信号分子对革兰氏阳性病原菌代谢和感染的调控机制

发布时间：2018-01-14 09:01

本文关键词：信号分子对革兰氏阳性病原菌代谢和感染的调控机制　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：细菌依赖不同的信号调节系统以响应不断变化的环境,实时调整胞内代谢途径。病原菌的代谢途径更为复杂,还需要高效的信号传导系统来介导细菌与宿主的相互作用,或逃避宿主的免疫系统,建立感染。核苷类第二信分子和生物素都是细菌重要的信号分子,调控着包括中心代谢途径、细胞组成、运动能力、毒力等重要的生理活动。(1)c-di-AMP对金黄色葡萄球菌小菌落突变体代谢和感染的调控机制研究金黄色葡萄球菌(Staphylococcus aureus)是人类重要的条件致病菌,可引发皮肤感染和呼吸道疾病。金黄色葡萄球菌小菌落突变体(S.aureus small colony variant,S.aureus SCV)通常是指甲萘醌、血红素和胸腺嘧啶核苷等营养缺陷型,其生长缓慢,具有耐药性,在固体培养基上形成微小菌落。胸腺嘧啶核苷依赖型小菌落突变体(S.aureus thymidine-dependent SCV,S.aureus TD-SCV)是由于胸腺嘧啶核苷酸合酶Thy A失活突变引起的,必须通过摄取外源的胸腺嘧啶核苷而生长,通常在经过磺胺甲恶唑和甲氧苄啶长期治疗的遗传性囊性纤维化(cystic fibrosis)肺炎病人体内出现,引起严重感染,并造成肺损伤。核苷类第二信使环二腺苷酸(c-di-AMP)是细菌特有的核苷类第二信使分子,由二腺苷酸环化酶合成,被特异性的磷酸二酯酶降解,真核生物缺乏合成或降解c-diAMP的相关蛋白。c-di-AMP参与调控细菌中心代谢、细胞壁合成、渗透压适应以及抗生素抗性等生理活动,并可与宿主细胞内质网接头蛋白STING结合,激活天然免疫反应。本研究通过分别敲除S.aureus Newman胸腺嘧啶核苷酸合酶基因thy A和胆色素原合酶编码基因hem B得到了胸腺嘧啶核苷依赖型-SCVΔthy A和血红素依赖型-SCVΔhem B。通过巨噬细胞、小鼠感染实验和Taqman免疫基因芯片分析,发现Δthy A相对于野生型菌株和SCV对照菌株Δhem B,在胸腺嘧啶核苷缺乏时,能够产生更高浓度的c-di-AMP,激活依赖于STING蛋白的天然免疫反应。而高浓度的c-diAMP会导致Δthy A基因组突变率升高,增强细菌的适应性。本研究很好地解释了TD-SCV能引起高炎症反应,并造成更严重的肺损伤的原因。Pst A是一个PII家族信号传递蛋白,同时也是c-di-AMP受体蛋白。本研究通过细菌双杂交实验和体外酶活测定发现,Pst A在c-di-AMP的存在下能够和胸腺嘧啶核苷酸激酶相互作用,并促进其激酶活性,促进胸腺嘧啶核苷的利用,帮助Δthy A在缺乏胸腺嘧啶核苷的环境下生存。本研究首次揭示了c-di-AMP通过调控胸腺嘧啶核苷利用而参与中心代谢的机制。(2)苏云金芽胞杆菌c-di-GMP核糖开关Bc2 RNA的调控机制研究苏云金芽胞杆菌(Bacillus thuringiensis)是一类杆状、产芽胞的革兰氏阳性细菌,也是昆虫致病菌。B.thuringiensis的生活周期主要分为营养期和芽胞形成期,在芽胞形成期可以产生由杀虫晶体蛋白组成的伴胞晶体,具有广谱杀虫活性。目前,B.thuringiensis制剂目前是世界上使用最广泛的微生物杀虫剂,研究B.thuringiensis生长代谢、生活周期等有很大的应用价值。环二鸟苷单磷酸c-di-GMP是广泛分布于细菌中的重要核苷类第二信使分子,通过结合不同的效应蛋白或核糖开关来调控细菌的毒力、细胞周期、生物被膜形成、运动性等多种生理活动。细菌通过二鸟苷酸环化酶和磷酸二酯酶来控制胞内c-diGMP的浓度。核糖开关是位于m RNA非翻译区的一段具有调控功能的s RNA,典型的核糖开关由适体区域和下游的表达平台区域紧密相连而成。本研究发现在B.thuringiensis BMB171编码胶原粘附蛋白(Cap)m RNA的5′-非翻译区存在一个c-di-GMP核糖关Bc2 RNA。本研究通过体外转录终止实验,并在cdi-GMP低浓度和高浓度突变株中,通过β-半乳糖苷酶实验、荧光定量PCR实验,揭示了Bc2 RNA表达平台区的终止子结构会强烈地抑制下游基因cap的转录,当结合c-di-GMP后,会引起Bc2 RNA变构,并形成抗终止子结构,解除对cap的转录抑制。敲除Bc2 RNA会导致cap超表达,从而抑制细菌的运动、胞外多糖的分泌和生物被膜的形成,同时促进细菌的沉降并影响细菌对棉铃虫的毒力。本研究首次提出了“抑制-去抑制”模型,并且Bc2 RNA是蜡样芽胞杆菌群中第一个被实验验证功能的c-di-GMP核糖开关。(3)转录调控因子Bio Q介导的生物素合成调控系统研究结核病是由结核分枝杆菌(Mycobacterium tuberculosis)感染引起的慢性传染病,可以感染人体的各种器官或组织,但主要侵染肺部。大多数抗结核药物只对生长期的结核分枝杆菌有效,而不能清除潜伏期的结核分枝杆菌,多耐药结核分枝杆菌的出现使得结核病的防控形势变得更加严峻,现有的抗结核药物已经不能满足医疗的需要,迫切需要从结核分枝杆菌鉴定新的靶标,开发出新型药物。生物素是所有生物都必须的维生素。它作为羧化酶的辅因子,对分枝杆菌的中心代谢和脂肪酸合成途径非常重要,活动期和潜伏期的结核分枝杆菌都必须依赖自身合成生物素而生存,破坏生物素合成途径会抑制结核分枝杆菌的生长并导致其丧失致病性。哺乳动物不具有生物素合成相关的基因,所以生物素合成途径已经成为新型抗结核药物的靶标。本研究以生长迅速、不致病的耻垢分枝杆菌(M.smegmatis)为模式菌株,系统地探究了生物素代谢相关基因的转录调控机制,为分枝杆菌生物素代谢研究奠定了基础。经典的生物调节系统依赖双功能酶Bir A,一方面作为生物素-蛋白连接酶调控生物素的利用,一方面作为转录调控因子控制生物的合成与转运。分枝杆菌的BirA缺乏转录调控因子的功能,我们在耻垢分枝杆菌中鉴定到了一个TetR家族的转录调控因子Bio Q,它补偿了Bir A转录调控因子的功能,本研究揭示了以Bir A和Bio Q协同介导的生物素代谢调节系统。BioQ是TetR家族的转录调控因子,本研究通过凝胶阻滞、DNase I足迹等实验鉴定了Bio Q识别的DNA序列为13 bp的保守的回文序列TGAAC-N3-GTTCA;并通过构建bio Q缺失菌株Δbio Q,利用荧光定量PCR和β-半乳糖苷酶实验探究了Bio Q作为转录抑制因子,调节生物素合成相关基因表达的机制。
[Abstract]:Bacteria rely on different signals in response to changing environmental regulation system, adjust the intracellular metabolic pathways. Metabolic pathways of pathogenic bacteria is more complex, the interaction also needs the signal transduction system mediated by high efficient bacteria and host, or evade the host immune system, the establishment of infection. Second nucleoside and biotin molecules is an important signal molecule of bacteria, including the regulation of central metabolic pathways, cell composition, exercise capacity, physical activity and other important virulence. (1) of small colony mutants of Staphylococcus aureus infection and metabolic mechanism of c-di-AMP regulation on Staphylococcus aureus (Staphylococcus aureus) is an important human pathogen, can cause skin infections and respiratory diseases. Staphylococcus aureus Petite mutant (S.aureus small colony variant, S.aureus