tRNA修饰酶GidA调控猪链球菌的生长和致病性的机制研究

发布时间：2018-06-25 01:11

本文选题：猪链球菌 + GidA蛋白　；参考：《华中农业大学》2016年博士论文

【摘要】：猪链球菌是一种人兽共患传染病的病原菌,不仅对养猪业造成重大的经济损失,而且威胁人类健康。根据荚膜多糖的抗原性,猪链球菌被分为33种血清型,其中猪链球菌2型(SS2)被认为是毒力最强的优势菌株。1998年和2005年,在中国爆发了两次由SS2感染引起的疫情,总共导致52人死亡。因此,探究SS2的致病机理进而研发有效的防控手段是当务之急。猪链球菌在感染宿主时,会遭受各种环境压力,为适应环境的改变,菌体的特异性基因表达也会发生相应改变,这些调控机制的研究对了解猪链球菌的致病机理具有重要意义。GidA是一种tRNA转录后修饰酶,与MnmE共同参与将羧基甲基氨甲基基团添加到tRNA第34位摇摆碱基尿嘧啶上。tRNA修饰酶对于蛋白质翻译的准确性以及高效性都至关重要。在大肠杆菌中,GidA参与调控细胞分裂;在变异链球菌中,GidA参与对环境压力的适应性调节;在嗜水气单胞菌中,GidA参与调控毒力蛋白的表达;在沙门氏菌中,GidA不仅参与调控细胞分裂还影响毒力蛋白的表达;而在丁香假单胞菌中,GidA是一个全局性的调控因子,调控丁香霉素等抗生素的产量、群游现象和毒力等。本研究对SS2的GidA的结构以及在SS2致病性中的作用进行了研究,并采用iTRAQ标记的定量蛋白组学方法鉴定了SS2中被GidA调控的蛋白质,揭示了GidA发挥各生物学功能的分子机制。取得的主要研究结果如下:本研究构建了gidA基因缺失突变菌株ΔgidA,对其表型进行了研究。结果发现,gidA基因缺失后,SS2细胞的生长速度减慢,菌体的荚膜厚度增加,对小鼠的致病性显著降低。其致病性降低主要表现为溶血能力减弱、抗小鼠巨噬细胞吞噬能力减弱、对HEp-2上皮细胞黏附与侵入能力减弱、对小鼠的致死性减弱以及在宿主体内生存能力减弱。为进一步解释上述表型产生的分子机制,运用iTRAQ标记的定量蛋白组学方法比较了ΔgidA及其亲本菌株的蛋白质表达谱,共鉴定出372个差异表达蛋白,其中182个蛋白表达上调,190个蛋白表达下调。上述差异表达蛋白占全菌蛋白数量的17.02%,并且这些差异蛋白分布广泛,涉及染色体复制、重组、修复、细胞分裂、信号转导、氨基酸代谢、碱基代谢等、细菌形态、病原性等。与DNA复制、重组和修复相关的蛋白质RnmV、GyrA、Gyr B、PcrA、XerS、XerD、RecN、RmuC、RnhB,、RNase H、RNase H、RNase E和Tag全都下调表达;与细胞分裂相关的蛋白质DivIVA、FtsQ、FtsX、Fts I、GpsB、StpK、PhpP、Cps2C和MurD,除GpsB(负调控因子)和FtsX上调表达外,其它均下调表达。与荚膜合成相关的蛋白质Cps2F、Cps2P、Cps2Q、Cps2R和Cps2S上调表达,Cps2C(负调控因子)下调表达。与毒力相关的蛋白质Sly、Enolase、GAPDH、ADS(ArcABC)、DltA、GlnA、GtfA、IMPDH、PurA和SadP全都下调表达。蛋白组学结果充分解释了突变菌株表型产生的分子机制。需要指出的是,本研究虽没有获得gidA基因缺失后的互补菌株,但RT-PCR实验在RNA水平排除了敲除gidA基因可能产生的极性效应。为了从结构角度解析GidA功能的机制,本研究对GidA蛋白的结构进行了初步研究,发现GidA晶体生长出来呈细棒状,两尖端稍有分岔,但衍射后分辨率过低,结晶条件还需继续优化。上述研究揭示了GidA在SS2生长和致病性中的调控功能,为深入理解猪链球菌的生长调控机制和致病机理提供了科学依据。
[Abstract]:Streptococcus suis is a pathogen of zoonosis, which not only causes serious economic loss to the pig industry, but also threatens human health. According to the antigenicity of capsule polysaccharide, Streptococcus suis is divided into 33 serotypes, of which Streptococcus suis type 2 (SS2) is considered to be the most virulent strain in.1998 and 2005, and two in China. An epidemic caused by SS2 infection results in a total of 52 deaths. Therefore, it is urgent to explore the pathogenesis of SS2 and to develop effective prevention and control methods. It is important to understand the pathogenesis of Streptococcus suis..GidA is a tRNA posttranscriptional modifier. It is essential to add the carboxyl methyl ammethylamino group to the tRNA thirty-fourth base uracil by adding the carboxyl methyl ammethylamino group to the.TRNA modifier for the accuracy and efficiency of the protein translation. In Escherichia coli, GidA participates in the modulation. Control cell division; in Streptococcus mutans, GidA participates in adaptive regulation of environmental pressure; in Aeromonas hydrophila, GidA participates in the regulation of the expression of virulence proteins; in Salmonella, GidA not only participates in the regulation of cell division but also affects the expression of virulence