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.     

Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌α,β,ε,τ毒素基因cpa、cpb,etx及iA序列合成了针对4种毒素基因的4对特异引物,建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法.结果本所保存的A,B,c,D,E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带,而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性;将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段.并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定,并与毒素中和试验鉴定结果进行了比较,结果表明两种方法具有较高的符合率.本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义.     

Plasmid transformation of Clostridium perfringens by electroporation methods

Two electroporation methods were compared and modified to improve the frequencies of transfer of plasmid DNA into Clostridium perfringens. A plasmid shuttle vector, pSB92A2, containing chloramphenicol and ampicillin resistance genes and a clostridial origin of replication isolated from a cryptic C. perfringens plasmid, was constructed and successfully introduced into C. perfringens by both electrotransformation methods. Modifications which improved frequencies by 15-28 fold are described and may improve frequencies sufficiently for some vector/host combinations to consider the future use of more direct cloning strategies for the clostridia.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌a, b, e, t 毒素基因cpa、cpb、etx 及iA序列合成了针对4种毒素基因的4对特异引物, 建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法。结果本所保存的A, B, C, D, E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带, 而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性; 将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段。并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定, 并与毒素中和试验鉴定结果进行了比较, 结果表明两种方法具有较高的符合率。本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义。     

Factors involved in the electroporation-induced transformation of Clostridium perfringens

The following factors were found to improve the efficiency of transformation of Clostridium perfringens 3624A Rifr Strr: (1) a reduction in cuvette sample volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 microgram/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) 3-fold concentration of cell density (3.0 x 10(8) CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the improved conditions resulted in transformation efficiencies for C. perfringens 3624A Rifr Strr ranging from 7.1 transformants/microgram DNA for plasmic pIP401 to 9.2 x 10(4) transformants per microgram DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 x 10(6) transformants/micrograms DNA for C. perfringens strain 13. Using the improved protocol, pAM beta 1 was transformed at a 42-fold greater level when compared with the values reported earlier [1]. In addition to C. perfringens 3624A Rifr Strr, strains 13, 10543A, 3628C, NTG-4, and 3624A were successfully transformed. Nuclease does not appear to be a factor in the C. perfringens strain-specific electro-transformation protocol.     

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.     

Stimulation of growth and sporulation of Clostridium perfringens by its homologous enterotoxin

C. perfringens enterotoxin shortened the lag phase and time of onset of sporulation of the same organism in a dose-dependent manner. The toxin stimulated macromolecular synthesis of pre-exponential phase cells.     

一起兔产气荚膜梭菌病爆发流行及病原体的分离鉴定

黑山县某兔场出现仔兔突然发病、大量死亡现象,根据临床症状和病理剖检变化疑似细菌感染,采取肝脏等样本进行病原分离、培养特性观察、菌落形态学观察、生化反应试验、致病性试验、药敏试验。结果从组织样本中分离获得1株病原菌,其培养特性、菌落形态、生化反应特性等均符合产气荚膜梭菌特征,药敏实验结果显示,该分离菌对氟苯尼考、头孢哌酮、哌拉西林等药物敏感。说明该兔场发生了产气荚膜梭菌病的疫情,采用敏感药物治疗后,收到明显效果。     

A型产气荚膜梭菌α毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从A型产气荚膜梭菌标准株染色体DNA中扩增出α毒素基因,构建了含α毒素基因的重组菌株BL21(DE3)(pXETA02)。经酶切鉴定和序列测定证实,构建的表达质粒pXETA02含有α毒素基因序列。经SDS-PAGE、Western blot分析和ELISA检测,重组菌株表达的α毒素蛋白能够被α毒素单抗识别。表达优化结果表明,以IPTG为诱导剂诱导α毒素表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.8mmol/L,菌体生长密度OD600达到0.8时加入IPTG,诱导时间5h,此时α毒素蛋白表达量为34.83%。以乳糖为诱导剂诱导α毒素表达的优化条件是:培养基pH7.5、培养温度37℃,乳糖浓度0.1g/L,菌体生长密度OD600达到0.8时加入乳糖,诱导时间5h,α毒素蛋白表达量为23.82%。动物实验结果表明,用重组菌株α毒素蛋白免疫的小鼠可以抵抗1MLD的A型产气荚膜梭菌标准株C57-1毒素攻击。     

The role of histidine residues in the alpha toxin of Clostridium perfringens

The α -toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Contraction induced by Clostridium perfringens epsilon toxin in the isolated rat ileum