SCV) is usually nail naphthaquinone, heme and thymus Such as pyrimidine auxotrophy, slow growth, resistance, small colonies formed in the medium of solid culture. Thymidine dependent small colony mutants (S.aureus thymidine-dependent SCV, S.aureus TD-SCV) is the thymidylate synthase Thy inactivation of A caused by mutations, must be grown by uptake of exogenous thymidine, usually in sulfamethoxazole and trimethoprim after long-term treatment of cystic fibrosis genetic (cystic fibrosis) appeared in the patients with pneumonia caused by serious infection, and cause lung injury. The nuclear glycosides second messenger cyclic adenylate (c-di-AMP) two is a nucleoside second messenger molecule specific bacteria, by two adenylate cyclase synthesis, degradation by specific phosphodiesterase the eukaryotes lack of synthesis or degradation of c-diAMP related protein.C-di-AMP is involved in the regulation of bacterial cell wall metabolism center, and To adapt to the osmotic pressure and antibiotic resistance and other physiological activities, and can be combined with the host cell endoplasmic reticulum adaptor protein STING, activate the innate immune response. This study by knockdown of S.aureus Newman thymidylate synthase gene thy A and porphobilinogen synthase encoding gene hem was obtained by B thymidine dependent -SCV thy A and heme dependent -SCV hem B. by macrophages, experiment and analysis of Taqman immune mice infected with gene chip, a thy A found that compared with wild type strains and control strains SCV delta hem B, in the absence of thymidine, can have a higher concentration of c-di-AMP, activation of the innate immune response depends on the high concentration of STING protein. The c-diAMP will lead to the delta thy A genome mutation rate increased, the enhancement of bacterial adaptability. This research is well explained by TD-SCV can cause inflammation, causing more serious The reasons of lung injury.Pst A is a family of PII signal transduction protein, but also the c-di-AMP receptor protein. This study was found by bacterial two hybrid assay and in vitro activity of Pst, A in the presence of c-di-AMP and thymidine kinase interaction, and promote the use of thymidine kinase activity, promote and help. Thy A survival in the absence of thymidine environment. This is the first study to reveal the mechanism of c-di-AMP involved in central metabolism through regulation of thymidine utilization. (2) of Bacillus thuringiensis c-di-GMP ribose switch Bc2 RNA regulatory mechanism of Bacillus thuringiensis (Bacillus thuringiensis) is a rod-shaped, gram positive bacteria sporiferous, insects are also the pathogen of.B.thuringiensis life cycle is divided into vegetative and spore formation stage in spore formation period can be produced by killing Composition of insect crystal protein parasporal crystal, with broad-spectrum insecticidal activity. At present, B.thuringiensis is currently using the preparation of microbial insecticides most widely in the world, on the growth and metabolism of B.thuringiensis, has great application value in life cycle. The cyclic Diguanylic monophosphate c-di-GMP nucleoside is an important second messenger molecules are widely distributed in bacteria, virulence. The regulation of bacteria by binding of effector proteins or riboswitches of different cell cycle, biofilm formation and motility and other physiological activities. The concentration of bacteria by Diguanylate cyclase and two phosphorus acid esterase to control intracellular c-diGMP. S RNA is a riboswitch located in the RNA untranslated region with m the regulation function of the typical riboswitch aptamer expression by the platform area and downstream region closely linked together. The