridium perfringens KZ1340 Isolated from Antarctic Soil

Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C. perfringens. Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc). To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340. The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined. Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity. Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level. Furthermore, the plc gene cloned from C. perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13. This indicates that the former strain represses plc gene expression in trans. When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C. perfringens strains, supporting the classification of the strain as a variant of C. perfringens.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Sequence Analysis of Flanking Regions of the pfoA Gene of Clostridium perfringens: β-Galactosidase Gene (pbg) Is Located in the 3′-Flanking Region

The 3′-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the β-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed β-galactosidase activities were selected from a λFIXII genomic library of C. perfringens by blue plaque screening using X-Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct β-galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a β-galactosidase of C. perfringens.     

Genomic Diversity in the pfoA Region of Theta-Toxin-Deficient Strains of Clostridium perfringens

The genomic structure of the pfoA-colA region in six theta-toxin-deficient strains of Clostridium perfringens was examined by Southern hybridization using the pfoR, pfoA, pbg, arcABDC and colA genes, encoding regulator for pfoA, theta-toxin, beta-galactosidase, arginine metabolism enzymes and kappa-toxin, respectively, as gene probes. It is suggested that the productivity of theta-toxin in these strains is diverse because of the multiple genetic backgrounds including single deletion of pfoA, large deletion of the pfoA-colA region and the putative point mutations.     

Characterization of two putative fibronectin-binding proteins of Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen that causes gas gangrene and food poisoning in humans and animals. Genomic analysis of C. perfringens strain 13 revealed that this bacterium contains two genes (CPE0737 and CPE1847) that encode putative fibronectin (Fn)-binding proteins (Fbps). These genes, named fbpA and fbpB, were found to be constitutively expressed in all three strains (13, NCTC8237, CPN50) of C. perfringens, isolated from gas gangrene of human, that were tested. Both fbpA and fbpB were cloned and His-tagged, recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) were purified by Ni²⁺-Sepharose column chromatography from transformed Escherichia coli. These recombinant Fbps were shown to bind to Fn, purified from human serum, in a ligand blotting assay. Additionally, Fn bound to these rFbps in a dose-dependent manner in an enzyme-linked immunosorbent assay. Furthermore, it was found that pre-incubation of Fn with either rFbpA or rFbpB inhibited the binding of Fn to C. perfringens cells.     

Cross-complementation of Clostridium perfringens PLC and Clostridium septicum α-toxin mutants reveals PLC is sufficient to mediate gas gangrene

Clostridium perfringens and Clostridium septicum are the most common causes of clostridial myonecrosis or gas gangrene. Although they mediate a similar disease pathology, they elaborate functionally very different α-toxins. We used a reciprocal complementation approach to assess the contribution of the primary toxin of each species to disease and found that C. perfringens α-toxin (PLC) was able to mediate the gross pathology of myonecrosis even in a C. septicum background, although it could not induce vascular leukostasis. Conversely, while C. septicum α-toxin restored some virulence to a C. perfringens plc mutant, it was less active than in its native background.     

Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.     

Comparison of Different Methods of Cell Lysis and Protein Measurements in Clostridium perfringens: Application to the Cell Volume Determination

Four cell lysis methods (NaOH-SDS solubilization, French press treatment, sonication, mutanolysin treatment) and three methods of protein assays (Lowry, Bradford, Pierce) were studied for their applicability to determination of cell volume in Clostridium perfringens NCTC 8798 cell suspensions. Protein contents were higher after a mechanical disruption of the cells than with the other techniques of lysis. The lowest concentrations of protein were obtained with the Bradford procedure. With each of the three protein assay methods, Clostridium perfringens NCTC 8798 protein cell contents were 45% to 58% of protein. Other factors possibly involved in variations of the intracellular volume measurements were examined. A control of the level of protein concentration in the test sample and the type of silicone oil used for the centrifugation were of prime importance during sample preparation. Under our conditions, an intracellular volume of 4 μl/(mg of protein) was routinely found for Clostridium perfringens NCTC 8798. Received: 11 April 1997 / Accepted: 6 August 1997     

