结核分枝杆菌PPE10（Rv0442c）通过线性泛素链组装复合物HOIP-NF-κB信号传导轴改变宿主细胞凋亡和细胞因子
发布时间：2024-06-10 23:49
结核分枝杆菌(Mycobacterium tuberculosis,简称结核杆菌)感染引起的结核病仍然是全世界十大高致死率疾病之一。结核杆菌成功之处在于能够主动改变宿主信号转导。结核杆菌基因组包含大量编码毒力因子的基因。PE/PPE家族蛋白广泛参与细菌-宿主互作,与结核杆菌毒力和抗原变异有关,在调控宿主细胞信号转导、帮助细菌逃避宿主免疫杀伤过程中发挥重要作用。结核分枝杆菌PPE10(Rv0442c)属于PPE家族,高度保守。PPE10能够干扰细胞壁完整性,增强海分枝杆菌的毒力并减少宿主细胞泛素的募集。BCG PPE10缺失菌株抑制吞噬体酸化的能力减弱。PPE10 BCG免疫小鼠可以激发强烈的T细胞反应。为了探索PPE10在细菌-宿主相互作用中的功能,我们构建了异源表达PPE10编码基因Rv0442c的非致病性耻垢分枝杆菌MsPPE10。Rv0442c过表达改变了耻垢分枝杆菌MsPPE10的单菌落形态,增强耻垢分枝杆菌MsPPE10生物膜形成能力。与仅携带空载的耻垢分枝杆菌Msvec相比,PPE10提...
【文章页数】：84 页
【学位级别】：博士
【文章目录】：
Abbreviation
摘要
ABSTRACT
CHAPTER ONE LITERATURE REVIEW
1.1 HISTORY OF TUBERCULOSIS
1.2 THE BURDEN OF TUBERCULOSIS
1.3 BIOLOGICAL CHARACTERISTIC OF M.TUBERCULOSIS
1.4 CLINICAL MANIFESTATIONS OF M.TUBERCULOSIS
1.5 THE PATHOGENICITY OF M.TUBERCULOSIS
1.6 THE VIRULENCE FACTORS OF M.TUBERCULOSIS
1.7 THE IMMUNE-MODULATORY EFFECTS OF M.TUBERCULOSIS
1.8 DIAGNOSIS,TREATMENT,AND VACCINE OF TUBERCULOSIS
1.8.1 Diagnosis of tuberculosis
1.8.2 Treatment of tuberculosis
1.8.3 Vaccines of tuberculosis
1.9 M.TUBERCULOSIS PE/PPE GENES FAMILY
1.10 THE SECRETION SYSTEM AND LOCATION OF PE/PPE PROTEINS FAMILY
1.11 THE ROLE OF PE/PPE PROTEINS IN PATHOGEN-HOST INTERACTIONS
1.12 THE ROLE OF PE/PPE GENES FAMILY IN IMMUNE EVASION
1.13 INTERFERING OF PE/PPE PROTEINS IN HOST CELL DEATH FATE
1.14 THE IMPLICATION OF M.TUBERCULOSIS PE/PPE GENES FAMILY IN VACCINE
CHAPTER TWO INTRODUCTION
AIMS AND HYPOTHESES
CHAPTER THREE MATERIAL AND METHODS
3.1 MATERIAL
3.1.1 Bacterial strains,plasmids,and culture conditions
3.1.2 Culture medium and growth conditions
3.1.3 Chemicals,Reagents,Kits,Media,other
3.1.3.1 Chemicals
3.1.3.2 Reagents and Kits
3.1.3.3 Media and others
3.1.4 SDS-PAGE and Western blotting Reagents
3.1.5 Antibodies used in this study
3.1.6 Instruments and Apparatus
3.2 METHODS
3.2.1 Construction of recombinant M.smegmatis strains
3.2.2 Expression of PPE10 in M.smegmatis
3.2.3 The survival of recombinant M.smegmatis under stresses
3.2.4 Biofilm formation assay
3.2.5 Ethidium bromide accumulation/efflux assay and Nile red uptake assays
3.2.6 Infection of macrophages
3.2.7 RT-PCR assay
3.2.8 Protein analysis by Western Blotting
3.2.9 Apoptosis analysis by fluorescence microscope
3.2.10 Cell signaling pathway inhibition
3.2.11 Flow cytometry
3.2.12 Statistical analysis
CHAPTER FOUR RESULTS
4.1 M.TUBERCULOSIS RV0442C(PPE10)SUCCESSFULLY EXPRESSED IN M.SMEGMATIS
4.2 PPE10 PROMOTES THE RESISTANCE OF M.SMEGMATIS TO STRESSES
4.3 PPE10 ALTERS COLONY MORPHOLOGY AND CELL WALL PERMEABILITY
4.4 PPE10 PROMOTES THE SURVIVAL OF RECOMBINANT M.SMEGMATIS WITHIN MACROPHAGES AND MODULATES THE TRANSCRIPTION LEVELS OF CYTOKINES
4.5 PPE10 DECREASES CASPASES EXPRESSION AND ALTERS APOPTOSIS IN INFECTED MACROPHAGE
4.6 PPE10 ALTERS CYTOKINE EXPRESSION AND CASPASES VIA NF-ΚB SIGNALING PATHWAY
