本文關(guān)鍵詞：信號分子對革蘭氏陽性病原菌代謝和感染的調(diào)控機制　出處：《華中農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 金黃色葡萄球菌 胸腺嘧啶核苷依賴型小菌落突變體 囊性纖維化 環(huán)二腺苷酸 天然免疫 蘇云金芽胞桿菌 環(huán)二鳥苷酸 核糖開關(guān) 膠原粘附蛋白 結(jié)核分枝桿菌 生物素 TetR家族的轉(zhuǎn)錄調(diào)控因子

【摘要】：細菌依賴不同的信號調(diào)節(jié)系統(tǒng)以響應(yīng)不斷變化的環(huán)境,實時調(diào)整胞內(nèi)代謝途徑。病原菌的代謝途徑更為復(fù)雜,還需要高效的信號傳導(dǎo)系統(tǒng)來介導(dǎo)細菌與宿主的相互作用,或逃避宿主的免疫系統(tǒng),建立感染。核苷類第二信分子和生物素都是細菌重要的信號分子,調(diào)控著包括中心代謝途徑、細胞組成、運動能力、毒力等重要的生理活動。(1)c-di-AMP對金黃色葡萄球菌小菌落突變體代謝和感染的調(diào)控機制研究金黃色葡萄球菌(Staphylococcus aureus)是人類重要的條件致病菌,可引發(fā)皮膚感染和呼吸道疾病。金黃色葡萄球菌小菌落突變體(S.aureus small colony variant,S.aureus SCV)通常是指甲萘醌、血紅素和胸腺嘧啶核苷等營養(yǎng)缺陷型,其生長緩慢,具有耐藥性,在固體培養(yǎng)基上形成微小菌落。胸腺嘧啶核苷依賴型小菌落突變體(S.aureus thymidine-dependent SCV,S.aureus TD-SCV)是由于胸腺嘧啶核苷酸合酶Thy A失活突變引起的,必須通過攝取外源的胸腺嘧啶核苷而生長,通常在經(jīng)過磺胺甲惡唑和甲氧芐啶長期治療的遺傳性囊性纖維化(cystic fibrosis)肺炎病人體內(nèi)出現(xiàn),引起嚴重感染,并造成肺損傷。核苷類第二信使環(huán)二腺苷酸(c-di-AMP)是細菌特有的核苷類第二信使分子,由二腺苷酸環(huán)化酶合成,被特異性的磷酸二酯酶降解,真核生物缺乏合成或降解c-diAMP的相關(guān)蛋白。c-di-AMP參與調(diào)控細菌中心代謝、細胞壁合成、滲透壓適應(yīng)以及抗生素抗性等生理活動,并可與宿主細胞內(nèi)質(zhì)網(wǎng)接頭蛋白STING結(jié)合,激活天然免疫反應(yīng)。本研究通過分別敲除S.aureus Newman胸腺嘧啶核苷酸合酶基因thy A和膽色素原合酶編碼基因hem B得到了胸腺嘧啶核苷依賴型-SCVΔthy A和血紅素依賴型-SCVΔhem B。通過巨噬細胞、小鼠感染實驗和Taqman免疫基因芯片分析,發(fā)現(xiàn)Δthy A相對于野生型菌株和SCV對照菌株Δhem B,在胸腺嘧啶核苷缺乏時,能夠產(chǎn)生更高濃度的c-di-AMP,激活依賴于STING蛋白的天然免疫反應(yīng)。而高濃度的c-diAMP會導(dǎo)致Δthy A基因組突變率升高,增強細菌的適應(yīng)性。本研究很好地解釋了TD-SCV能引起高炎癥反應(yīng),并造成更嚴重的肺損傷的原因。Pst A是一個PII家族信號傳遞蛋白,同時也是c-di-AMP受體蛋白。本研究通過細菌雙雜交實驗和體外酶活測定發(fā)現(xiàn),Pst A在c-di-AMP的存在下能夠和胸腺嘧啶核苷酸激酶相互作用,并促進其激酶活性,促進胸腺嘧啶核苷的利用,幫助Δthy A在缺乏胸腺嘧啶核苷的環(huán)境下生存。本研究首次揭示了c-di-AMP通過調(diào)控胸腺嘧啶核苷利用而參與中心代謝的機制。(2)蘇云金芽胞桿菌c-di-GMP核糖開關(guān)Bc2 RNA的調(diào)控機制研究蘇云金芽胞桿菌(Bacillus thuringiensis)是一類桿狀、產(chǎn)芽胞的革蘭氏陽性細菌,也是昆蟲致病菌。B.thuringiensis的生活周期主要分為營養(yǎng)期和芽胞形成期,在芽胞形成期可以產(chǎn)生由殺蟲晶體蛋白組成的伴胞晶體,具有廣譜殺蟲活性。目前,B.thuringiensis制劑目前是世界上使用最廣泛的微生物殺蟲劑,研究B.thuringiensis生長代謝、生活周期等有很大的應(yīng)用價值。