MsmK调控猪链球菌生长和致病性的研究

发布时间：2018-04-16 09:33

本文选题：猪链球菌 + msmK基因　；参考：《华中农业大学》2016年博士论文

【摘要】：猪链球菌(Streptococcus suis)是具有荚膜的革兰氏阳性球菌,通常定植于猪的上呼吸道,主要是鼻腔和扁桃体。根据荚膜抗原成分,可将猪链球菌分成33个血清型,其中2型猪链球菌(SS2)被认为是毒力最强的血清型,是临床上分离的主要血清型。猪链球菌可引起感染猪的败血症、脑膜炎、心内膜炎、关节炎等症状,给养猪业造成了巨大的经济损失。同时,猪链球菌引起人的脑炎和中毒样休克综合症,严重时导致死亡,威胁着人类的健康。对于病原菌来说,在宿主体内获取营养物质是其生存和繁殖进而损伤动物机体的前提。碳源的利用与病原菌的生存和致病性息息相关。碳源的获取和利用影响SS2在宿主口咽腔的定植平衡态,调控毒力因子的表达,促进致病进程。一些碳源代谢相关的蛋白质也是SS2的毒力因子。本研究对ATP酶MsmK在SS2的碳源转运、致病性以及细胞分裂三个方面的功能进行了研究,主要研究结果如下:1、猪链球菌MsmK是多种碳水化合物ABC转运子的ATP酶所有异养型细菌必须从外界摄取营养物质,以保证其生命活动和生长繁殖。α-葡聚糖的利用对于维持SS2在感染的各个阶段的生存、繁殖和毒力尤为重要。细菌转运碳水化合物的方式有两种:磷酸转移酶系统(PTS)和ABC转运子。关于α-葡聚糖降解产物的转运在猪链球菌中还缺乏研究。本实验以SS2菌株SC-19为研究对象,分析比较了两对ABC转运子msmEFG和malXCD基因簇在SC-19基因组中的位置,实验证明了MsmEFG负责转运棉籽糖和蜜二糖,而MalXCD负责α-葡聚糖降解产物的转运。在SC-19中,SSUSC84_1724编码蛋白质Msm K具有ATP酶活性。将MsmK缺失突变株用含不同碳源的人工培养基培养,发现突变株不能再利用棉籽糖、蜜二糖以及麦芽糊精。此外,MsmK在这些碳源中的表达水平明显升高,说明MsmK属于MsmEFG和MalXCD转运子的组分,其生理功能是为这两个转运子水解ATP供能。2、MsmK促进猪链球菌在小鼠体内的定植α-葡聚糖的代谢与SS2侵入宿主的生理过程紧密关联,猪链球菌在宿主口咽腔以α-葡聚糖为主要碳源,α-葡聚糖代谢通路中的蛋白质活跃表达,粘附和侵入的毒力基因也随之表达上调,使猪链球菌侵入宿主上皮细胞或扩散至软组织。感染后期,受损组织和细胞释放的糖原也是SS2可利用的碳源之一。本研究用野生株SC-19和msmK基因缺失株ΔmsmK进行了小鼠致病性实验,发现突变株的致死率下降。小鼠竞争性定植定植实验表明,MsmK影响SS2在小鼠脑、肺、肝等组织中感染后期的定植,可能与突变株失去利用糖原的能力相关。体内实验和体外实验都证明MsmK有助于增强猪链球菌在血液中的存活能力。突变株对全血杀伤的敏感性更高,也更容易被吞噬细胞所吞噬。细菌应激实验表明,突变株对高渗和过氧化氢的耐受能力减弱,生物被膜的形成能力也明显下降。本实验证实了MsmK对猪链球菌的致病性有关,进一步证实了病原细菌的碳源利用与其致病性密切相关。3、MsmK影响猪链球菌细胞分裂过程研究致病性细菌的细胞分裂过程不仅让我们更好的理解细菌的基础生理过程,更是为开发新的抗菌药物寻找潜在的目标蛋白质。细菌细胞分裂过程是开发新型抗菌药物的良好靶点,目前针对大肠杆菌和枯草芽孢杆菌为代表的杆菌的生长机制已研究的比较深入,但是对球菌(特别是链球菌)的细胞分裂机制了解甚少。本研究以猪链球菌细胞分裂骨架蛋白FtsZ为诱饵蛋白,利用细菌双杂交筛选可与之互作的蛋白质。实验结果显示,ABC转运子组分ATP酶Msm K可与FtsZ相互作用。MsmK可在体外促进FtsZ多聚体的形成。和野生株SC-19标准的椭球形细胞形态相比,突变株ΔmsmK由于细菌的分裂面与细菌长轴方向不垂直,导致分裂后的细菌失去正常细胞形态;而且缺失株中细胞分裂相关基因的表达量也发生了变化,预示着MsmK参与猪链球菌的细胞分裂过程。Msm K促进和维持FtsZ多聚体的形成的具体机制还有待进一步的研究。
[Abstract]:Streptococcus suis (Streptococcus suis) is a gram positive coccus with capsule, usually planted in the upper respiratory tract of pigs, mainly nasal and tonsils. According to capsular antigens, Streptococcus suis can be divided into 33 serotypes, including Streptococcus suis serotype 2 (SS2) is considered to be the most virulent serotype, is the main serotypes of clinical isolates. Streptococcus suis can cause sepsis, infection of pig meningitis, endocarditis, arthritis and other symptoms, caused huge economic losses to the pig industry. At the same time, people like poisoning caused by Streptococcus suis encephalitis and shock syndrome, leading to serious death threats to human health. The pathogenic bacteria, in vivo nutrients the material is the premise of survival and reproduction and damage the body of the animal. The carbon source and pathogen survival and pathogenicity are closely related. Obtaining carbon source and utilization effect of SS2 in the night The main planting equilibrium of oropharyngeal cavity, regulate the expression of virulence factors, promote the pathogenic process. Some virulence factors of carbon metabolism related protein is SS2. The research on the ATP enzyme MsmK in carbon source transport SS2, pathogenicity and cell division three functions are studied, the main results are as follows: 1 MsmK ATP, Streptococcus suis enzyme multiple carbohydrate ABC transporters all heterotrophic bacteria must be from outside the intake of nutrients, in order to guarantee the life activity and growth. Alpha glucan used to maintain SS2 at various stages of infection of survival, reproduction and virulence is particularly important. There are two kinds of bacteria: carbohydrate transport phosphotransferase system (PTS) and ABC transporters. A degradation product of alpha glucan transport is still lack of research. In this experiment in Streptococcus suis SS2 strain SC-19 as the research object, analysis and comparison of the two The position of the ABC transporter msmEFG and