study found in the B.thuringiensis BMB171 encoding collagen adhesion Protein (Cap) region of the existence of a c-di-GMP Bc2 ribose RNA. this study by in vitro transcription termination experiment of non m RNA 5 '- cdi-GMP, and in the low and high concentration of the mutant strain, the beta galactosidase experiments, fluorescence quantitative PCR experiments revealed that the Bc2 expression of RNA transcription termination platform area the sub structure will strongly inhibit the downstream genes of cap, when combined with c-di-GMP, RNA and Bc2 will cause the allosteric, form the anti terminator structure, lift the transcription inhibition of cap. Knockdown of Bc2 RNA will lead to the over expression of cap, thus inhibiting the movement of bacteria, extracellular polysaccharide secretion and biofilm formation at the same time, promote the settlement and the influence of bacterial virulence of bacteria on the cotton bollworm. This study first proposed the "inhibition to suppress" model, and the Bc2 RNA is Bacillus cereus group in the first experimental verification function of the c-di-GMP riboswitch. (3) transcription factor Bio Q mediated biotin synthesis regulation system of TB is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) infection caused by chronic infectious diseases, can infect various organs or tissues of the body, but the main pulmonary infection. Most anti tuberculosis drugs are only effective against Mycobacterium tuberculosis mycobacterium tuberculosis growth stage, but can not eliminate the latency multi drug resistant Mycobacterium tuberculosis, the tuberculosis prevention and control situation is becoming more and more serious, the existing anti tuberculosis drugs has been unable to meet the medical needs, the urgent need from Mycobacterium tuberculosis identification of new targets, develop new drugs. Biotin is all creatures must vitamin cofactor. As carboxylase center, metabolism and fatty acid synthesis pathway of Mycobacterium tuberculosis is very important, activity and incubation period must rely on their own as a student Biotin and biotin synthesis way of survival, destruction will inhibit the growth of Mycobacterium tuberculosis and cause its pathogenicity. No mammals with biotin synthesis related genes, so the biotin biosynthesis pathway has become a new anti tuberculosis drug targets. Based on the growth of the fast, non pathogenic Mycobacterium smegmatis (M.smegmatis) as a model strain and systematically explore the mechanism of transcriptional regulation of biotin metabolism related genes, which laid the foundation for the study on metabolism of mycobacteria biotin. The classic biological regulation system depends on the bifunctional enzyme Bir A, as a biotin protein ligase by regulation of biotin, as a transcriptional regulation of synthesis and transport the biological control factor. Mycobacterium BirA lack of transcription factor function, we get a TetR family transcription factor Bio Q in identification of Mycobacterium smegmatis And it compensates for the transcription factor Bir A gene transcription, this study reveals to Bir A and Bio Q co mediated biotin metabolism regulation system.BioQ transcription factors of the TetR family, the DNase I by gel retardation, footprinting experiments identified DNA sequence Bio Q identification of conservative palindrome TGAAC-N3-GTTCA 13 BP; and through the construction of bio Q mutant strain bio Q, using fluorescence quantitative PCR and beta galactosidase experiments to explore the Bio Q as a transcriptional repressor, regulating biotin synthesis mechanism related gene expression.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378

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