環(huán)二鳥苷單磷酸c-di-GMP是廣泛分布于細菌中的重要核苷類第二信使分子,通過結(jié)合不同的效應(yīng)蛋白或核糖開關(guān)來調(diào)控細菌的毒力、細胞周期、生物被膜形成、運動性等多種生理活動。細菌通過二鳥苷酸環(huán)化酶和磷酸二酯酶來控制胞內(nèi)c-diGMP的濃度。核糖開關(guān)是位于m RNA非翻譯區(qū)的一段具有調(diào)控功能的s RNA,典型的核糖開關(guān)由適體區(qū)域和下游的表達平臺區(qū)域緊密相連而成。本研究發(fā)現(xiàn)在B.thuringiensis BMB171編碼膠原粘附蛋白(Cap)m RNA的5′-非翻譯區(qū)存在一個c-di-GMP核糖關(guān)Bc2 RNA。本研究通過體外轉(zhuǎn)錄終止實驗,并在cdi-GMP低濃度和高濃度突變株中,通過β-半乳糖苷酶實驗、熒光定量PCR實驗,揭示了Bc2 RNA表達平臺區(qū)的終止子結(jié)構(gòu)會強烈地抑制下游基因cap的轉(zhuǎn)錄,當結(jié)合c-di-GMP后,會引起B(yǎng)c2 RNA變構(gòu),并形成抗終止子結(jié)構(gòu),解除對cap的轉(zhuǎn)錄抑制。敲除Bc2 RNA會導(dǎo)致cap超表達,從而抑制細菌的運動、胞外多糖的分泌和生物被膜的形成,同時促進細菌的沉降并影響細菌對棉鈴蟲的毒力。本研究首次提出了“抑制-去抑制”模型,并且Bc2 RNA是蠟樣芽胞桿菌群中第一個被實驗驗證功能的c-di-GMP核糖開關(guān)。(3)轉(zhuǎn)錄調(diào)控因子Bio Q介導(dǎo)的生物素合成調(diào)控系統(tǒng)研究結(jié)核病是由結(jié)核分枝桿菌(Mycobacterium tuberculosis)感染引起的慢性傳染病,可以感染人體的各種器官或組織,但主要侵染肺部。大多數(shù)抗結(jié)核藥物只對生長期的結(jié)核分枝桿菌有效,而不能清除潛伏期的結(jié)核分枝桿菌,多耐藥結(jié)核分枝桿菌的出現(xiàn)使得結(jié)核病的防控形勢變得更加嚴峻,現(xiàn)有的抗結(jié)核藥物已經(jīng)不能滿足醫(yī)療的需要,迫切需要從結(jié)核分枝桿菌鑒定新的靶標,開發(fā)出新型藥物。生物素是所有生物都必須的維生素。它作為羧化酶的輔因子,對分枝桿菌的中心代謝和脂肪酸合成途徑非常重要,活動期和潛伏期的結(jié)核分枝桿菌都必須依賴自身合成生物素而生存,破壞生物素合成途徑會抑制結(jié)核分枝桿菌的生長并導(dǎo)致其喪失致病性。哺乳動物不具有生物素合成相關(guān)的基因,所以生物素合成途徑已經(jīng)成為新型抗結(jié)核藥物的靶標。本研究以生長迅速、不致病的恥垢分枝桿菌(M.smegmatis)為模式菌株,系統(tǒng)地探究了生物素代謝相關(guān)基因的轉(zhuǎn)錄調(diào)控機制,為分枝桿菌生物素代謝研究奠定了基礎(chǔ)。經(jīng)典的生物調(diào)節(jié)系統(tǒng)依賴雙功能酶Bir A,一方面作為生物素-蛋白連接酶調(diào)控生物素的利用,一方面作為轉(zhuǎn)錄調(diào)控因子控制生物的合成與轉(zhuǎn)運。分枝桿菌的BirA缺乏轉(zhuǎn)錄調(diào)控因子的功能,我們在恥垢分枝桿菌中鑒定到了一個TetR家族的轉(zhuǎn)錄調(diào)控因子Bio Q,它補償了Bir A轉(zhuǎn)錄調(diào)控因子的功能,本研究揭示了以Bir A和Bio Q協(xié)同介導(dǎo)的生物素代謝調(diào)節(jié)系統(tǒng)。BioQ是TetR家族的轉(zhuǎn)錄調(diào)控因子,本研究通過凝膠阻滯、DNase I足跡等實驗鑒定了Bio Q識別的DNA序列為13 bp的保守的回文序列TGAAC-N3-GTTCA;并通過構(gòu)建bio Q缺失菌株Δbio Q,利用熒光定量PCR和β-半乳糖苷酶實驗探究了Bio Q作為轉(zhuǎn)錄抑制因子,調(diào)節(jié)生物素合成相關(guān)基因表達的機制。
[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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R378

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本文編號：1422891