Clostridium perfringens epsilon toxin caused contraction of the isolated ileum of the rat in a dose-dependent manner. The contraction caused by the toxin was inhibited by a low Na medium, tetrodotoxin (TTX), atropine, mecamylamine or tetraethylammonium (TEA). Furthermore, the contractile response induced by the toxin was abolished by incubation in Ca-free medium, and completely restored by and addition of Ca2+. In addition, verapamil inhibited contraction induced by the toxin in a dose-dependent manner. These data suggest that epsilon toxin induces contraction of the isolated ileum and that the toxin-elicited contraction is the result of an indirect action mediated through the nervous systems.     

食品中产气荚膜梭菌LAMP快速检测方法的建立

应用环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP), 针对产气荚膜梭菌特有的α毒素(CPa)基因序列分析设计特异性引物, 建立食品中产气荚膜梭菌特异性快速检测方法。研究结果表明, 所设计的引物具有良好的特异性, 5株产气荚膜梭菌均能扩增出特异性片段, 而13株非产气荚膜梭菌均未扩增出相应片段, 无假阴性或假阳性情况出现。同时, 该方法可在1 h内完成反应, 且检测灵敏度达到10 fg/μL。该方法为产气荚膜梭菌的快速检测提供了一种重要的技术手段。     

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.     

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.     

Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.     

A型产气荚膜梭菌经小鼠传代的毒力变化

目的:探讨A型产气荚膜梭菌经小鼠腹腔传代后的毒力变化。方法:选择10只小鼠,腹腔注射浓度为5.0×108 cfu/mL的A型产气荚膜梭菌菌液1 mL,记录第一代小鼠存活时间;收集第一代死亡最早的小鼠腹腔内的细菌,经血平板厌氧培养24 h后配制5.0×108 cfu/mL浓度的菌液,并腹腔注射10只小鼠,每只1 mL,记录第二代小鼠存活时间;重复实验并记录第三代、第四代小鼠的存活时间;死亡小鼠腹腔收集的细菌以革兰染色涂片镜检,血平板培养,用API 20A试剂条进行生化鉴定;用SPSS 13.0软件分析每代小鼠的存活时间组间差异。结果:小鼠腹腔收集的标本经革兰染色后均可见两端钝圆棒状的革兰阳性杆菌,血平板厌氧培养均为边缘呈锯齿状有双溶血环的较大菌落,API 20A的结果为产气荚膜梭菌;小鼠存活时间随小鼠腹腔传代数不断延长,第一代与第二代、第三代、第四代之间的存活时间差异有统计学意义,第二代、第三代与第四代两两之间的存活时间差异无统计学差异。结论:A型产气荚膜梭菌经小鼠腹腔第一次传代后毒力显著减弱,随后传代中毒力基本不变,与传统的细菌经动物腹腔培养后毒力增强的观点不一致。     

Detection of Clostridium perfringens in tsunami deposits after the Great East Japan Earthquake

The Great East Japan Earthquake struck off the Tohoku and caused a tsunami in 2011. Most of the microbial characteristics of tsunami‐affected soil remain unknown and no published study has shown how a tsunami affects the risk of infection by Clostridium perfringens living in soil. In 2011 and 2015, C. perfringens was assessed in deposits in soil from tsunami‐damaged areas and undamaged areas of Miyagi. It was found that the number of C. perfringens was overwhelmingly greater in 2011 than in 2015 in the tsunami‐damaged areas. According to real‐time PCR, the prevalence C. perfringens organisms (%) was 10³ fold greater in the damaged than in the undamaged areas.     

Factors involved in the transformation of previously non-transformable Clostridium perfringens type B

Abstract The pre-shock incubation of cells plus DNA and the methylation state of plasmid DNA were found to play a role in the electroporation-based transformation of Clostridium perfringens 3626B. Following pre-shock incubation, the highest number of C. perfringens 3626B transformants was obtained when plasmid pGK201 was both dam⁺ dcm⁺ modified, while no transformants were obtained when pGK201 was not methylated or only dcm methylated. This is consistent with the observation that plasmid pGK201 was protected against digestion by C. perfringens 3626B cell-associated nucleases for up to 3 min when methylated by both methylases. C. perfringens 3626B was successfully transformed only within a narrow cell recovery rate window. The erm AM gene associated with pGK201 and pAK102 was found to integrate into the chromosome of C. perfringens strains 13A and 3626B.