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。     

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。     

艰难梭菌致病机制研究进展

艰难梭菌(Clostridium difficile)属于革兰阳性厌氧菌,可以导致艰难梭菌感染(Clostridium difficile infection, CDI),是伪膜性结肠炎和抗生素相关性腹泻的主要病原体,并与结肠癌、帕金森病、子痫前期等疾病相关,严重影响患者的健康。研究表明,艰难梭菌可以通过毒素影响MAPK、NF-κB、TLR和ERS等信号转导,由鞭毛和膜囊泡介导炎症反应,与肠道微生物相互作用调节菌群代谢产物水平等途径参与致病。本文总结了艰难梭菌感染引起的相关疾病、致病机理和治疗现状。     

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。     

艰难梭菌耐药性及耐药机制研究进展

艰难梭菌(Clostridium difficile)是医疗保健相关性腹泻最主要的病原菌。2002年起欧美地区艰难梭菌感染发病率和病死率均明显增高,耐药艰难梭菌的出现和传播更给临床治疗和预防带来了挑战。绝大多数临床分离菌对甲硝唑及万古霉素仍呈高度敏感,但已有异质性耐药或最低抑菌浓度上升的报道;对红霉素和莫西沙星等其他抗菌药物的耐药率在不同国家和地区则有较大差异。艰难梭菌对甲硝唑或万古霉素敏感性下降产生的耐药机制尚不明确,而对红霉素、氟喹诺酮类、四环素和利福霉素形成的耐药机制主要是因为作用靶点发生了改变。文章简述了近年来国际上艰难梭菌耐药性及耐药机制方面的研究进展。     

艰难梭菌的研究进展

艰难梭菌为革兰阳性厌氧芽胞杆菌,可引起艰难梭菌相关性腹泻,导致一系列肠道感染症状和相关临床表现。近年来由于高致病株的出现、菌株耐药性的增加,艰难梭菌感染在全球呈蔓延趋势。本文就艰难梭菌的耐药机制、检测技术、防治及国内感染现状等作一简要综述。     

123例腹泻患儿粪便中艰难梭菌毒素基因特征及感染危险因素分析

探讨腹泻患儿粪便中艰难梭菌毒素基因特征, 并分析产毒艰难梭菌感染的危险因素。

采集2019年1月至2021年3月嘉兴市第二医院儿科收治的腹泻患儿的粪便标本共123份, 其中社区获得性腹泻患儿60例, 医院获得性腹泻患儿63例。标本进行厌氧培养, 采用实时荧光PCR鉴定艰难梭菌tpi基因, 并检测tcd A、tcd B毒素基因。收集患儿临床资料, 采用Logistic回归分析产毒艰难梭菌感染的危险因素。

123例粪便标本共检出艰难梭菌tpi基因阳性35例(28.46%), 毒素基因中tcd A⁺ B⁺为19例(15.45%), tcd A⁺B^-为3例(2.44%), tcd A^-B⁺为2例(1.63%), tcd A^-B^-为11例(8.94%)。社区获得性腹泻患儿中tcd A/B产毒艰难梭菌感染率为28.33%, 高于医院获得性腹泻患儿的11.11%(P < 0.05)。产毒艰难梭菌感染腹泻患儿与非产毒艰难梭菌感染腹泻患儿的性别、年龄、居住地、是否早产、有无先天性疾病、既往有无胃肠道手术、抗生素应用种类、近1个月内有无机械通气、临床症状、血液白细胞计数、血红蛋白、粪便白细胞计数、血清C反应蛋白、血清白细胞介素-6、血清白蛋白、血肌酐、血清总胆红素水平比较差异无统计学意义(均P > 0.05)。产毒艰难梭菌感染腹泻患儿中的非母乳喂养、近1个月内抗生素用药史、抗生素持续用药时间 > 7 d、近1个月内糖皮质激素用药史、近1个月内抑酸药用药史、近1个月内肠内营养者的比例显著高于非产毒艰难梭菌感染腹泻患儿(均P < 0.05)。非母乳喂养(OR=2.433)、近1个月内抗生素用药史(OR=3.040)、近1个月内肠内营养(OR=2.330)是腹泻患儿产毒艰难梭菌感染的独立危险因素(均P < 0.05)。

嘉兴地区儿童艰难梭菌毒素基因主要为tcd A/B基因, 患儿以社区获得性腹泻为主, 非母乳喂养、抗生素用药史、肠内营养的儿童更容易感染艰难梭菌。

     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.     

