大肠杆菌不耐热肠毒素B亚单位研究进展

不耐热肠毒素B亚单位(heat-labile enterotoxin B subunit,LTB)是产肠毒素性大肠杆菌分泌到细胞周质的肠毒素的组成部分,具有黏膜免疫佐剂活性,可介导细菌与肠上皮细胞结合.此文对LTB的结构、重组表达、作为黏膜佐剂的免疫学评价和作用机制等方面进行综述.     

重组金葡菌肠毒素O的克隆表达及生物学活性分析

克隆金葡菌肠毒素O(SEO)的全长基因，实现其可溶性表达,并对纯化的表达产物进行生物学活性分析。从金葡菌FRI 100菌株基因组中得到SEO基因，克隆至谷胱甘肽S-转移酶(GST)融合表达载体pGEX-4T-1，转化大肠杆菌，获高效表达。融合蛋白GST-SEO经Glutathione Sepharose 4B亲和纯化和凝血酶消化获重组SEO(rSEO)后，MTT法检测脾淋巴细胞的增殖作用，分析纯化后rSEO的生物学活性。测序结果表明，得到正确的肠毒素SEO基因序列，并获高效表达的融合蛋白；MTT结果表明，rSEO具有与SEC相当的显著的促淋巴细胞增殖以及抑制肿瘤细胞生长的能力。本研究成功克隆、表达、纯化了具有抗肿瘤生物学活性的rSEO蛋白，为进一步研究该蛋白的抗肿瘤机制奠定了基础，并有望成为一种新的超抗原制剂用于肿瘤的临床治疗。     

克林霉素毒副作用与艰难梭状芽孢杆菌的关系

目的探讨克林霉素与艰难梭状芽孢杆菌的关系。方法2008年10月至2009年1月对应用克林霉素患者320例进行观察,了解记录出现腹泻出现,病例数、出现时间、临床症状并进行培养确诊艰难梭状芽孢杆菌感染。结果应用克林霉素患者320例68例腹泻患者艰难梭状芽孢杆菌感染31例,艰难梭状芽孢杆菌率9.68%,占腹泻发生例数45.58%。结论应用克林霉素容易造成艰难梭状芽孢杆菌引起高度重视,在临床应用中应严格掌握克林霉素的抗菌谱、适应证和用法、用量,避免滥用、过量、长时间应用等措施以免引起条件致病菌艰难梭状芽孢杆菌感染。     

PPI引起艰难梭状芽孢杆菌性腹泻的危险因素

目的:评价治疗酸相关性疾病患者的质子泵抑制剂是引起艰难梭状芽孢杆菌性腹泻的另类危险因素。方法:查阅近期文献进行评价和分析。结果:队列研究表明艰难梭状芽孢杆菌性腹泻发生于约6.8%用过抗生素的住院患者,多自变量因素分析表明艰难梭状芽孢菌性腹泻与质子泵抑制剂的应用明显相关。结论:抗生素破坏了正常肠道菌群,公认是引起艰难梭状芽孢杆菌性腹泻的危险因素,而胃酸度降低一直被认为是其危险因素,质子泵抑制剂因为其强力的抑酸作用,可能是致艰难梭状芽孢杆菌性腹泻的独立危险因素。     

60例脓毒性休克患者并发艰难梭状芽孢杆菌感染的危险因素分析及其防治措施

目的:分析脓毒性休克患者并发艰难梭状芽孢杆菌感染的危险因素及其预防措施。方法:选取医院2016年8月-2019年8月间收治的脓毒性休克患者60例资料,分析其艰难梭状芽孢杆菌感染的发生与相关因素关系。结果:60例脓毒性休克患者中,并发艰难梭状芽孢杆菌感染发生率为75.00%;经Logistic分析,急性与慢性健康评分(APACHEⅡ)的评分值≥20分、实施机械通气、血乳酸水平≥4 mmol/L、抗菌药物使用时长≥14 d均为影响艰难梭状芽孢杆菌感染发生的危险因素,而实施血液净化对预防艰难梭状芽孢杆菌感染发生具有保护因素(P<0.05)。结论:APACHEⅡ评分值≥20分、实施机械通气、血乳酸水平≥4 mmol/L、抗菌药物使用时长≥14 d均属于脓毒性休克并发艰难梭状芽孢杆菌感染的危险因素;而血管活性药物使用时长≥7 d、实施血液净化为保护因素,临床应密切关注导致其感染的危险因素,以降低其感染的发生率。     

