Detection of α- and ε-toxigenic Clostridium perfringens Type D in sheep and goats using a DNA amplification technique (PCR)

Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the α-, β- and ε-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the α- and ε-toxin genes but were devoid of the β-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the α-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the α- and ε-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the ε-toxin gene, whereas the majority of the colonies were of type A with the α-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens . The β-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation.     

Action of staphylococcal α-toxin on membranes: Some recent advances

Summary Recent developments in the area of Staphylococcal -toxin studies are presented which modify the concepts previously held with respect to both biological and physical properties of -toxin. New data concerning the nature of the binding site for -toxin on rabbit erythrocyte membranes and a model to explain the various observed complexes of -toxin and membrane receptor are discussed. Finally, evidence suggesting that Staphylococcal -toxin is a potent demyelinating agent is presented.The work was supported in part by NSF grant PCM 77-19307     

The carboxy-terminal C2-like domain of the α-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition

The lethal, cytolytic α-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain. Calcium ions are essential for the binding of α-toxin to lipid films. Sixteen α-toxin variants with single amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native α-toxin under calcium-limiting conditions. Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C₂β-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C-terminal region of α-toxin mediates interactions with membrane phospholipids in a calcium-dependent manner. Mutations to this domain may account for the natural lack of toxicity of the α-toxin homologue, phospholipase C of Clostridium bifermentans .     

Typing of Clostridium perfringens by in vitro amplification of toxin genes

G. DAUBE, B. CHINA, P. SIMON, K. HVALA AND J. MAINIL. 1994. The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of α-toxin, α-toxin, β-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the *** l -toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the α-toxin gene was obtained from direct enrichments of animal intestinal contents.     

The anti-haemolysin activity of tea and coffee

Extracts of tea and coffee inhibited the haemolytic activities of Staphylococcus aureus α-toxin and Vibrio parahaemolyticus thermostable direct haemolysin (Vp-TDH). Black tea had the strongest anti-haemolysin activity. Green tea was more active than pu-erh tea. Coffee had anti-Vp-TDH activity but not anti-α-toxin activity.     

Ref cell hydrolases: Glycosidase activities in human erythrocyte plasma membranes

Summary Human erythrocyte plasma membranes were found to contain the following glycosidases: α- and β-glucosidase, α- and β-galactosidase, α- and β-fucosidase, β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase, β-xylosidase and α-mannosidase. All the enzymes except β-fucosidase had activity interpreted to be on the external surface of the plasma membrane. The enzymes had optimum pH values of 5.2 to 5.0 and temperatures of 37 to 40°C. The enzymes were not greatly activated by divalent cations but Hg⁺⁺ and Pb⁺⁺ were inhibitory. The enzyme extract of the human erythrocyte plasma membranes liberated carbohydrate from intact red cells, which lead to the speculation that the glycosidases might function to modify the erythrocyte plasma membrane. The author is a Research Career Development Awardee of the National Institute of General Medical Sciences.     

Perturbation of endothelial junction proteins by Staphylococcus aureus α-toxin: inhibition of endothelial gap formation by adrenomedullin

Endothelial hyperpermeability is a hallmark of an inflammatory reaction and contributes to tissue damage in severe infections. Loss of endothelial cell–cell adhesion leads to intercellular gap formation allowing paracellular fluid flux. Although Staphylococcus aureus α-toxin significantly contributed to staphylococci disease, little is known about its mechanism of endothelial hyperpermeability induction. Here, we demonstrate that in a model of isolated perfused rat ileum discontinuation of capillary vascular-endothelial-cadherin (VE-cadherin) was observed after bolus application of S. aureus α-toxin being inhibited by the endogenous peptide adrenomedullin (ADM). In vitro, α-toxin exposure induced loss of immunoreactivity of VE-cadherin and occludin in human cultured umbilical vein endothelial cells. Likewise, ADM blocked α-toxin-related junctional protein disappearance from intercellular sites. Additionally, cyclic AMP elevation was shown to stabilize endothelial barrier function after α-toxin application. Although no RhoA activation was noted after endothelial α-toxin exposure, inhibition of Rho kinase and myosin light chain kinase blocked loss of immunoreactivity of VE-cadherin and occludin as well as intercellular gap formation. In summary, stabilization of endothelial junctional integrity as indicated by interendothelial immunostaining might be an interesting approach to stabilize endothelial barrier function in severe S. aureus infections.Andreas C. Hocke and Bettina Temmesfeld-Wollbrueck have contributed equally to this article.     

