結(jié)核病,特別是耐多藥結(jié)核病,仍然是一個(gè)巨大的公共衛(wèi)生威脅。硫化氫(H_2S)是目前在結(jié)核分枝桿菌(M.tuberculosis)的病理生理學(xué)中出現(xiàn)的一種重要的氣體介質(zhì)。結(jié)核分枝桿菌Rv3340(metC)基因在甲硫氨酸生物合成中的倒數(shù)第二個(gè)步驟是將半胱氨酸分解為同型半胱氨酸,這個(gè)過程會(huì)產(chǎn)生硫化氫。我們?cè)趷u垢分枝桿菌中克隆并表達(dá)了結(jié)核分枝桿菌Rv3340(metC),然后以半胱氨酸、硫酸鹽、抗生素和NaHS作為常氧或缺氧的唯一硫源進(jìn)行處理。我們報(bào)道了恥垢分枝桿菌(Ms_Rv3340/Ms_metC)中Rv3340的過表達(dá)誘導(dǎo)了硫化氫(H_2S)的產(chǎn)生,以幫助其在惡劣條件下獲得能量。我們發(fā)現(xiàn),當(dāng)Ms_Rv3340暴露于H_2O_2時(shí),其存活能力降低,我們發(fā)現(xiàn)Ms_Rv3340通過調(diào)節(jié)H_2S增強(qiáng)對(duì)鏈霉素的耐藥性。重組Ms_Rv3340通過芬頓反應(yīng)啟動(dòng)DNA損傷,過表達(dá)Ms_Rv3340下調(diào)三個(gè)鏈霉素應(yīng)答基因的表達(dá)水平。在本研究的范圍內(nèi),我們的結(jié)果表明,在缺氧條件下,Ms_metC增強(qiáng)了H_2S的產(chǎn)生。我們證明了在硫酸鹽存在下重組的Ms_metC能刺激半胱氨酸的外排,而半胱氨酸能促進(jìn)正常條件下Ms_metC的快速生長。pH曲線表明,在H_2S供體存在的情況下,Ms_metC呈pH依賴性。半胱氨酸存在時(shí),Ms_metC對(duì)缺氧敏感,暴露于H_2S供體后,Ms_metC生長迅速,這可能與H_2S有關(guān)。據(jù)我們所知,之前沒有研究報(bào)道過結(jié)核分枝桿菌Rv3340(metC)能產(chǎn)生H_2S調(diào)節(jié)對(duì)鏈霉素的耐藥性。這些研究結(jié)果表明,在不同的環(huán)境下,無論是正常缺氧還是缺氧,Ms_metC誘導(dǎo)H_2S的產(chǎn)生對(duì)重組Ms_metC的存活具有不同的潛在的生物學(xué)作用。
【學(xué)位單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2020
【中圖分類】：R378.911
【文章目錄】：
Abstract
摘要
Chapter Ⅰ:Literature Review
    1.1 BACKGROUND
    1.2 GLOBAL BURDEN AND TB IMPACT
    1.3 DRUG RESISTANT TB BURDEN
    1.4 Difference between active TB and LTBI
    1.5 TREATMENT OF MDR-TB
    1.6 TB TRANSMISSION AND PREVENTIVE TREATMENT
    1.7 INCUBATION PERIOD
    1.8 TUBERCULOSIS DIAGNOSIS
    1.9 EMERGING DRUGS AND DRUG TARGETS AGAINST TUBERCULOSIS
    1.10 METABOLIC PATHWAYS
        1.10.1 Targeting the M.tuberculosis fatty acid
        1.10.2 TARGETING THE M.TUBERCULOSIS LONG-CHAIN FATTY ALCOHOLS
        1.10.3 TARGETING THE M.TUBERCULOSIS TCA CYCLE
        1.10.4 TARGETING THE M.TUBERCULOSIS IRON HOMEOSTASIS
        1.10.5 TARGETING THE M.TUBERCULOSIS DORMANCY
    1.11 TARGETING THE M.TUBERCULOSIS TOXIN-ANTITOXIN
    1.12 Targeting the M.tuberculosis cell wall
    1.13 TARGETING THE M.TUBERCULOSIS EFFLUX PUMP
    1.14 Targeting the M.tuberculosis ATP synthase
    1.15 Potential novel drug targets against M.tuberculosis
    1.16 New paradigm for novel antibiotics discovery and improvement
    1.17 Therapeutic application
Chapter Ⅱ:Introduction
    2.1 RESEARCH SIGNIFICANCE
    2.2 GENERAL AIM
    2.3 SPECIFIC OBJECTIVES
Chapter Ⅲ:Mycobacterium tuberculosis metC（Rv3340）derived hydrogen sulfide conferring bacteria stress survival
    3.1 INTRODUCTION
    3.2 MATERIALS AND METHODS
        3.2.1 INSTRUMENTS
        3.2.2 Reagents
    3.3 SDS-PAGE REAGENTS
    3.4 OTHER REAGENTS
    3.5 MEDIA AND BUFFER PREPARATION
    3.6 BACTERIAL STRAINS AND GROWTH CONDITIONS
    3.7 Plasmid construction
    3.8 Restriction enzyme digestion and detection by gel
