Characterization of immunogenic p230 as the toxin A of Clostridium difficile

Abstract For the identification of toxin A of Clostridium difficile , a 2-dimensional gel system was used. In its first dimension, samples were separated in the absence of reducing and dissociating agents, conditions which maintained the activity of the enterotoxin. This was followed by reduction and dissociation in the second dimension where a 230 kDa polypeptide was electroeluted. Rabbits were immunized with polyacrylamide gel slices containing entrapped native toxin A and the denatured 230 kDa protein. As revealed by immunoblotting, neutralizing antisera derived from native protein samples recognized the native toxin, the denatured 230 kDa protein and another polypeptide of about M _r 35 000. Using both types of antisera as probes the pI of the enterotoxin was about 5.9. Preliminary evidence suggests that the enterotoxin is a multimeric protein of 230 kDa and 35 kDa subunits.     

Clostridium difficile toxins A and B: Receptors,pores, and translocation into cells

The most potent toxins secreted by pathogenic bacteria contain enzymatic moieties that must reach the cytosol of target cells to exert their full toxicity. Toxins such as anthrax, diphtheria, and botulinum toxin all use three well-defined functional domains to intoxicate cells: a receptor-binding moiety that triggers endocytosis into acidified vesicles by binding to a specific host-cell receptor, a translocation domain that forms pores across the endosomal membrane in response to acidic pH, and an enzyme that translocates through these pores to catalytically inactivate an essential host cytosolic substrate. The homologous toxins A (TcdA) and Toxin B (TcdB) secreted by Clostridium difficile are large enzyme-containing toxins that for many years have eluded characterization. The cell-surface receptors for these toxins, the non-classical nature of the pores that they form in membranes, and mechanism of translocation have remained undefined, exacerbated, in part, by the lack of any structural information for the central ～1000 amino acid translocation domain. Recent advances in the identification of receptors for TcdB, high-resolution structural information for the translocation domain, and a model for the pore have begun to shed light on the mode-of-action of these toxins. Here, we will review TcdA/TcdB uptake and entry into mammalian cells, with focus on receptor binding, endocytosis, pore formation, and translocation. We will highlight how these toxins diverge from classical models of translocating toxins, and offer our perspective on key unanswered questions for TcdA/TcdB binding and entry into mammalian cells.     

Signal transduction pathways and cellular intoxication with Clostridium difficile toxins

In cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin-induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4-bromophenacylbromide. Agents affecting certain G-proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C-dependent pathway, but not pertussis toxin-sensitive G-proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.     

艰难梭菌相关性腹泻的治疗进展及对我国的启示

艰难梭菌相关性腹泻（Clostridium difficile associated diarrhea,CDAD）是艰难梭菌感染（Clostridium difficile Infection,CDI）引起的一种机体严重疾病。随着艰难梭菌感染率的增加及高毒力株027／NAP1／BI的出现,导致该病在全球特别是北美、及欧洲等地区爆发性流行,对于该病的控制引起全球研究者的高度重视。本文就其治疗进展进行综述,并结合我国实际分析该病防治中存在的问题并提出建议。     

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo . In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.     

重组艰难梭菌毒素B对小鼠结肠癌CT26细胞的诱导凋亡作用

本文探讨了重组艰难梭菌毒素B(rTcd B)对小鼠结肠癌CT26细胞的诱导凋亡作用。采用不同浓度rTcd B处理CT26细胞, 通过MTT法检测细胞增殖抑制率; 比色法测定Caspase 3活性; 细胞形态学和流式细胞技术检测细胞凋亡。结果表明, rTcd B显著抑制了CT26细胞的增殖, 并呈时间?剂量依赖性; Caspase 3活性在处理6 h后显著升高, 至18 h达到最大值, 与对照组相比差异显著, 具有统计学意义(P<0.05); 荧光显微镜观察到典型细胞凋亡形态学变化, 细胞膜内侧的磷脂酰丝氨酸(PS)异位到了膜外侧, 细胞膜呈明亮的绿色荧光; 通过流式细胞仪检测结果表明, 细胞凋亡率呈时间?剂量依赖性增加。实验结果表明, 重组艰难梭菌毒素B能够诱导小鼠结肠癌CT26细胞凋亡。     

Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from Equidae

Clostridium difficile is an antibiotic-associated emerging pathogen of humans and animals. Thus far three toxins of C. difficile have been described: an enterotoxin (ToxA), a cytotoxin (ToxB) and an ADP-ribosyltransferase (CDT). In the present work we describe the first isolation of CDT producing C. difficile from Equidae with gastro-intestinal disease. Out of 17 C. difficile strains isolated from Equidae, 11 were positive for the genes tcdA and tcdB encoding ToxA and ToxB. In addition four of these 11 isolates were positive for the cdtA gene encoding the catalytic subunit of the ADP-ribosyltransferase CDT. Interestingly none of the isolates derived from canines (41 isolates) and felines (4 isolates) harboured the cdtA gene. In C. difficile field isolates which contained the cdtA gene, ADP-ribosyltransferase activity could also be detected in culture supernatants indicating expression and secretion of CDT. All strains were associated with intestinal disorders, but no association was found for the occurrence of toxins with a specific clinical diagnosis.     