金黄色葡萄球菌肠毒素SEC2的纯化与活性研究

从金黄色葡萄球菌发酵液中纯化金黄色葡萄球菌肠毒素C2（SEC2）。发酵液经超滤浓缩、硫酸铵沉淀、阴离子交换色谱和分子筛色谱，获得了纯度为90％的SEC2，总收率23％。体外刺激脾淋巴细胞增殖实验以及体外肿瘤细胞增殖抑制试验表明该蛋白具备良好的超抗原活性。     

重组金黄色葡萄球菌肠毒素A突变体D227A蛋白的表达、纯化和鉴定

建立从金黄色葡萄球菌肠毒素A突变体D227A原核表达系统pET32a-SEAD227A-E.coliBL21DE3中,表达、纯化和鉴定重组金黄色葡萄球菌肠毒素A突变体D227A(rSEA-D227A)蛋白的方法。采用不同浓度的IPTG(0.1、0.5和1.0mmol.L-1)诱导原核表达系统表达rSEA-D227A,10%SDS-PAGE检测表达产量,Ni-NTA亲和层析法纯化rSEA-D227A,采用HPLC检测其纯度,Western-blotting进行免疫学鉴定。从已构建的金黄色葡萄球菌肠毒素A突变体D227A原核表达系统中成功表达和纯化了rSEA-D227A蛋白,产量约占细菌总蛋白的38.2%,纯度为88.7%。为后续进一步研究该蛋白的生物活性奠定基础。     

金葡菌肠毒素C2的克隆表达及其生物学活性

目的克隆金葡菌肠毒素C₂全长基因，构建SEC₂的表达载体，实现其可溶性表达，并对纯化的rSEC₂蛋白的生物学活性进行研究。方法通过聚合酶链式反应(polymerase chain reaction，PCR)从金葡菌FRI1230菌株基因中得到肠毒素SEC₂的基因，将其克隆至融合表达载体pGEX-4T-1，转化大肠杆菌进行表达并对融合蛋白进行亲和色谱纯化。通过考察重组SEC₂对淋巴细胞的增殖作用及其对肿瘤细胞杀伤活性的影响，对其超抗原活性和免疫学活性进行研究。结果得到正确的肠毒素SEC₂基因序列并得到高效表达的融合蛋白，MTT法结果表明，重组SEC₂表现出良好的促淋巴细胞增殖活性，且能够增强淋巴细胞对肿瘤细胞的杀伤活性。结论本研究成功克隆了SEC₂基因，表达并纯化出具有抗肿瘤生物学活性的重组SEC₂蛋白，为进一步对其分子抗肿瘤作用机制进行研究以及构建靶向抗肿瘤融合蛋白奠定了基础。     

野生型金黄色葡萄球菌肠毒素C2在E.coli中的表达和纯化研究

目的克隆金黄色葡萄球菌肠毒素C2(SEC2)基因在E.coli重组表达,纯化重组SEC2(rSEC2)并进行生物学活性分析。方法PCR获得正确编码SEC2的基因片断,构建表达质粒pET-28a-sec2在E.coliBL21中表达。利用离子交换和分子筛色谱纯化rSEC2,四甲基偶氮唑盐(MTT)法对rSEC2和天然SEC2(nSEC2)生物学活性进行分析和比较。结果可溶性rSEC2占菌体总蛋白质的40%,两步纯化后蛋白质纯度约达95%,rSEC2和nSEC2均具有显著的超抗原活性。结论成功获得与nSEC2有着类似活性的高纯度rSEC2,为进一步研究该蛋白质的抗肿瘤机理奠定物质基础。     

大肠杆菌不耐热肠毒素的粘膜免疫机理

大肠杆菌不耐热肠毒素（ＬＴ）是由Ａ、Ｂ两种亚单位组成的ＡＢ５型蛋白，其中Ａ亚单位具有ＡＤＰ－核糖基化活性，Ｂ亚单位具有与肠粘膜上皮细胞膜上的神经节苷脂结合的作用。它能引起人和猪等动物的腹泻。另外，它还是强有力的粘膜免疫原和粘膜免疫佐剂。了解其粘膜免疫机理，对于发展ＥＴＥＣ菌苗和粘膜免疫佐剂具有重要意义。本文分别从ＬＴ的粘膜免疫原性和佐剂效应两方面对其粘膜免疫机理作一讨论。     

Autoproteolytic cleavage mediates cytotoxicity of <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin A

Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC₅₀ regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.     

