【作者】 张建；

【导师】 邓旭明；

【作者基本信息】 吉林大学 ， 基础兽医学， 2020， 硕士

【摘要】 产气荚膜梭菌(Clostridium perfringens,C.perfringens),又称魏氏梭菌,广泛存在于自然界内,是存在于人和动物体内的正常肠道菌群,属条件致病菌。依据其所分泌的外毒素将其分为A-E五种类型,A型产气荚膜梭菌可导致人的食物中毒及气性坏疽等多种疾病,其中气性坏疽在多种动物身上均有发生,在遭受深度伤害或术后护理不当的情况下,患者通常会感染致病菌引起坏疽,并且气体坏疽的潜伏期短,通常从发作到死亡,仅需48 h。因此,一旦错过感染早期的黄金治疗期,将导致感染宿主死亡,此外,如果不能及时进行有效的治疗,将对公众健康造成威胁并导致严重的经济损失。梭菌性气体坏疽是一种暴发性传染病,主要由产气荚膜梭菌所分泌的α毒素与θ毒素两种毒素引起。α毒素(Phospholipase C,CPA or PLC)属于细菌锌-金属磷脂酶家族,是产气荚膜梭菌的主要毒素之一,同时具有磷脂酶C和鞘磷脂酶的活性,是引起气性坏疽的主要毒素。θ毒素(perfringolysin O)又称PFO,是成孔胆固醇依赖性溶细胞素家族(CDCs)成员之一,可与α毒素共同作用,对感染部位聚集的中性粒细胞造成损伤,内皮细胞功能失调,进而为产气荚膜梭菌的生长繁殖提供有力的厌氧环境。本研究以引起梭菌性气性坏疽的两种毒素CPA及PFO为研究靶点,通过溶血试验建立抑制剂筛选平台,最终从多种中药化合物中筛选出了潜在的毒力因子抑制剂,并对其作用机制进行了阐述,从而为探索梭菌性气性坏疽的新治疗方法提供新的解决思路及一定的理论依据。在本研究中,通过溶血试验、生长曲线及最小抑菌浓度等多种试验方法,筛选出的天然化合物分子——毛蕊花糖苷能够在不影响产气荚膜梭菌正常生长的浓度范围内(2-32μg/ml)便可有效抑制CPA及PFO的溶血活性。体外细胞试验以及小鼠感染模型试验研究结果表明,毛蕊花糖苷对因CPA或PFO侵袭而产生损伤的Caco-2细胞具有很好的保护作用,同时降低了气性坏疽模型小鼠的死亡率。分子动力学模拟以及定点残基突变试验结果表明,毛蕊花糖苷通过与PFO及CPA相结合抑制了两种毒素的活性,其中与PFO的结合主要残基为ASN199、SER55及ASN197,与CPA结合的主要残基为ALA89及TYR90。综上所述,本研究表明毛蕊花糖苷可有效抑制CPA和PFO的溶血活性,降低细菌致病力,是一种治疗气性坏疽的潜在先导化合物,为气性坏疽的临床治疗提供了一定的理论依据。更多还原

【Abstract】 Clostridium perfringens,known as Clostridium weisserii,is widely present in nature.It is a regular intestinal flora found in humans and animals.It is a conditional pathogen and divided into five serotypes of AE according to the exotoxin secreted from it.Clostridium perfringens type A could induce food poisoning and a variety of diseases such as gas gangrene,among which gas gangrene is presented in multiple types of animals.In the case of deep injury or inadequate postoperative care,patients generally get infected by pathogens and induced gangrene,which possess a short incubation period.The infection it will merely take 48 hours from episoede to death.Therefore,once the early golden treatment period of infection is passed up,it often induces to death because of the infection.In addition,if the treatment is not performed on time and effectively,it will also threaten public health and induce to severe economic losses.Clostridium gas gangrene is an outbreak of infectious disease,which is mainly caused by alpha toxin and theta toxin secreted by Clostridium perfringens.Alpha toxin(C.perfringens alpha toxin or Phospholipase C,CPA or PLC)which belongs to the bacterial zinc-metal phospholipase family,is one of the main toxins of Clostridium perfringens,and possesses both phospholipase C and sphingomyelinase activities.It is the crucial toxin induced to gangrene gas.Theta toxin,known as PFO,is a member of the pore-forming cholesterol-dependent cytolysin family.It could be associated with alpha toxins to induce neutrophils injuries aggregated at the site of infection,endothelial cells dysfunction,and subsequently offers a anaerobic environment to airborne capsules.In this study,two toxins that cause clostridial gas gangrene were used as research targets.An inhibitor screening platform was established through hemolysis experiments.Finally,potential virulence factor inhibitors were screened out from traditional Chinese medicine compounds and their mechanism of action was determined.It expounded to provide new solutions and theoretical basis for exploring new treatment methods of Clostridium gas gangrene.In this study,through various test methods such as hemolysis test,growth curve and MIC test,the natural compound molecule verbascoside was screened in a concentration range that does not affect the normal growth of Clostridium perfringens(2-32 μg / ml)could effectively inhibit the hemolytic activity of CPA and PFO.In vitro cell tests and rats infection model tests implied that Verbascoside performes a very good protective effect on Caco-2 cells damaged by CPA or PFO invasion.Meanwhile,it reduces the mortality of gas gangrene rats.Molecular dynamics simulation and site-specific residue mutation test results showed that Verbascoside inhibits two toxins activities by combining with PFO and CPA.The main residues which bind to PFO are ASN199,SER55 and ASN197,while the main residues that bind to CPA are ALA89 and TYR90.In summary,this study showed that Verbascoside could effectively inhibit the Hemolytic activities of CPA and PFO,and reduce the bacterial pathogenicity.It is a potential lead compound for the treatment of gas gangrene and provides a certain theory basis for the gas gangrene therapy..更多还原