艰难拟梭菌毒素致病机制及毒素基因调控的研究进展

艰难拟梭菌(Clostridioides difficile)是一种产芽孢的革兰氏阳性专性厌氧杆菌,是引起抗生素相关性腹泻的主要致病菌。艰难拟梭菌产生的毒素A和毒素B在其致病过程中发挥关键作用。毒素发挥毒性作用依赖其4个功能结构域:葡萄糖基转移酶结合域、半胱氨酸蛋白酶结合域、易位域和受体结合域。毒素的受体结合域识别并结合细胞表面受体,介导毒素内吞并形成内体。经过自体催化切割,毒素将真正的毒性片段——葡萄糖基转移酶结合域释放到胞浆中,葡萄糖基转移酶能够失活宿主肠上皮细胞内的GTP酶导致细胞骨架解聚和坏死,进而引起腹泻和伪膜性结肠炎等临床症状。艰难拟梭菌毒素产生受致病基因座内及基因座外许多调控因子的调节。tcdR和tcdC基因位于致病基因座内,对毒素基因的表达分别起促进和抑制作用,而基因座外如spo0A、codY等基因则通过抑制tcdR的表达从而间接影响毒素蛋白产生。本文将重点介绍艰难拟梭菌毒素的致病过程和影响毒素基因表达的分子调控机制,以期为开发针对毒素的治疗手段提供新思路。     

艰难梭菌基因编辑技术研究进展

艰难梭菌Clostridioesdifficile是一种革兰氏阳性、产芽孢、专性厌氧细菌,是医院相关性腹泻的主要病原体。近年来,随着强毒株的出现(如核糖体027型),其流行性与致死率逐年上升,因此对艰难梭菌生理、生化特征及致病机制的研究受到广泛重视。艰难梭菌生理、生化特征及致病机制研究又以建立其稳定、高效的基因编辑方法为必要前提。借助基因编辑工具,研究者可以扰动艰难梭菌核心生物学过程,在分子水平研究其分子致病机制。如Clos Tron技术在艰难梭菌毒素A (Toxin A)和毒素B (Toxin B)与其致病力关系的研究中起到了关键作用。文中以时间为主线综述了艰难梭菌基因编辑技术的发展历程和最新进展,并对艰难梭菌基因编辑技术未来的研究方向进行展望。     

中药复方对艰难梭菌毒素基因tcd A/tcd B体外表达的影响

目的探讨中药复方治疗抗生素相关性腹泻(antibiotic associated diarrhea,AAD)的药理机制。方法应用荧光定量PCR技术检测2种中药复方(参苓白术散和理中汤)对2株艰难梭菌临床分离株(A+B+和A-B+)体外培养下毒素基因表达的影响。结果在A+B+艰难梭菌中,参苓白术散组对tcd A和tcd B基因的表达均有显著或是完全抑制,理中汤也有显著的抑制;而对于A-B+艰难梭菌,2种中药复方则完全抑制tcd B基因的表达。结论通过影响毒素基因的表达可能是中药复方治疗AAD的药理学机制之一。     

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。     

人艰难梭菌感染的致病机制与防治策略研究进展

艰难梭菌是一种非常重要的医院感染病原菌,其感染占抗菌素相关腹泻的10．25％,占抗菌素相关肠炎的50．75％,占抗菌素相关伪膜炎的90．100％。而且越来越多的证据表明艰难梭菌的感染与其它疾病,如活性关节炎、婴儿突发性死亡、溶血性尿毒症、坏死性肠炎、希施斯普龙病等有关。更糟糕的是艰难梭菌存在着复发性感染,15—20％的病人在成功治愈后会复发。近年来,随着强毒株的出现,艰难梭菌的感染率和发病率逐年上升,病情也越来越严重,由艰难梭菌感染引起的死亡率也成倍增加。到目前为止,艰难梭菌发病率升高的原因及其致病机制还有待进一步研究。由于艰难梭菌是一种孢子产生菌,对大部分抗菌素都有抗性,目前用于艰难梭菌防治的主要抗菌素甲硝唑和万古霉素的治疗效果也在不断下降,所以正确认识艰难梭菌的感染与流行,进一步了解其致病机制,寻找新的替代疗法已是迫在眉睫。本文对艰难梭菌感染和流行的病因进行了较为全面的分析,对其致病机制进行了深入的总结,并探讨了艰难梭菌防治的最新策略和方法,将有利于更好的认识和研究艰难梭菌,为艰难梭菌的防治提供新思路。     