4.7 PPE10 REGULATES NF-ΚB PATHWAY THROUGH LUBAC AND C-IAP1
4.8 PPE10 reduces expression and phosphorylation of RelA/p65 NF-κB
CHAPTER FIVE DISCUSSION AND CONCLUSION
5.1 DISCUSSION
5.2 Conclusion
REFERENCE
Contributions
ACKNOWLEDGEMENT
本文编号：3992081
【文章页数】：84 页
【学位级别】：博士
【文章目录】：
Abbreviation
摘要
ABSTRACT
CHAPTER ONE LITERATURE REVIEW
1.1 HISTORY OF TUBERCULOSIS
1.2 THE BURDEN OF TUBERCULOSIS
1.3 BIOLOGICAL CHARACTERISTIC OF M.TUBERCULOSIS
1.4 CLINICAL MANIFESTATIONS OF M.TUBERCULOSIS
1.5 THE PATHOGENICITY OF M.TUBERCULOSIS
1.6 THE VIRULENCE FACTORS OF M.TUBERCULOSIS
1.7 THE IMMUNE-MODULATORY EFFECTS OF M.TUBERCULOSIS
1.8 DIAGNOSIS,TREATMENT,AND VACCINE OF TUBERCULOSIS
1.8.1 Diagnosis of tuberculosis
1.8.2 Treatment of tuberculosis
1.8.3 Vaccines of tuberculosis
1.9 M.TUBERCULOSIS PE/PPE GENES FAMILY
1.10 THE SECRETION SYSTEM AND LOCATION OF PE/PPE PROTEINS FAMILY
1.11 THE ROLE OF PE/PPE PROTEINS IN PATHOGEN-HOST INTERACTIONS
1.12 THE ROLE OF PE/PPE GENES FAMILY IN IMMUNE EVASION
1.13 INTERFERING OF PE/PPE PROTEINS IN HOST CELL DEATH FATE
1.14 THE IMPLICATION OF M.TUBERCULOSIS PE/PPE GENES FAMILY IN VACCINE
CHAPTER TWO INTRODUCTION
AIMS AND HYPOTHESES
CHAPTER THREE MATERIAL AND METHODS
3.1 MATERIAL
3.1.1 Bacterial strains,plasmids,and culture conditions
3.1.2 Culture medium and growth conditions
3.1.3 Chemicals,Reagents,Kits,Media,other
3.1.3.1 Chemicals
3.1.3.2 Reagents and Kits
3.1.3.3 Media and others
3.1.4 SDS-PAGE and Western blotting Reagents
3.1.5 Antibodies used in this study
3.1.6 Instruments and Apparatus
3.2 METHODS
3.2.1 Construction of recombinant M.smegmatis strains
3.2.2 Expression of PPE10 in M.smegmatis
3.2.3 The survival of recombinant M.smegmatis under stresses
3.2.4 Biofilm formation assay
3.2.5 Ethidium bromide accumulation/efflux assay and Nile red uptake assays
3.2.6 Infection of macrophages
3.2.7 RT-PCR assay
3.2.8 Protein analysis by Western Blotting
3.2.9 Apoptosis analysis by fluorescence microscope
3.2.10 Cell signaling pathway inhibition
3.2.11 Flow cytometry
3.2.12 Statistical analysis
CHAPTER FOUR RESULTS
4.1 M.TUBERCULOSIS RV0442C(PPE10)SUCCESSFULLY EXPRESSED IN M.SMEGMATIS
4.2 PPE10 PROMOTES THE RESISTANCE OF M.SMEGMATIS TO STRESSES
4.3 PPE10 ALTERS COLONY MORPHOLOGY AND CELL WALL PERMEABILITY
4.4 PPE10 PROMOTES THE SURVIVAL OF RECOMBINANT M.SMEGMATIS WITHIN MACROPHAGES AND MODULATES THE TRANSCRIPTION LEVELS OF CYTOKINES
4.5 PPE10 DECREASES CASPASES EXPRESSION AND ALTERS APOPTOSIS IN INFECTED MACROPHAGE
4.6 PPE10 ALTERS CYTOKINE EXPRESSION AND CASPASES VIA NF-ΚB SIGNALING PATHWAY
4.7 PPE10 REGULATES NF-ΚB PATHWAY THROUGH LUBAC AND C-IAP1
4.8 PPE10 reduces expression and phosphorylation of RelA/p65 NF-κB
CHAPTER FIVE DISCUSSION AND CONCLUSION
5.1 DISCUSSION
5.2 Conclusion
REFERENCE
Contributions
ACKNOWLEDGEMENT
本文编号：3992081
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