四川地区猪源艰难梭菌分子分型调查

[背景] 艰难梭菌是一种重要的人畜共患肠道病原菌,可引起人和多种动物抗生素相关性腹泻或假膜性肠炎。四川作为我国主要的生猪产区,还未有猪源艰难梭菌流行病学调查的相关报道,对猪源艰难梭菌的防控及保障猪肉安全带来挑战。[目的] 调查四川地区猪源艰难梭菌的感染、流行情况,并对分离出的艰难梭菌进行分子分型研究。[方法] 收集来自四川生猪主要产区6个养殖场中猪的粪便标本（n=110）,采用厌氧培养技术在艰难梭菌鉴别培养基上进行分离培养;采用PCR方法扩增艰难梭菌4个毒素基因（tcdA、tcdB、cdtA、cdtB）和7个管家基因（adk、atpA、dxr、glyA、recA、sodA、tpi）,对分离株进行毒素基因分型和多位点序列分型。[结果] 从110份样品中,经革兰氏染色镜检及PCR鉴定,共分离出20株艰难梭菌,分离率高达18.18%;毒素基因分型结果显示共获得3种毒素基因型,包括tcdA⁺tcdB⁺cdtA/cdtB⁺（n=3）、tcdA⁺tcdB⁺cdtA/cdtB^—（n=6）、tcdA^—tcdB^—cdtA/cdtB^—（n=11）;多位点序列分型结果显示获得5个ST型,包括ST11（n=3）、ST3（n=1）、ST35（n=2）、ST36（n=4）、ST109（n=10）;进化树结果显示,所有分离株聚集为2个大群,分别为3个分支和17个分支。[结论] 四川主要生猪产区猪群存在艰难梭菌感染,分离株的分子分型呈多样性,主要流行型为ST11、ST3、ST35、ST36、ST109型,并且存在ST11型高毒力菌株流行的风险。     

24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

[目的]旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估.[方法]利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化,挑选可疑菌落进行微生物质谱鉴定,进一步通过16S rRNA基因测序进行鉴定,16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析;同时,进行所有...     

fliL基因显著影响艰难拟梭菌运动功能及产孢能力

鞭毛基底体相关FliL家族蛋白(flagellar basal body-associated FliL family protein,fliL)基因编码FliL蛋白,FliL是一种与鞭毛基体相结合的单跨膜蛋白。为研究艰难拟梭菌fliL基因功能,使用非等长同源臂偶联等位交换(allele-coupled exchange,ACE)方法成功构建了fliL基因缺失(ΔfliL)和回补(::fliL)突变株,研究突变菌株与野生型菌株(CD630)生长曲线、抗生素敏感性、pH耐受性、运动能力及产孢能力等表型的差异。结果显示,菌株ΔfliL生长速率及最大生物量均小于菌株CD630,::fliL回补菌株生长情况回复至野生型。与CD630菌株相比,ΔfliL对阿莫西林、氨苄青霉素、诺氟沙星的敏感性提高,对卡那霉素、四环素敏感性降低,::fliL抗生素敏感性部分回复至野生型水平。与CD630菌株相比,ΔfliL游泳运动能力显著降低,::fliL运动能力超越野生型菌株CD630。相比菌株CD630,菌株ΔfliL在pH值为5时耐受能力显著提高,在pH值为9时,耐受能力显著降低。除此之外,ΔfliL产孢能力较CD630显著降低,::fliL产孢能力部分恢复。以上结果表明,艰难拟梭菌fliL基因与其运动能力、抗生素敏感性、环境耐受能力和产孢能力密切相关,可能进一步影响艰难拟梭菌菌株的致病力。     

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.     

Efficient expression and purification of methyltransferase in acetyl-coenzyme a synthesis pathway of the human pathogen Clostridium difficile

The Wood-Ljungdahl pathway is responsible for acetyl-CoA biosynthesis and used as a major mean of generating energy for growth in some anaerobic microbes. Series of genes, from the anaerobic human pathogen Clostridium difficile, have been identified that show striking similarity to the genes involved in this pathway including methyltetrahydrofolate- and corrinoid-dependent methyltransferase. This methyltransferase plays a central role in this pathway that transfers the methyl group from methyltetrahydrofolate to a cob(I)amide center in the corrinoid iron-sulfur protein. In this study, we developed two efficient expression and purification methods for methyltransferase from C. difficile for the first time with two expression vectors MBPHT-mCherry2 and pETDuet-1, respectively. Using the latter vector, more than 50mg MeTr was produced per liter Luria-Bertani broth media. The recombinant methyltransferase was well characterized by SDS-PAGE, gel filtration chromatography, enzyme assay and far-UV circular dichroism (CD). Furthermore, a highly effective approach was established for determining the methyl transfer activity of the methyltetrahydrofolate- and cobalamin-dependent methyltransferase using exogenous cobalamin as a substrate by stopped-flow method. These results will provide a solid basis for further study of the methyltransferase and the Wood-Ljungdahl pathway.