艰难梭菌荚膜致病作用的初步研究

本文在我室先前研究的基础上,以3株艰难梭菌的荚膜多糖提取物、全菌悬液以及全菌免疫血清分别在小鼠中进行半数致死量和保护试验等动物实验,以探讨艰难梭菌荚膜的致病作用。结果测得其中一株艰难梭菌的荚膜多糖对小鼠有致死毒性,其LD50为2.83mg,特异性抗血清保护试验证实艰难梭菌免疫血清对动物有保护作用。实验结果可能为进一步研究艰难梭菌荚膜的致病作用提供了参考资料。     

上海部分地区儿童艰难梭菌相关性腹泻的临床分析

本文旨在监测儿童腹泻中艰难梭菌相关性腹泻(CDAD)的发病情况,并对其进行回顾性临床分析。对复旦大学附属儿科医院2007年2月～9月111例腹泻患儿的粪便标本进行艰难梭菌毒素A检测及粪便厌氧菌培养,对所有患儿进行回顾性病史采集及分析,并对粪便培养所得的4株艰难梭菌菌株用多位点可变数目串联重复序列分析(MLVA)技术进行同源性分析。111例患者中,艰难梭菌毒素A检测及艰难梭菌培养均为阳性者无,艰难梭菌毒素A阳性而艰难梭菌培养阴性者16例,艰难梭菌毒素A阴性而艰难梭菌培养阳性者4例,艰难梭菌毒素A及艰难梭菌培养均阴性者91例。比较院内、院外组3种不同病程腹泻CDAD的发病率,无显著差异。MLVA分析发现粪便培养得到的4株艰难梭菌菌株有部分同源性。结果提示,目前上海部分地区儿童CDAD发病情况为散发,但彼此之间有部分同源性;院内、外获得性腹泻的CDAD发病率无显著差异;艰难梭菌毒素A阳性或艰难梭菌培养阳性的病例在临床表现上与艰难梭菌毒素A阴性且艰难梭菌培养阴性的腹泻病例无显著差异。     

艰难梭菌及其致病力

艰难梭菌(Clostridium difficile)被发现于1935年,命名为艰难芽孢杆菌(Bacillus difficilis)。1938年由Prevot根据其厌氧性质,将其划入梭菌属,改为现名。发现该菌时就发现它能产生毒素,引起实验动物死亡,但长期未引起重视。     

艰难梭菌研究现状

艰难梭菌(Clostridium difficile,CD)是产芽胞革兰阳性厌氧菌,产毒素艰难梭菌可导致艰难梭菌感染(Clostridium difficile infection,CDI)。产毒素艰难梭菌已被公认是抗菌药物相关性腹泻最常见的病原体,也是发达国家最常见的导致住院患者腹泻感染的原因之一。高毒力菌株导致全世界范围内CDI的发病率和死亡率增加。如何快速而准确地检测CD一直是备受关注的问题,国内外多推荐两步法和三步法进行检测。艰难梭菌治疗的一线用药是甲硝唑、万古霉素以及非达霉素,应用粪便微生物移植(faecal microbiota transplantation,FMT)也可作为治疗轻度或严重复发CDI(recurrence Clostridium difficile infection,rCDI)的可选方法。艰难梭菌的芽胞能抵抗不良环境,但抗菌药物的使用,医院环境的复杂性和医务工作者的手等因素会导致艰难梭菌易于传播,因此医疗机构应加强艰难梭菌的预防。本文主要对艰难梭菌的流行病学、致病机制、检测及治疗进行综述。     

ICU患者艰难梭菌分离培养与 流行病学特征研究

目的了解ICU患者艰难梭菌的定植和感染情况,为预防艰难梭菌的流行提供参考。方法收集2016年9月至2017年6月福建医科大学附属第一医院ICU中139例住院时间7d的患者的粪便样本,对其进行选择性厌氧培养和质谱鉴定。对艰难梭菌培养阳性标本进行毒素基因(tcdA、tcdB、cdt A、cdtB)的PCR检测以及毒素A、B表型检测。收集所有患者的临床资料,并对艰难梭菌培养阳性患者的临床特征和实验室检查结果进行单因素分析和多因素回归分析。结果艰难梭菌检出率为17.27%(24/139)。其中,14株艰难梭菌的tcdA和tcdB基因检测阳性,占58.3%(14/24);10株为tcdA和tcdB基因检测阴性,占41.7%(10/24)。所有菌株二元毒素基因(ctdA/ctdB)均未检出。单因素分析提示,高龄、长时间住院、高淋巴细胞数、使用β-内酰胺类抗生素是艰难梭菌定植的高危因素;多因素回归分析提示,使用β-内酰胺类抗生素是艰难梭菌定植的独立危险因素(OR=3.881,P=0.039)。结论我院ICU可能存在艰难梭菌感染和传播的风险,对具有高龄、长期住院以及使用抗生素等高危因素的患者应进行艰难梭菌的监测,以防艰难梭菌的传播、感染和艰难梭菌相关性腹泻的发生。     

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ²⁸) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.     