A recombinant carboxy‐terminal domain of alpha‐toxin protects mice against Clostridium perfringens

Clostridium perfringens alpha‐toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C‐terminal domain (CP251‐370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S‐transferase (GST) fusion proteins. Antibodies that cross‐reacted with alpha‐toxin were produced after immunization with recombinant proteins including GST‐CP251‐370, GST‐CP281‐370, GST‐CP311‐370, CB1‐372 and GST‐CB251‐372. Anti‐GST‐CP251‐370, anti‐GST‐CP281‐370 and anti‐GST‐CP311‐370 sera neutralized both the PLC and hemolytic activities of alpha‐toxin, whereas anti‐CB1‐372 and anti‐GST‐CB251‐372 weakly neutralized these activities. Immunization with GST‐CP251‐370 and GST‐CP281‐370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST‐CP311‐370 and CB1‐372. GST‐CP251‐370 and GST‐CP281‐370 are promising candidates for vaccines for clostridial‐induced gas gangrene.     

Genetic Characterization of Type A Enterotoxigenic Clostridium perfringens Strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.     

Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of <Emphasis Type="Italic">Clostridium perfringens</Emphasis>

Clostridium perfringens produces potent toxins and histolytic enzymes, causing various diseases including life-threatening fulminant diseases in humans and other animals. Aiming at utilizing a phage endolysin as a therapeutic alternative to antibiotics, we surveyed the genome and bacteriophage sequences of C. perfringens. A phiSM101 muramidase gene (psm) revealed by this study can be assumed to encode an N-acetylmuramidase, since the N-terminal catalytic domain deduced from the gene shows high homology of those of N-acetylmuramidases. The psm gene is characteristic in that it is present in phiSM101, an episomal phage of enterotoxigenic C. perfringens type A strain, SM101, and also in that homologous genes are present in the genomes of all five C. perfringens toxin types. The psm gene was cloned and expressed in Escherichia coli as a protein histidine-tagged at the N-terminus (Psm-his). Psm-his was purified to homogeneity by nickel-charged immobilized metal affinity chromatography and anion-exchange chromatography. The purified enzyme lysed cells of all C. perfringens toxin types but not other clostridial species tested, as was shown by a turbidity reduction assay. These results indicate the Psm-his is useful as a cell-wall lytic enzyme and also suggest that it is potentially useful for biocontrol of this organism.     

A simple method for the isolation and determination of Clostridium perfringens

Summary A method for the isolation and determination of small numbers of vegetative cells or spores of Clostridium perfringen has been developed based on enrichment under anaerobic conditions in a fluid thioglycollate medium without dextrose, containing 400 g of D-cycloserine/ml at 46°C for 18 h (PEM). It allows virtually complete recovery of vegetative cells of all strains of Clostridium perfringens tested, whereas facultative anaerobes present in food are inhibited. Undamaged Clostridium perfringens spores can also be detected by this procedure. After enrichment, isolation of Clostriduum perfringens is carried out on iron sulphite agar at 46°C for 18 h. Typical black colonies are picked and confirmed by the following tests: neutralization of the -toxin by a specific diagnostic antiserum and absence of indole, motility, and ability to liquify gelatin.     

Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239

Clostridium perfringens type A is a common source of food‐borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food‐borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co‐cultured with Caco‐2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co‐cultured in Roswell Park Memorial Institute‐1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO₃]₂) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO₃)₂‐deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO₃)₂ down‐regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down‐regulating Spo0A‐regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation.     

Genome mapping ofClostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne

The intron-encoded endonuclease I-CeuI fromChlamydomonas eugametos was shown to cleave the circular chromosomes of allClostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins,β, ε, and?, in addition to the enterotoxin andλ-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins,? andμ, were missing.