福氏2a痢疾杆菌htpG基因功能的研究
发布时间：2018-11-28 08:20
【摘要】： 志贺氏菌属(Shigella spp.)细菌是一类不形成芽孢的革兰氏阴性致病菌,能够通过侵袭大肠引起患者典型菌痢症状(发热、腹痛、腹泻)。根据生化反应和O-抗原结构的不同,志贺氏菌可以分为四个群。其中,福氏志贺氏菌(S. flexneri)是发展中国家卫生条件较差地区痢疾流行的主要病原菌,感染者多为5岁以下儿童。近三年来我国每年大约有2000万人次感染痢疾,年累计发病数一直位于第3位,仅次于肺结核和乙肝。由于志贺氏菌感染剂量极低(10-100个细菌),加之多重耐药菌株的不断出现,常规的抗生素治疗方式遇到了巨大的挑战。由于人们对痢疾杆菌的致病机理和宿主的免疫保护机制还不十分清楚,所以迄今为止仍未研究出能有效控制痢疾的理想疫苗。 志贺氏菌的HtpG蛋白是真核生物中Hsp90蛋白家族的同源蛋白。研究表明,细菌中的HtpG蛋白在热休克和酸休克时被诱导;在缓和的热休克条件下,HtpG参与新合成蛋白的折叠;而在氮源饥饿时HtpG蛋白的表达被抑制。此外,Mason CA等人发现一个有趣的现象,大肠杆菌HtpG蛋白的表达受到生长环境的影响:在LB复合培养基中培养时,可以观察到HtpG蛋白在菌体热休克时被诱导表达;而在含葡萄糖的基础培养基中培养时,HtpG蛋白的表达与温度无关。这种现象暗示HtpG蛋白的表达除受温度调控外,还受到其他信号分子的调控;其功能不仅仅与热休克相关,还可能具有未知的功能。 为了探索福氏2a志贺氏菌2457T中htpG基因的功能,本研究将由Datsenko和Wanner建立的λ噬菌体Red重组系统稍加改进,成功地敲除了福氏2a志贺氏菌2457T株的htpG基因,并利用低拷贝质粒构建了回复体加以验证,对HtpG进行了初步的功能研究。对野生株、突变株和回复株的生长曲线、生化反应、豚鼠角膜试验进行了比较分析,结果表明,野生株、突变株和回复株的生长曲线基本一致;突变株与野生株的生化反应没有明显差异;野生株、突变株与回复株均能够引起豚鼠角膜强烈炎症反应。这些结果表明HtpG蛋白功能与福氏志贺氏菌的基本生化代谢无关,也不影响侵袭蛋白的毒力。同时考察了野生株、突变株和回复株腹腔注射引起小鼠炎症反应的强弱,结果表明,HtpG蛋白能够引起小鼠强烈的炎症反应,可能与细菌的免疫致病性相关。 利用双向电泳和质谱技术对野生株、突变株和回复株进行了全菌体蛋白的比较蛋白组学研究。在蛋白质组的比较分析中我们发现,htpG基因敲除后表达上调的蛋白为16个,表达下调的有13个。结果表明HptG作为热休克蛋白家族的一员,参与了多种体内代谢活动,尤其是氧化防御机制和嘌呤合成途径。缺失HtpG后,双功能过氧化物酶KatG表达上调,而超氧化物歧化酶SodA表达下调;嘌呤合成相关蛋白PurH、PurB、PurC的表达减少。这些结果加深了我们对痢疾杆菌中HtpG蛋白的认识,有助于痢疾杆菌致病机理的研究。
[Abstract]:Shigella (Shigella spp.) Bacteria are a class of Gram-negative bacteria that do not form spores and can cause typical bacterial dysentery (fever, abdominal pain, diarrhea) by invading the large intestine. Shigella can be divided into four groups according to biochemical reaction and O-antigen structure. Among them, Shigella flexneri (S. flexneri) is the main pathogen of dysentery in developing countries, most of which are children under 5 years old. In recent three years, there are about 20 million people infected with dysentery every year in our country, and the cumulative incidence of dysentery is always in the third place, second only to tuberculosis and hepatitis B. Due to the extremely low infection dose of Shigella (10-100 bacteria) and the continuous emergence of multidrug resistant strains, the routine antibiotic therapy has met great challenges. Since the pathogenesis of Shigella dysenteriae and the immune protection mechanism of the host have not been fully understood, so far no ideal vaccine has been developed to effectively control dysentery. The HtpG protein of Shigella is the homologous protein of Hsp90 protein family in eukaryotes. The results showed that HtpG protein in bacteria was induced during heat shock and acid shock, HtpG was involved in the folding of newly synthesized protein under mild heat shock, and the expression of HtpG protein was inhibited during nitrogen starvation. In addition, Mason CA et al. found an interesting phenomenon that the expression of HtpG protein in Escherichia coli was affected by the growth environment: when cultured in LB compound medium, it was observed that the expression of HtpG protein was induced by heat shock. However, the expression of HtpG protein was not related to temperature when cultured in basic medium containing glucose. This phenomenon implies that the expression of HtpG protein is not only regulated by temperature, but also by other signaling molecules, and its function is not only related to heat shock, but also has unknown function. In order to explore the function of htpG gene in Shigella flexneri 2457T, the 位 phage Red recombinant system established by Datsenko and Wanner was improved to successfully knockout the htpG gene of Shigella flexneri 2457T strain. The low copy plasmid was used to construct the reverberant to verify the function of HtpG. The growth curve, biochemical reaction and cornea test of wild strain, mutant and recovery strain were compared and analyzed. The results showed that the growth curve of wild strain, mutant plant and recovery strain was basically the same. There was no significant difference in biochemical reaction between the mutant strain and the wild strain, mutant strain and recovery strain could cause intense inflammation in the cornea of guinea pigs. These results indicated that the HtpG protein function had no relationship with the basic biochemical metabolism of Shigella flexneri and did not affect the virulence of the invading protein. At the same time, the intensity of inflammatory reaction induced by intraperitoneal injection of wild strain, mutant strain and recovery strain was investigated. The results showed that HtpG protein could induce a strong inflammatory reaction in mice, which might be related to the immunogenicity of bacteria. The comparative proteomics of whole cell proteins of wild, mutant and recovery strains was studied by two-dimensional electrophoresis and mass spectrometry. In the comparative analysis of proteome we found that 16 proteins were up-regulated after htpG knockout and 13 were down-regulated. The results showed that HptG, as a member of the heat shock protein family, was involved in many metabolic activities in vivo, especially the oxidative defense mechanism and purine synthesis pathway. After deletion of HtpG, the expression of bifunctional peroxidase KatG was up-regulated, while the expression of superoxide dismutase (SOD) SodA was down-regulated, while the expression of purine synthesis-associated protein PurH,PurB,PurC was decreased. These results deepen our understanding of the HtpG protein in Shigella and contribute to the study of the pathogenic mechanism of Shigella dysenteriae.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378