Oligomer formation of staphylococcal α-toxin analyzed by electron microscopy and image processing

Abstract The 12S oligomeric form of Staphylococcus aureus α-toxin has been studied with electron microscopy after incubation of the toxin with membrane preparations or liposomes. The target material originated from human platelet. Different electron microscopic preparation techniques were used including negative staining, freeze-fracture and vitrification in liquid ethane. Analysis of micrographs with image processing methods revealed two groups of ring-like structures corresponding to α-toxin oligomers. One form measured 75 Å in diameter and had a high stain density in the central protein deficient part while the other was larger with a diameter of 100 Å and less stain accumulation in the center. The conditions under which the latter were formed suggest that this corresponds to an inactive loosely-bound form of the toxin. The high stain density in the smaller particle is consistent with the presence of a penetrating pore in this structure.     

Separation and properties of α-mannosidase and mannanase from the basidiomycetePhellinus abietis

Proteins of a crude enzyme preparation obtained from the cultivation medium of the basidiomycetePhellinus abietis were separated by gel filtration and ion-exchange chromatography. The preparation contained a minimum of three enzymes capable of splitting α-d-mannosidic bonds: α-mannosidase, exomannanase, and endomannanase, which were separated. Some properties of the mannanase complex of the crude enzyme preparation, and of a partially purified α-mannosidase were examined. The mannanase complex exhibited two pH optima, its temperature optimum being at 46 °C The pH optimum of purified α-mannosidase was at pH 5.0, the temperature optimum was at 60 °C; the enzyme had a relatively high heat stability. The K_m of α-mannosidase forp-nitrophenyl α-d-mannopyranoside was 1.5 x 10⁻⁵ M. Pure α-mannosidase did not split mannan.     

Extracellular α-glucosidase of an alkalophilic microorganism, Bacillus sp. ATCC 21591

Abstract Bacillus sp. ATCC 21591, an alkalophilic bacterium, produces 3 enzymes associated with degradation of starch-α-amylase, pullulanase and α-glucosidase. The latter reached a maximum after 24 h growth. Highest activities of α-glucosidase and pullulanase were obtained when the initial pH of the medium was 9.7 and although at pH 10.4 highest biomass was attained after 48 h no α-glucosidase was present. The pH optimum for activity with maltose as substrate was 7.0, which is surprisingly low for an alkalophilic organism. The enzyme was substrate specific for p -nitrophenyl- α -D-glucoside, maltose and maltotriose in that order. Forty eight times the activity was located in the cell-free supernatant, relative to that found intracellulary. Transferase activity was detected - the major end-product formed from maltose was a compound with an R _f -value similar to isomaltose.     

Channel-Forming Abilities of Spontaneously Occurring α-Toxin Fragments from Staphylococcus aureus

Pore formation by four spontaneously occurring α-toxin fragments from Staphylococcus aureus were investigated on liposome and erythrocyte membranes. All the isolated fragments bound to the different types of membranes and formed transmembrane channels in egg-phosphatidyl glycerol vesicles. Fragments of amino acids (aa) 9–293 (32 kD) and aa 13–293 (31 kD) formed heptamers, similar to the intact toxin, while the aa 72–293 (26 kD) fragment formed heptamers, octamers, and nonamers, as judged by gel electrophoresis of the liposomes. All isolated fragments induced release of chloride ions from large unilamellar vesicles. Channel formation was promoted by acidic pH and negatively charged lipid head groups. Also, the fragments’ hemolytic activity was strongly decreased under neutral conditions but could be partially restored by acidification of the medium. We paid special attention to the 26-kD fragment, which, despite the loss of about one-fourth of the N-terminal part of α-toxin, did form transmembrane channels in liposomes. In light of the available data on channel formation by α-toxin, our results suggest that proteolytic degradation might be better tolerated than previously reported. Channel opening could be inhibited and open channels could be closed by zinc in the medium. Channel closure could be reversed by addition of EDTA. In contrast, digestion at the C terminus led to premature oligomerization and resulted in species with strongly diminished activity and dependent on protonation.     