Production of an enterotoxin different from toxin A by Clostridium difficile

Abstract Clostridium difficile has been demonstrated to produce at least two toxins: an enterotoxin (toxin A) which elicits haemorrhagoc fluid accumulation in the rabbit ileal loop (RIL) test and a potent cytotoxin (toxin B). We report the isolation of an enterotoxic factor inducing a positive response in the RIL test without haemorrhage. This factor was separated by ion-exchange chromatography and its molecular weight, as estimated by SDS-polyacrylamide gel electrophoresis, was about 45 000.     

艰难梭菌毒素B羧基端的表达与卵黄抗体制备简

目的：在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端（TcdB-e）,免疫产蛋鸡,获得针对TcdB-c的卵黄抗体（IgY）。方法：人工合成TcdB-c的基因,将其克隆至pET32b（＋）载体中,转化大肠杆菌BL21（DE3）,诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果：构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79000,经凝血酶酶切后的相对分子质量约65000;目的蛋白免疫产蛋鸡后获得效价为1：20000的抗TcdB-C卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论：获得了具有生物学活性的TcdB-C,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。     

Detection of toxin B of Clostridium difficile based on immunomagnetic separation and aptamer-mediated colorimetric assay

Clostridium difficile can cause antibiotic-associated diarrhoea or pseudo-membranous colitis in humans and animals. Currently, the various methods such as microbiological culture, cytotoxic assay, ELISA and polymerase chain reaction have been used to detect Clostridium difficile infection (CDI). These conventional methods, however, require long detection time and professional staff. The paper is to describe a simple strategy which employs immunomagnetic separation and aptamer-mediated colorimetric assay for the detection of toxin B of C. difficile (TcdB) in the stool samples. HRP-labelled aptamer against TcdB selected by SELEX was firstly captured on the surface of magnetic beads (MB) by DNA hybridization with a complementary strand. In the presence of TcdB, aptamer specifically recognized and bound TcdB, disturbing the DNA hybridization and causing the release of HRP-aptamer from MB. This reduced the catalytic capacity of HRP and consequently the absorption intensity. As there was a relationship between the decrease in the absorption intensity and target concentration, a quantitative analysis of TcdB can be accomplished by the measurement of the absorption intensity. Under the optimal conditions, the assay system is able to detect TcdB at a concentration down to 5 ng ml⁻¹. Moreover the method had specificity of 97% and sensitivity of 66% and the system remained excellent stability within 4 weeks. The proposed method is a valuable screening procedure for CDI and can be extended readily to detection of other clinically important pathogens.     

艰难梭菌细胞毒素B功能区的克隆及序列分析

目的克隆艰难梭菌(Clostridium difficile,C.d)细胞毒素B羧基末端功能区(CDB3)基因,并对其进行测序及生物信息学分析。方法利用PCR技术扩增CDB3基因,并将其定向插入pET-22b( )载体中,以DNA自动分析仪进行序列测定,并以生物信息学软件分析其生物学特性。结果成功克隆了艰难梭菌CDB3基因,经测序表明与GenBank中分布的Clostridium difficile VPI10463的ToxinB3基因序列完全一致。DNAstar软件预测其蛋白质的相对分子量(Mr)约为71.3 kD,并显示出良好的抗原性。结论研究获得了序列正确的CDB3基因,为其重组表达及其相关研究奠定了良好基础。     

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin

Abstract A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay ( r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining ( r = 0.995).     

腹泻患者艰难梭菌及其毒素A/B检出率分析

目的了解住院腹泻患者艰难梭菌及其毒素A/B的检出情况,为临床诊断抗生素相关性腹泻提供参考依据。方法收集2015年9月至2016年8月崇州市人民医院疑似抗生素相关性腹泻住院患者的粪便标本163份,用艰难梭菌快速检测试剂盒检测艰难梭菌特异性抗原谷氨酸脱氢酶(GDH);用酶联免疫荧光法检测GDH阳性标本中艰难梭菌毒素A/B的产生情况。结果 163份粪便标本中GDH阳性为34份,阳性率为20.86%。34份GDH阳性标本中艰难梭菌毒素A/B阳性率为41.18%(14/34),可疑阳性率为17.65%(6/34),阴性率为41.18%(14/34)。结论疑似抗生素相关性腹泻住院患者艰难梭菌GDH检出率较高,毒素A/B阳性率也比较高,提示临床应重视抗生素相关性腹泻患者艰难梭菌感染的诊断。     