本文编号：2362329
[Abstract]:Shigella (Shigella spp.) Bacteria are a class of Gram-negative bacteria that do not form spores and can cause typical bacterial dysentery (fever, abdominal pain, diarrhea) by invading the large intestine. Shigella can be divided into four groups according to biochemical reaction and O-antigen structure. Among them, Shigella flexneri (S. flexneri) is the main pathogen of dysentery in developing countries, most of which are children under 5 years old. In recent three years, there are about 20 million people infected with dysentery every year in our country, and the cumulative incidence of dysentery is always in the third place, second only to tuberculosis and hepatitis B. Due to the extremely low infection dose of Shigella (10-100 bacteria) and the continuous emergence of multidrug resistant strains, the routine antibiotic therapy has met great challenges. Since the pathogenesis of Shigella dysenteriae and the immune protection mechanism of the host have not been fully understood, so far no ideal vaccine has been developed to effectively control dysentery. The HtpG protein of Shigella is the homologous protein of Hsp90 protein family in eukaryotes. The results showed that HtpG protein in bacteria was induced during heat shock and acid shock, HtpG was involved in the folding of newly synthesized protein under mild heat shock, and the expression of HtpG protein was inhibited during nitrogen starvation. In addition, Mason CA et al. found an interesting phenomenon that the expression of HtpG protein in Escherichia coli was affected by the growth environment: when cultured in LB compound medium, it was observed that the expression of HtpG protein was induced by heat shock. However, the expression of HtpG protein was not related to temperature when cultured in basic medium containing glucose. This phenomenon implies that the expression of HtpG protein is not only regulated by temperature, but also by other signaling molecules, and its function is not only related to heat shock, but also has unknown function. In order to explore the function of htpG gene in Shigella flexneri 2457T, the 位 phage Red recombinant system established by Datsenko and Wanner was improved to successfully knockout the htpG gene of Shigella flexneri 2457T strain. The low copy plasmid was used to construct the reverberant to verify the function of HtpG. The growth curve, biochemical reaction and cornea test of wild strain, mutant and recovery strain were compared and analyzed. The results showed that the growth curve of wild strain, mutant plant and recovery strain was basically the same. There was no significant difference in biochemical reaction between the mutant strain and the wild strain, mutant strain and recovery strain could cause intense inflammation in the cornea of guinea pigs. These results indicated that the HtpG protein function had no relationship with the basic biochemical metabolism of Shigella flexneri and did not affect the virulence of the invading protein. At the same time, the intensity of inflammatory reaction induced by intraperitoneal injection of wild strain, mutant strain and recovery strain was investigated. The results showed that HtpG protein could induce a strong inflammatory reaction in mice, which might be related to the immunogenicity of bacteria. The comparative proteomics of whole cell proteins of wild, mutant and recovery strains was studied by two-dimensional electrophoresis and mass spectrometry. In the comparative analysis of proteome we found that 16 proteins were up-regulated after htpG knockout and 13 were down-regulated. The results showed that HptG, as a member of the heat shock protein family, was involved in many metabolic activities in vivo, especially the oxidative defense mechanism and purine synthesis pathway. After deletion of HtpG, the expression of bifunctional peroxidase KatG was up-regulated, while the expression of superoxide dismutase (SOD) SodA was down-regulated, while the expression of purine synthesis-associated protein PurH,PurB,PurC was decreased. These results deepen our understanding of the HtpG protein in Shigella and contribute to the study of the pathogenic mechanism of Shigella dysenteriae.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378
【引证文献】
相关博士学位论文 前1条
1 王芳;志贺氏菌Ⅲ型分泌系统效应子蛋白IpaH4.5功能研究[D];中国人民解放军军事医学科学院;2010年
，本文编号：2362329
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