鸭疫里默氏杆菌AS8708785基因编码IX型分泌系统的功能组分GldK

论文摘要

鸭疫里默氏杆菌（Riemerella anatipestifer）是引起水禽败血症和渗出性疾病的重要致病菌。毒力因子的研究将有助于我们了解该病原菌的致病机制,并制定有效的防控措施。鸭疫里默氏杆菌具有称为IX型分泌系统的蛋白质分泌系统。前期研究中,我们发现AS8708785基因的缺失显著降低了鸭疫里默氏杆菌Yb2株的细菌毒力,然而,其机制仍然未知。本研究构建并鉴定了鸭疫里默氏杆菌AS8708785基因突变和互补菌株。序列分析表明,AS8708785基因编码GldK蛋白,通过蛋白质印迹分析鉴定该蛋白位于细胞膜组分。液相色谱-串联质谱分析显示,在Yb2ΔgldK培养物中不存在具有典型T9SS CTD的四种蛋白质。进一步的测定显示突变菌株Yb2ΔgldK在蛋白质消化中是有缺陷的,支持了这些聚合物消化所涉及的酶由T9SS分泌的观点。互补菌株cYb2ΔgldK恢复蛋白水解活性。突变菌株Yb2ΔgldK在琼脂平板上表现出有缺陷的滑动运动,蛋白酶活性和蛋白质分泌能力降低。转录组测序和qPCR分析表明,突变株Yb2ΔgldK中有13个基因的下调表达。动物实验表明,与野生型Yb2感染的鸭相比,Yb2ΔgldK感染鸭血液中的载菌量显著降低。互补菌株cYb2ΔgldK恢复了大部分缺陷的生物学特性。综上所述,鸭疫里默氏杆菌AS8708785基因编码IX型分泌系统的关键组分GldK,其作用于细菌滑动运动,蛋白质分泌和细菌毒力。

论文目录

• 附表
• ABSTRACT
• 摘要
• List of abbreviations
• CHAPTER 1.Research progress on the Riemerella anatipestifer
•   1.1 INTRODUCTION
•     1.1.1 The Riemerella anatipestifer genus'taxonomy
•     1.1.2 Growth conditions and phenotypes of Riemerella anatipestifer
•     1.1.3 Molecular biology and fingerprints of Riemerella anatipestifer
•     1.1.4 Sequences analysis and virulence factors of Riemerella anatipestifer
•     1.1.5 Serological Features
•     1.1.6 Epidemiology of Riemerella anatipestifer
•     1.1.7 Pathogenesis,clinical evidence and disease of Riemerella antipestifer infection
•     1.1.8 Type IX secretion system overview
•     1.1.9 Type IX secretion system
•     1.1.10 Gliding motility of bacteriodetes
•   1.2 Literature Review
•   1.3 Goal of the project
•     1.3.1 Research objectives
•     1.3.2 Specific purpose of the project
• 08785 gene encodes a functional component GldK of the type IX secretion system'>CHAPTER 2 Riemerella anatipestifer AS87₀8785 gene encodes a functional component GldK of the type IX secretion system
•   2.1 Introduction
•   2.2 Materials and methods
•     2.2.1 Bacterial strains,plasmids and culture conditions
•     2.2.2 Medium
•     2.2.3 Buffer
•     2.2.4 Equipment
•     2.2.5 Consumables
•     2.2.6 Chemical and reagents
•     2.2.7 Enzymes
•     2.2.8 Cloning kit
•     2.2.9 Isolation of bacterial genomic DNA
•     2.2.10 Concentration and quality of DNA determination
•     2.2.11 Identification of the mutant strain Yb2ΔgldK
•     2.2.12 Complementation of the Yb2ΔgldK mutant strain
•     2.2.13 Bacterial growth,adherence and invasion assays
•     2.2.14 GldK expression antibody production and western blotting analysis
•     2.2.15 Subcellular fraction
•     2.2.16 Western blot
•     2.2.17 Microscopic observation of colony spreading
•     2.2.18 Measurement of protease activity
•     2.2.19 Liquid chromatography-tandem mass spectrometry（LC-MS/MS）analysis of the protein secretion
•     2.2.20 RNA-sequencing and differential expression analysis
•     2.2.21 Extraction of Bacterial Total RNA
•     2.2.22 Real-time quantitative PCR（qPCR）analysis
•     2.2.23 Assessment of virulence in vivo
•     2.2.24 Statistical analysis
•   2.3 RESULTS
•     2.3.1 Bioinformatics analysis of the R.anatipestifer gldK gene
•     2.3.2 Expression of R.anatipestifer recombinant GldK
•     2.3.3 Identification of the mutant strain Yb2ΔgldK
•     2.3.4 Deletion of the gldK gene decreases bacterial adherence and invasion capacities
•     2.3.5 Subcellular location of the GldK
•     2.3.6 The gldK deletion mutant is defective in gliding motility
•     2.3.7 Deletion of the gldK gene significantly attenuates bacterial virulence
•     2.3.8 The gldK deletion mutant exhibits defects in protein secretion
•     2.3.9 The gldK disruption affects gene expression
•   2.4 DISCUSSION AND CONCLUSION
• REFERENCES
• ACKNOWLEDGEMENTS
• CURRICULUM VITAE

文章来源

类型: 博士论文

作者: Kanwar Kumar

导师: 于圣青

关键词: 鸭疫里默氏杆菌,细菌滑行运动,型分泌系统,细菌毒力

来源: 中国农业科学院

年度: 2019

分类: 基础科学,农业科技

专业: 生物学,畜牧与动物医学

单位: 中国农业科学院

基金: National Key R&D Program (2016YFD0500805),the Shanghai Key Project on Agricultural Development through Science and Technology (2016HNG4-1),Co-innovation of Science and Technology Innovation Project in Chinese Academy of Agricultural Sciences (CAAS-XTCX2016011-04-8)

分类号: S852.61

总页数: 81

文件大小: 2880K

下载量: 78