福氏2a痢疾桿菌htpG基因功能的研究
發(fā)布時間：2018-11-28 08:20
【摘要】： 志賀氏菌屬(Shigella spp.)細(xì)菌是一類不形成芽孢的革蘭氏陰性致病菌,能夠通過侵襲大腸引起患者典型菌痢癥狀(發(fā)熱、腹痛、腹瀉)。根據(jù)生化反應(yīng)和O-抗原結(jié)構(gòu)的不同,志賀氏菌可以分為四個群。其中,福氏志賀氏菌(S. flexneri)是發(fā)展中國家衛(wèi)生條件較差地區(qū)痢疾流行的主要病原菌,感染者多為5歲以下兒童。近三年來我國每年大約有2000萬人次感染痢疾,年累計發(fā)病數(shù)一直位于第3位,僅次于肺結(jié)核和乙肝。由于志賀氏菌感染劑量極低(10-100個細(xì)菌),加之多重耐藥菌株的不斷出現(xiàn),常規(guī)的抗生素治療方式遇到了巨大的挑戰(zhàn)。由于人們對痢疾桿菌的致病機理和宿主的免疫保護機制還不十分清楚,所以迄今為止仍未研究出能有效控制痢疾的理想疫苗。 志賀氏菌的HtpG蛋白是真核生物中Hsp90蛋白家族的同源蛋白。研究表明,細(xì)菌中的HtpG蛋白在熱休克和酸休克時被誘導(dǎo);在緩和的熱休克條件下,HtpG參與新合成蛋白的折疊;而在氮源饑餓時HtpG蛋白的表達被抑制。此外,Mason CA等人發(fā)現(xiàn)一個有趣的現(xiàn)象,大腸桿菌HtpG蛋白的表達受到生長環(huán)境的影響:在LB復(fù)合培養(yǎng)基中培養(yǎng)時,可以觀察到HtpG蛋白在菌體熱休克時被誘導(dǎo)表達;而在含葡萄糖的基礎(chǔ)培養(yǎng)基中培養(yǎng)時,HtpG蛋白的表達與溫度無關(guān)。這種現(xiàn)象暗示HtpG蛋白的表達除受溫度調(diào)控外,還受到其他信號分子的調(diào)控;其功能不僅僅與熱休克相關(guān),還可能具有未知的功能。 為了探索福氏2a志賀氏菌2457T中htpG基因的功能,本研究將由Datsenko和Wanner建立的λ噬菌體Red重組系統(tǒng)稍加改進,成功地敲除了福氏2a志賀氏菌2457T株的htpG基因,并利用低拷貝質(zhì)粒構(gòu)建了回復(fù)體加以驗證,對HtpG進行了初步的功能研究。對野生株、突變株和回復(fù)株的生長曲線、生化反應(yīng)、豚鼠角膜試驗進行了比較分析,結(jié)果表明,野生株、突變株和回復(fù)株的生長曲線基本一致;突變株與野生株的生化反應(yīng)沒有明顯差異;野生株、突變株與回復(fù)株均能夠引起豚鼠角膜強烈炎癥反應(yīng)。這些結(jié)果表明HtpG蛋白功能與福氏志賀氏菌的基本生化代謝無關(guān),也不影響侵襲蛋白的毒力。同時考察了野生株、突變株和回復(fù)株腹腔注射引起小鼠炎癥反應(yīng)的強弱,結(jié)果表明,HtpG蛋白能夠引起小鼠強烈的炎癥反應(yīng),可能與細(xì)菌的免疫致病性相關(guān)。 利用雙向電泳和質(zhì)譜技術(shù)對野生株、突變株和回復(fù)株進行了全菌體蛋白的比較蛋白組學(xué)研究。在蛋白質(zhì)組的比較分析中我們發(fā)現(xiàn),htpG基因敲除后表達上調(diào)的蛋白為16個,表達下調(diào)的有13個。結(jié)果表明HptG作為熱休克蛋白家族的一員,參與了多種體內(nèi)代謝活動,尤其是氧化防御機制和嘌呤合成途徑。缺失HtpG后,雙功能過氧化物酶KatG表達上調(diào),而超氧化物歧化酶SodA表達下調(diào);嘌呤合成相關(guān)蛋白PurH、PurB、PurC的表達減少。這些結(jié)果加深了我們對痢疾桿菌中HtpG蛋白的認(rèn)識,有助于痢疾桿菌致病機理的研究。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R378
【引證文獻】
相關(guān)博士學(xué)位論文 前1條
1 王芳;志賀氏菌Ⅲ型分泌系統(tǒng)效應(yīng)子蛋白IpaH4.5功能研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年
，本文編號：2362329
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