malXCD gene cluster in the genome of SC-19. The experiment proves that MsmEFG is responsible for the transport of raffinose and honey sugar two, while MalXCD is responsible for the degradation products of alpha glucan transport. In SC-19, SSUSC84_1724 Msm K ATP encoding protein with enzyme activity. The MsmK mutant strains with artificial medium different carbon source culture, found that mutant can no longer use cottonseed sugar, two honey sugar and maltodextrin. In addition, the expression level of MsmK in these carbon sources were significantly increased, indicating that MsmK belongs to the MsmEFG and MalXCD transporter component, its physiological function is the two transporter for hydrolysis of ATP.2. MsmK promotes the metabolism and physiological process of Streptococcus suis in mice SS2 colonization of alpha glucan invades the host is closely related to, Streptococcus suis in host oropharyngeal cavity with alpha glucan as the main carbon source, the protein alpha glucan metabolic pathways in living Step expression, adhesion and invasion of virulence genes are also up-regulated, the Streptococcus suis invade host epithelial cells or spread to the soft tissue. One of the later stage of infection, impaired release of tissue and cell glycogen is SS2 available carbon source. This study used the SC-19 and msmK genes of wild-type mice were lack of pathogenic strains was lost msmK, found that the mutant death rate decreased. The competitive colonization experiment showed that mice colonized effects of SS2 in mouse brain, MsmK, lung, liver and other tissues in late infection and colonization, may lose the ability to use mutant glycogen. In vivo experiments and in vitro experiments have proved that MsmK is helpful to the enhancement of Streptococcus suis in blood the survival ability of the mutant. The sensitivity of blood kill more, are more likely to be engulfed by phagocytes. Show that bacterial mutant to hyperosmotic stress test, and hydrogen peroxide tolerance ability is abate, life is The film forming ability also decreased significantly. This experiment confirmed that the pathogenic MsmK of Streptococcus suis, further confirmed the pathogenic bacteria using carbon source and its pathogenicity is closely related to.3, MsmK influence the process of cell division of Streptococcus suis pathogenic bacteria cell division process not only let us better understand the physiological basis of bacteria, and more to find the target protein potential for the development of new antimicrobial agents. The process of bacterial cell division is a good target for development of new antimicrobial drugs, have been studied for the growth mechanism of Escherichia coli and Bacillus subtilis as the representative bacteria more thorough, but for aureus (especially Streptococcus) cell division mechanism is poorly understood. This study on Streptococcus suis cell division skeleton protein FtsZ as a bait protein, screening the interaction with the protein using bacterial two hybrid experiment results show. , ABC transporter component ATP enzyme Msm K interacts with FtsZ in vitro.MsmK can promote the formation of FtsZ multimers. Compared with ellipsoidal cell morphology and wild strain SC-19, the mutant msmK due to bacterial surface bacteria and the direction of the long axis is not perpendicular to the division of bacteria to normal cells the expression of mutant forms; and cell division related genes have also changed, the formation of the specific mechanism indicates that MsmK is involved in the process of cell division.Msm of Streptococcus suis K to promote and maintain the FtsZ multimer remains to be further studied.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28

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