The Role of Rho GTPases in Toxicity of Clostridium difficile Toxins

Clostridium difficile (C. difficile) is the main cause of antibiotic-associated diarrhea prevailing in hospital settings. In the past decade, the morbidity and mortality of C. difficile infection (CDI) has increased significantly due to the emergence of hypervirulent strains. Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of C. difficile, are the major virulence factors of CDI. The common mode of action of TcdA and TcdB is elicited by specific glucosylation of Rho-GTPase proteins in the host cytosol using UDP-glucose as a co-substrate, resulting in the inactivation of Rho proteins. Rho proteins are the key members in many biological processes and signaling pathways, inactivation of which leads to cytopathic and cytotoxic effects and immune responses of the host cells. It is supposed that Rho GTPases play an important role in the toxicity of C. difficile toxins. This review focuses on recent progresses in the understanding of functional consequences of Rho GTPases glucosylation induced by C. difficile toxins and the role of Rho GTPases in the toxicity of TcdA and TcdB.     

The Enterotoxicity of Clostridium difficile Toxins

The major virulence factors of Clostridium difficile infection (CDI) are two large exotoxins A (TcdA) and B (TcdB). However, our understanding of the specific roles of these toxins in CDI is still evolving. It is now accepted that both toxins are enterotoxic and proinflammatory in the human intestine. Both purified TcdA and TcdB are capable of inducing the pathophysiology of CDI, although most studies have focused on TcdA. C. difficile toxins exert a wide array of biological activities by acting directly on intestinal epithelial cells. Alternatively, the toxins may target immune cells and neurons once the intestinal epithelial barrier is disrupted. The toxins may also act indirectly by stimulating cells to produce chemokines, proinflammatory cytokines, neuropeptides and other neuroimmune signals. This review considers the mechanisms of TcdA- and TcdB-induced enterotoxicity, and recent developments in this field.     

Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration

Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD.     

Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl₂ and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.     

Difference in F-Actin Depolymerization Induced by Toxin B from the Clostridium difficile Strain VPI 10463 and Toxin B from the Variant Clostridium difficile Serotype F Strain 1470

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin’s capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon Rac1 inactivation).     

Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network. Received: 28 October 1997 / Accepted: 19 January 1998     

Difference in the biological effects of <Emphasis Type="Italic">Clostridium difficile</Emphasis> toxin B in proliferating and non-proliferating cells

Toxin A (TcdA) and toxin B (TcdB) from Clostridium difficile are the causative agents of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis. TcdA and TcdB both glucosylate and thereby inactivate low molecular weight GTP-binding proteins of the Rho, Rac, and Cdc42 subfamilies. In cultured cell lines, TcdB induces actin re-organization and bi-nucleation (“cytopathic effects”) and cell death (“cytotoxic effects”). In this study, the role of cell cycle progression in the cytopathic and the cytotoxic effects of TcdB is evaluated by a differential analysis of these effects in proliferating and non-proliferating cells. Density-synchronized murine fibroblasts and confluent HT29 colonocytes are exploited as cell culture models for non-proliferating cells. Cell death is analyzed in terms of a loss of cell viability, phosphatidylserine exposure, and DNA fragmentation. In proliferating cells, TcdB blocks cell proliferation and induces apoptotic cell death. In contrast, TcdB induces non-apoptotic cell death in non-proliferating cells. TcdB-induced cell rounding turns out to be independent of cell cycle progression. Cell cycle progression is an important determinant in the biological effects of TcdB. With respect to the pathology of CDAD, this study leads to the new hypothesis that necrotic cell death of terminally differentiated colonocytes and inhibition of epithelial renewal of the colon contribute to the pathogenesis of CDAD.     

An Update on Clostridioides difficile Binary Toxin

Infection with Clostridioides difficile (CDI), a common healthcare-associated infection, includes symptoms ranging from mild diarrhea to severe cases of pseudomembranous colitis. Toxin A (TcdA) and toxin B (TcdB) cause cytotoxicity and cellular detachment from intestinal epithelium and are responsible for CDI symptomatology. Approximately 20% of C. difficile strains produce a binary toxin (CDT) encoded by the tcdA and tcdB genes, which is thought to enhance TcdA and TcdB toxicity; however, the role of CDT in CDI remains controversial. Here, we focused on describing the main features of CDT and its impact on the host, clinical relevance, epidemiology, and potential therapeutic approaches.