结核分枝杆菌Rv3340（metC）可通过增强硫化氢的产生来提高重组菌低氧存活

发布时间：2020-11-02 22:59

　　 结核病,特别是耐多药结核病,仍然是一个巨大的公共卫生威胁。硫化氢(H_2S)是目前在结核分枝杆菌(M.tuberculosis)的病理生理学中出现的一种重要的气体介质。结核分枝杆菌Rv3340(metC)基因在甲硫氨酸生物合成中的倒数第二个步骤是将半胱氨酸分解为同型半胱氨酸,这个过程会产生硫化氢。我们在耻垢分枝杆菌中克隆并表达了结核分枝杆菌Rv3340(metC),然后以半胱氨酸、硫酸盐、抗生素和NaHS作为常氧或缺氧的唯一硫源进行处理。我们报道了耻垢分枝杆菌(Ms_Rv3340/Ms_metC)中Rv3340的过表达诱导了硫化氢(H_2S)的产生,以帮助其在恶劣条件下获得能量。我们发现,当Ms_Rv3340暴露于H_2O_2时,其存活能力降低,我们发现Ms_Rv3340通过调节H_2S增强对链霉素的耐药性。重组Ms_Rv3340通过芬顿反应启动DNA损伤,过表达Ms_Rv3340下调三个链霉素应答基因的表达水平。在本研究的范围内,我们的结果表明,在缺氧条件下,Ms_metC增强了H_2S的产生。我们证明了在硫酸盐存在下重组的Ms_metC能刺激半胱氨酸的外排,而半胱氨酸能促进正常条件下Ms_metC的快速生长。pH曲线表明,在H_2S供体存在的情况下,Ms_metC呈pH依赖性。半胱氨酸存在时,Ms_metC对缺氧敏感,暴露于H_2S供体后,Ms_metC生长迅速,这可能与H_2S有关。据我们所知,之前没有研究报道过结核分枝杆菌Rv3340(metC)能产生H_2S调节对链霉素的耐药性。这些研究结果表明,在不同的环境下,无论是正常缺氧还是缺氧,Ms_metC诱导H_2S的产生对重组Ms_metC的存活具有不同的潜在的生物学作用。
【学位单位】：西南大学
【学位级别】：博士
【学位年份】：2020
【中图分类】：R378.911
【文章目录】：
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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