protein, but in Pseudomonas Syringa, GidA is a global regulator. The production of antibiotics such as syringomycin, the phenomenon of group travel and virulence. This study studied the structure of GidA and the role of SS2 in the pathogenicity of SS2. The quantitative proteomics method of iTRAQ markers was used to identify the protein regulated by GidA in SS2, and the molecular mechanism of GidA to play various biological functions was revealed. The results are as follows: in this study, a mutant strain of gidA gene, Delta gidA, was constructed, and its phenotype was studied. The results showed that after the deletion of gidA gene, the growth rate of SS2 cells was slow, the capsule thickness of the mycelium increased and the pathogenicity of the mice decreased significantly. The main manifestation of the lower pathogenicity was the weakening of hemolysis and anti mouse macrophage. The ability of phagocytosis weakened, the adhesion and invasion ability of HEp-2 epithelial cells weakened, the lethality of the mice and the survival ability of the host weakened in the host. In order to further explain the molecular mechanism of the above phenotypes, the protein expression profiles of delta gidA and their parent strains were compared with the quantitative proteomics method of iTRAQ markers, and 37 of them were identified. 2 differentially expressed proteins, of which 182 proteins are up-regulated, and 190 proteins are down regulated. The above differential proteins account for 17.02% of the total number of proteins, and these differential proteins are widely distributed, involving chromosome replication, recombination, repair, cell division, signal transduction, amino acid metabolism, base metabolism, bacteria morphology, pathogen and so on. And DNA RnmV, GyrA, Gyr B, PcrA, XerS, XerD, RecN, RmuC, RnhB, RNase H. Cps2F, Cps2P, Cps2Q, Cps2R and Cps2S are up-regulated, and Cps2C (negative regulatory factors) down regulated. The proteins Sly, Enolase, GAPDH, ADS (ArcABC) are all down regulated by the virulence related proteins. This study did not obtain the complementary strain after the deletion of the gidA gene, but the RT-PCR experiment eliminated the possible polar effect of the knockout gidA gene at the RNA level. In order to analyze the mechanism of the GidA function from the structural angle, the structure of the GidA protein was preliminarily studied in this study. The growth of the GidA crystal showed a fine bar, and the two tip was slightly However, the resolution is too low and the crystallization conditions need to be optimized. The above studies reveal the regulatory function of GidA in the growth and pathogenicity of SS2, and provide a scientific basis for understanding the growth regulation mechanism and pathogenesis mechanism of Streptococcus suis.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28

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