Purification and characterization of an extracellular acid trehalase fromLentinula edodes

Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules. The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and was defined as an acid trehalase.     

Novel, thermostable family-13-like glycoside hydrolase fromMethanococcus jannaschii

A novel glycoside hydrolase from the hyperthermophilic archaeonMethanococcus jannaschii has been cloned intoEscherichia coli. Extremely thermoactive and thermostable amylolytic activity was confirmed in partially purified enzyme solution. This enzyme exhibited a temperature optimum of 100 °C and a pH optimum pH 5.0–8.0. Hydrolysis of large 1,6-α- and 1,4-α-linked polysaccharides yielded glucose polymers of 1–7 units. Incubation with amylose displayed the highest activity. The catalyst was activated and stabilized by Ca²⁺ and exhibited extreme thermostability at 100 °C with a half-life of 78 h.     

Simultaneous saccharification and fermentation of cellulose for lactic acid production

Lactic acid production from α-cellulose by simultaneous saccharification and fermentation (SSF) was studied. The cellulose was converted in a batch SSF using cellulase enzyme Cytolase CL to produce glucose sugar andLactobacillus delbrueckii to ferment the glucose to lactic acid. The effects of temperature, pH, yeast extract loading, and lactic acid inhibition were studied to determine the optimum conditions for the batch processing. Cellulose was converted efficiently to lactic acid, and enzymatic hydrolysis was the rate controlling step in the SSF. The highest conversion rate was obtained at 46°C and pH 5.0. The observed yield of lactic acid from α-cellulose was 0.90 at 72 hours. The optimum pH of the SSF was coincident with that of enzymatic hydrolysis. The optimum temperature of the SSF was chosen as the highest temperature the microorganism could withstand. The optimum yeast extract loading was found to be 2.5 g/L. Lactic acid was observed to be inhibitory to the microorganisms’ activity.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

Staphylococcus aureus in chronic and recurrent infections

Ninety-fourStaphylococcus aureus strains isolated from chronic and recurrent skin and respiratory tract infections were investigated for several virulence factor expressions. Production of protein A was noticed in all of the tested strains in amounts from less than 0.1 to more than 2.5 ng per 10⁶ bacterial cells. The percentage of the extracellularly produced protein A was found to lie between 4.5 and 27.8%. Two strains (both from the respiratory tract) produced more than 50 % of protein A in the extracellular form and one strain did not produce any detectable amount of the extracellular protein A; 99 % of the tested strains produced the clumping factor, 96% staphylocoagulase, 79 % staphylokinase and 90 % gelatinolytic activity; 79 % produced α-toxin exclusively or in combination with δ- or β-toxin; 8 % of strains produced β-toxin. There were differences in β-toxin production between strains from the respiratory tract (5 %) and skin infections (25 %). δ-Toxin was produced by 53 % of the strains. In each of the tested strains a complex of virulence factors was detected. The importance of inactivated extracellular products (especially α- and δ-toxin and in the case of skin infections also β-toxin) as components of staphylococcal whole-cell vaccine was suggested. Dedicated to Professor C. John on the occasion of his 75th birthday     

Purification and properties of the endocellular β-glucosidase of Candida cacaoi Buckley and Van Uden CBS 2020

D. DRIDER, P. POMMARES, P. CHEMARDIN, A. ARNAUD AND P. GALZY. 1993. The endocellular enzyme β-glucosidase of Candida cacaoi was purified by ion-exchange chromatography and gel filtration. The molecular weight was 220 ± 10 kDa; its optimum pH was between 4 and 5.5 and its optimum temperature was 60C. This enzyme was active against soluble glucosides tested with β(1–2), β(1–3), β(1–4) and even α(1–4) and α(1–6) and was inhibited by D-glucono-δ-lactone. The enzyme was constitutive but its synthesis was repressed by glucose.     

Amylase activity ofTorulopsis ingeniosa Di Menna

Torulopsis ingeniosaDi Menna was found to possess an α-amylase strongly attached to the cell wall, its pH optimum being at 5.5, optimum temperature at 50 °C, highly sensitive to thermal inactivation. The enzyme was found to be induced by starch but the synthesis is not subject to a glucose effect.