Purification and kinetic properties of sialidase from Clostridium perfringens

Clostridium perfringens sialidase was isolated from a culture medium of bacterial cells by ammonium sulfate precipitation (42-85%), followed by purification through Sephadex G-75 gel chromatography, DEAE A-50 anion exchange chromatography, FPLC medium pressure anion exchange chromatography and finally FPLC medium pressure isochromatofocussing. From 9 l culture medium 1.17 mg sialidase was isolated with a specific activity of 295 U/mg. The enzyme appeared to be homogeneous by analytical polyacrylamide gel electrophoresis. The molecular mass was measured to be 66 kDa. Km values ranging from 0.6 to 1.6mM were determined for several oligosaccharides as substrates. The enzyme catalyzed transglycosylation reactions with methanol as a nucleophilic reagent competitive with water. In the enzymatic hydrolysis of the (3'-methoxyphenyl)glycoside of alpha-N-acetylneuraminic acid, increase of methanol concentration had no effect on the release of 3-methoxyphenol. This finding suggests that the formation of the enzyme-glycon intermediate is the rate-determining step for this substrate.     

Clostridium perfringens alpha-toxin: characterization and mode of action

Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C. perfringens. The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin. Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin. To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens

Abstract The C-terminal domain of the alpha-toxin (cpa_247–370) of Clostridium perfringens has been expressed in Escherichia coli and purified. Antiserum raised against cpa_247–370 reacted in an identical manner to anti-alpha-toxin serum when used to map epitopes in the C-terminal domain, suggesting that cpa_247–370 was immunologically and structurally identical to this region in the alpha-toxin. The isolated cpa_247–370 was devoid of sphingomyelinase activity or haemolytic activity and was not cytotoxic for mouse lymphocytes. Haemolytic activity was detected when cpa_247–370 was tested with the N-terminal domain of the alpha-toxin (cpa_1–249), confirming that cpa_247–370 confers haemolytic properties on the phospholipase C activity of the alpha-toxin. Haemolytic activity was not detected if cpa_247–370 was tested with the Bacillus cereus phosphatidylcholine phospholipase C, nor if cpa_1–249 and cpa_247–370 were incubated sequentially with erythrocytes.     

Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens.

1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.     

A型产气荚膜梭菌α-毒素基因的克隆与核苷酸序列分析

利用聚合酶链式反应(PCR)技术,从A型产气莫膜梭菌染色体基因组中扩增了1．2kb的α毒素基因。通过T4 DNA连接酶,将纯化的PCR产物与载体pGEM-T连接,转化受至体菌JM109中,经NcoI/EcoRI和BamHI/EcoRI双酶切分析,证明重组质粒pXCPA02中含有A型产气荚膜棱菌α毒素全基因。经核苷酸序列分析,明确了克隆的α毒素基因在重组质粒中的连接向位且核苷酸序列是正确的。     

Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea

Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxin-encoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 toxin producing strains. Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea.     

Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli]

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.     

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10² bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.     

Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.     

Purification and some properties of nitrite reductase from Clostridium perfringens

Nitrite reductase from Clostridium perfringens was purified by chromatographies on DEAE-cellulose, DEAE-Sephadex, Sephadex G-150, and hydroxylapatite and by isoelectric focussing to a homogeneous state, showing essentially a single protein band in disc gel electrophoresis and a single immuno-precipitation line in double diffusion against antiserum obtained from immunized rabbits. The reductase was induced in the presence of nitrate. It had a molecular weight of 54,000 and showed no absorption peak in the visible region. The pH optimum was 6.2 and Km for nitrite was 5 mM. Ferredoxin, as well as viologen dyes, was found to be an electron donor. The product of nitrite reduction was hydroxylamine. This reductase was inhibited by o-phenanthroline and azide but not by cyanide or diethyldithiocarbamate.