    3.9 Agarose gel electrophoresis
    3.10 Gel extraction
    3.11 Ligation
    3.12 Transformation
    3.13 EXPRESSION OF RV3340 IN M.SMEGMATIS
    3.14 THE MIC DETERMINATION FOR ANTIBIOTICS
    3.15 H2S ACTIVITY AND H2O2 SENSITIVITY
    3.16 HYDROGEN SULPHIDE INHIBITOR ASSAY
    3.17 FENTON REACTION
    3.18 RNA PREPARATION AND RT-PCR
    3.19 STATISTICAL ANALYSIS
    3.20 RESULTS
        3.20.1 The Rv3340 gene is conserved among mycobacteria
        3.20.2 Overexpression of Rv3340 decrease streptomycin susceptibility
        3.20.3 Docking analysis of Rv3340 protein
        3.20.4 Hydrogen sulfide production
Rv3340 to growth'>        3.20.5 Exogenous h2s facilitated Ms_Rv3340 to growth
Rv340'>        3.20.6 Thiourea delayed streptomycin mediated killing Ms_Rv340
Rv340'>        3.20.7 The effective of streptomycin and H2O2 against Ms_Rv340
Rv3340 survival against inhibitor'>        3.20.8 H2S is necessary for Ms_Rv3340 survival against inhibitor
        3.20.9 H2O2 mediated cysteine induction of dna damage
Rv3340 to diamide stress'>        3.20.10 Response of Ms_Rv3340 to diamide stress
        3.20.11 Rv3340 transcriptional regulator of mRNA expression levels of H2S and streptomycin responsive genes
        3.20.12 Discussion
Chapter Ⅳ:Mycobacterium tuberculosis Rv3340（metC）improving recombinants hypoxia survival via enhanced hydrogen sulfide production
    4.1 INTRODUCTION
    4.2 MATERIALS AND METHODS
        4.2.1 INSTRUMENTS
        4.2.2 Reagents
    4.3 SDS-PAGE REAGENTS
    4.4 OTHER REAGENTS
    4.5 RNA ISOLATION AND RT-PCR ANALYSES
    4.6 CHEMICALS AND REAGENTS
    4.7 MEDIA AND GROWTH CONDITIONS
    4.8 SURVIVAL CURVES
METC UNDER HARSH CONDITIONS'>    4.9 IN VITRO GROWTH OF MS_METC UNDER HARSH CONDITIONS
    4.10 PH MEASUREMENT
    4.11 MEASUREMENTS OF CYSTEINE EXCRETION
    4.12 STATISTICAL ANALYSIS
    4.13 RESULTS
metC survival in hypoxia'>        4.13.1 The increases of nicotinamide concentration enhance Ms_metC survival in hypoxia
metC growth'>        4.13.2 Glucose reduces the recombinant Ms_metC growth
metC via endogenous H2S in hypoxia'>        4.13.3 NaHS restores the growth of recombinant Ms_metC via endogenous H2S in hypoxia
metC'>        4.13.4 Acidification state of h2s produced by Ms_metC
metC in hypoxia'>        4.13.5 Sulfur compound does effectively the cysteine excreted by Ms_metC in hypoxia
metC growth in hypoxia'>        4.13.6 Systeine sensitizes Ms_metC growth in hypoxia
metC mRNA treated with NaHS in hypoxia'>        4.13.7 Relative transcription levels of Ms_metC mRNA treated with NaHS in hypoxia
    4.14 Discussion
Chapter Ⅴ:Conclusion
    5.1 CONCLUSION
References
Acknowledgements
Contributions
Abbreviations

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