CD630＿27900基因缺失显著降低艰难拟梭菌自溶速率、毒力及对酸和抗生素的耐受性

艰难拟梭菌(Clostridioidesdifficile)CD630＿27900基因位于slpA-cwp66基因座上,CD630＿27900基因属于假定的Lmbe家族的酶,但基因功能尚未明确。【目的】本研究通过构建艰难拟梭菌CD630＿27900基因敲除菌株,比较野生型菌株(CD630)与突变株表型差异,探讨CD630＿27900基因对艰难拟梭菌感染的影响。【方法】用非等长同源臂偶联等位交换(allele-coupled exchange, ACE)构建CD630＿27900基因缺失菌株与回补菌株。比较它们在生长曲线、自溶素(cwp19,Acd)基因表达、细胞毒力、主要毒素基因表达、抗生素及pH敏感性差异,以研究CD630＿27900基因的功能。【结果】成功构建△CD630＿27900突变菌株和::CD630＿27900回补菌株。菌株△CD630＿27900在衰亡期自溶速率显著低于菌株CD630,::CD630＿27900自溶速率恢复。实时荧光定量聚合酶链反应(real-timefluorescencequantitative polymerasechainreaction,RT-q...     

Four Distinct Structural Domains in Clostridium difficile Toxin B Visualized Using SAXS

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of ∼ 275 Å. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.     

Intestinal lactoflora in Estonian and Norwegian patients with antibiotic associated diarrhea

The disruption of intestinal microbiota is an important risk factor for the development of Clostridium difficile caused antibiotic associated diarrhea (AAD). The role of intestinal lactoflora in protection against C. difficile is unclear. Fecal samples (n = 74) from AAD patients were investigated for C. difficile and lactobacilli by culture and real-time PCR. Lactobacilli were identified by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and sequencing of 16S rRNA. In C. difficile negative cases we found somewhat higher counts of intestinal Lactobacilli (5.02 vs. 2.15 CFU log₁₀/g; p = 0.053) by culture and more frequently Lactobacillus plantarum (33.3% vs. 9.4%; p = 0.03) as compared with positive ones. Results of total counts of lactobacilli comparing Estonian and Norwegian samples were conflicting by culture and PCR. We found higher colonization of Norwegian AAD patients with L. plantarum (21% vs. 5%, p = 0.053) and Estonians with Lactobacillus gasseri (19% vs. 2%, p = 0.023). Particular lactobacilli (e.g. L. plantarum) may have a role in protection against C. difficile, whereas the meaning of total counts of lactobacilli remains questionable. In different persons and nations, different lactobacilli species may have a protective role against C. difficile.     

Clostridium difficile toxin and faecal lactoferrin assays in adult patients

Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea. The faecal lactoferrin (FL) assay is a simple in vitro test which is highly sensitive to the presence of a marker of polymorphonuclear cells. We evaluated the use of the FL assay in conjunction with the C. difficile toxin assay in faecal samples obtained from 231 adult patients. The relationship between C. difficile toxin and FL in both negative and positive status was highly significant statistically (P < 0.001). Therefore, the FL assay performed simultaneously with the C. difficile toxin assay can help rule out asymptomatic carriage of C. difficile.     

Large clostridial cytotoxins

The large clostridial cytotoxins are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B), C. sordellii (lethal and hemorrhagic toxin) and C. novyi (-toxin). The exotoxins are major pathogenicity factors which in addition to their in vivo effects are cytotoxic to cultured cell lines causing reorganization of the cytoskeleton accompanied by morphological changes. The exotoxins are single-chain protein toxins, which are constructed of three domains: receptor-binding, translocation and catalytic domain. These domains reflect the self-mediated cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and execution of their cytotoxic activity by an inherent enzyme activity. Enzymatically, the toxins catalyze the transfer of a glucosyl moiety from UDP-glucose to the intracellular target proteins which are the Rho and Ras GTPases. The covalent attachment of the glucose moiety to a conserved threonine within the effector region of the GTPases renders the Rho-GTPases functionally inactive. Whereas the molecular mode of cytotoxic effects is fully understood, the mechanisms leading to inflammatory processes in the context of disease (e.g., antibiotic-associated pseudomembranous colitis caused by Clostridium difficile) are less clear.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.