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.     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

Purification of the recombinant beta2 toxin (CPB2) from an enterotoxaemic bovine Clostridium perfringens strain and production of a specific immune serum

Overgrowth of Clostridium perfringens clones with production of one or more of its toxin(s) results in diverse digestive and systemic pathologies in human and animals, such as cattle enterotoxaemia. The so-called beta2 toxin (CPB2) is the most recently described major toxin produced by C. perfringens. In this study, the cpb2 ORF (cpb2FM) from a cattle C. perfringens-associated enterotoxaemia was cloned and sequenced. The cpb2FM and its deduced nucleotide sequence clearly corresponded to the cpb2 allele considered as "consensus" and not to "atypical" allele, despite its "non-porcine" origin. Expression assays of the recombinant toxin CPB2FM were performed in Escherichia coli and Bacillus subtilis with the expression vector pBLTS72, and by genomic integration by double recombination in B. subtilis. Highest level of production was obtained with the expression vector in B. subtilis 168 strain. The recombinant CPB2FM protein was purified and a specific rabbit polyclonal antiserum was produced. Polyclonal antibodies could detect CPB2 production in supernatants of C. perfringens from enterotoxaemic cattle.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant

Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation.     

Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model

Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin.

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.     

Cloning and molecular characterization of a unique hemolysin gene of Vibrio pommerensis sp. nov.: development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp. from the Baltic Sea

A group of hemolytic Vibrio strains was isolated from surface water of the Baltic Sea in 1995. A typical representative strain, CH-291, was found to lyse washed human and animal erythrocytes. Hemolysis was found to be calcium-dependent and occurred over a temperature range from 25 to 37 degrees C. The hemolysin-encoding genes were identified by screening a genomic library of total DNA from strain CH-291. A cloned chromosomal DNA fragment of 15.6 kb conferred to Escherichia coli DH5alpha a hemolytic phenotype. Hybridization and sequence analysis showed the cloned sequence to be unique to these Baltic Sea Vibrio isolates and therefore provides a useful marker for their identification. Moreover, the cloned 15.6-kb DNA fragment possessed structural features typical for genetic islands, including a decreased GC content and a flanking cryptic insertion sequence element.     

Method for Estimating the Presence of Clostridium perfringens in Food

The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 10(6)C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed.     

Genomic map of Clostridium perfringens strain 13

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1-Mb chromosome and a large 50-kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (approximately 400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50-kb plasmid.     

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.     

Gnotobiotic, athymic mice; a possible system for the study of the role of bacteria in human amoebiasis

Two groups of 12 + 14 gnotobiotic, athymic mice were intracaecally injected with Entamoeba histolytica strain HK9 and NIH:200, respectively. Two groups of 16 and 15 mice were given amoebae together with a pure strain of Escherichia coli and a further two groups of 16 and 27 were given amoebae with a pure strain of Clostridium perfringens. Batches of 3-7 mice from each group were killed at intervals of 1-4 weeks. All the mice given NIH:200 alone were found to be infected with trophozoites. Of those given HK9 alone, 20% of the first and 57% of the second group to be examined were infected. Groups of mice given either strain of amoeba monocontaminated with E. coli were all found to be infected at post-mortem examination with no apparent clinical signs and little histological change. The group given HK9 and C. perfringens, although all were infected, failed to produce clinical signs or histological lesions, though some died expectedly. In the group given NIH:200 with C. perfringens the amoebae showed a change of activity and there was evidence of both caecal and liver lesions after 120 days. The usefulness of the system in studying the effect of individual species of bacteria on invasive amoebae is discussed.