Comparison of methods for the production and purification of toxin A from Clostridium difficile

Abstract The production and purification of toxin A from Clostridium difficile were studied. When the toxin was produced in dialysis culture it preicipitated quantitatively at pH 5.5 and after purification it appeard homogeneous in polyacrylamide gel electrophoresis (PAGE). The toxin probably consists of two noncovalently bound peptides, each with a molecular mass of about 250 dDa. It is resistant to trypsin but sensitive to papain and chymotrypsin. In contrast, toxin A produced in anaerbic chamber culture precipitated poorly at pH 5.5 (yield 14%) and easily formed aggregates as observed in gel filtration and PAGE Accordingly, dialysis culture seems to be a better method for producing and purifying toxin A.     

酶联免疫吸附试验检测艰难梭菌A毒素

实验用兔单特异抗艰难梭菌Ａ毒素ＩｇＧ包被酶标板,以羊抗艰难梭菌Ａ毒素ＩｇＧ标记辣根过氧化物酶作为第二抗体,采用双抗体夹心ＥＬＩＳＡ法检测艰难梭菌Ａ毒素,可检测出０．９４ｎｇ的精制Ａ毒素,对６１株菌的培养液及６５份健康人粪便标本检测发现此法具有较高的特异性。用平行线定量法对几份典型产毒培养物进行了定量测定,结果表明,在一定剂量范围内线性及平行性好,结果准确、可靠。可用于临床粪便标本中艰难梭菌Ａ毒素的筛查及定量检测。     

住院腹泻患者艰难梭菌快速检测结果分析

目的 了解住院腹泻患者艰难梭菌带菌情况,为抗生素相关腹泻患者的诊断和治疗提供参考。方法 收集2015年9月至2016年8月住院腹泻患者粪便标本163份,用艰难梭菌快速检测试剂盒检测艰难梭菌。结果 艰难梭菌检出总阳性率为20.86%（34/163）,其中春季16.67%、夏季15.79%、秋季17.86%、冬季27.87%,经比较差异无统计学意义（2=2.943,P＞0.05）;成人粪便标本阳性率为22.52%（25/111）,儿童粪便标本阳性率为17.31%（9/52）,差异无统计学意义（2=0.583,P＞0.05）;男性粪便标本阳性率为21.69%（18/83）,女性粪便标本阳性率为20.0%（16/80）,差异无统计学意义（2=0.202,P＞0.05）;艰难梭菌阳性患者抗菌药物平均使用天数为12.74 d,阴性患者为8.21 d,差异有统计学意义（t=－7.81,P＜0.01）。结论 住院腹泻患者艰难梭菌阳性率高低与抗菌药物使用时间长短有关。     

Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B

The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied. ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1–5 mM range) delay in the appearance of the cytopathogenic effect. With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h. Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction. The protective effect of ATP did not depend on its energy producing ability. Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells. ATP was protective even upon addition 10 min after the toxin binding step. In the presence of ATP, the toxin remained accessible to neutralization with antitoxin. In analogy with the P-site on diphtheria toxin, we postulate that C. difficile toxin B contains a polyphosphate-binding site. This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step.     

Enzyme electrophoresis combined with serogrouping for improved differentiation of Clostridium difficile strains

Abstract We used enzyme electrophoresis to study a set of epidemiologically related and unrelated isolates of Clostridium difficile . The 53 strains belonged to the most frequent serogroups (A1, C, G, H and K). Nine electrophoretic profiles were defined on the basis of five enzymes, and two were characteristic of a single strain. Each serogroup was resolved into two or three different enzyme patterns. By combining the two methods we were able to resolve the strains into 12 types. There was an excellent correlation between enzyme electrophoresis and serogrouping data. This method may be of use in investigating nosocomial transmission.     

Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes

Clostridium difficile toxinotypes are groups of strains defined by changes in the PaLoc region encoding two main virulence factors: toxins TcdA and TcdB. Currently, 24 variant toxinotypes (I-XXIV) are known, in addition to toxinotype 0 strains, which contain a PaLoc identical to the reference strain VPI 10463. Variant toxinotypes can also differ from toxinotype 0 strains in their toxin production pattern. The most-studied variant strains are TcdA-, TcdB+ (A-B+) strains and binary toxin CDT-producing strains. Variations in toxin genes are also conserved on the protein level and variant toxins can differ in size, antibody reactivity, pattern of intracellular targets (small GTPases) and consequently in their effects on the cell. Toxinotypes do not correlate with particular forms of disease or patient populations, but some toxinotypes (IIIb and VIII) are currently associated with disease of increased severity and outbreaks worldwide. Variant toxinotypes are very common in animal hosts and can represent from 40% to 100% of all isolates. Among human isolates, variant toxinotypes usually represent up to 10% of strains but their prevalence is increasing.