肺炎链球菌DnaJ对细菌毒力的影响及其在诱导宿主天然免疫反应中的作用

发布时间：2018-08-08 15:54

【摘要】：肺炎链球菌（Streptococcus pneumoniae，S.pn）是一种常见的革兰阳性条件致病菌，其引起的肺炎、脑膜炎和败血症等疾病在全球有很高的发病率和死亡率。近20年研究发现S.pn的许多蛋白，可作为炎症介质或直接损伤宿主组织，，且编码这些蛋白的基因突变后，细菌的毒力显著下降，因此S.pn的毒力蛋白在肺炎链球菌感染的发病机制中起重要作用。细菌由外界环境进入宿主后必然面临生存条件的改变，它们会应激性地调整基因表达以适应环境的选择压力，细菌的热休克蛋白（HeatShock Protein，HSP）正是应激诱导产生的一组对细菌本身具有保护作用的相关蛋白。从理论上讲，这些应激表达的HSP往往是细菌潜在的毒力因子。因此全面理解肺炎链球菌HSP对自身毒力的影响及其与宿主免疫细胞间的相互作用将有助于抗肺炎球菌感染的新型治疗试剂的开发。 DnaJ/Hsp40蛋白就是一种高度保守并广泛存在的热休克蛋白，作为DnaK的共分子伴侣，激活DnaK的ATPase活性。S.pn的DnaJ蛋白是一种表面蛋白，已有研究显示该蛋白具有疫苗的潜力，腹腔或粘膜接种使宿主产生抵抗多种血清型S.pn感染的获得性免疫反应。但是,DnaJ蛋白是否参与细菌致病以及DnaJ蛋白与宿主天然免疫细胞的相互作用尚不清楚。本研究分别从体内、体外两方面观察肺炎链球菌dnaJ缺陷对自身毒力的影响及DnaJ在诱导宿主天然免疫反应中的作用——促炎因子的分泌，模式识别受体（PRR）对DnaJ蛋白的识别，信号通路的激活。研究内容包括以下三个部分： 1、构建S.pn的dnaJ基因缺失的突变体，观察温度应激时其体外生长情况，为后续研究DnaJ在细菌和与宿主相互作用中所扮演的角色提供实验菌株和技术平台。采用长臂同源多聚酶链式反应（LFH-PCR）方法，制备中间为红霉素耐药基因（erm），两侧为dnaJ基因上、下游同源序列的连接片段，转化肺炎链球菌D39或R6菌，在含红霉素的血平板上筛选并采用PCR及测序鉴定缺陷菌株。结果显示缺陷菌株的dnaJ基因完全被erm基因替代；革兰染色后形态无变化，但其菌落明显变小；30℃和37℃时体外生长曲线与野生菌大体一致，但20℃和40℃时生长明显受抑制，几乎不能生长。 2、分别从体内、体外两方面观察肺炎链球菌dnaJ缺陷对自身毒力的影响。腹腔攻毒后直接观察感染野生菌或缺陷菌小鼠的生存情况；滴鼻感染S.pn建立小鼠肺炎模型，计数易感组织的细菌载量，明确dnaJ缺陷对细菌自身定植能力的影响；将S.pn感染小鼠肺上皮细胞MLE12，比较野生和缺陷菌株黏附及侵袭能力的差异。毒力实验显示dnaJ缺陷菌组的小鼠可耐受致死剂量的S.pn并长期存活；定植实验发现缺陷菌组小鼠鼻咽部和肺部的细菌载量均显著低于野生菌组，并迅速清除入血的缺陷菌；dnaJ缺陷使S.pn对MLE12细胞的黏附侵袭能力显著减弱，而且用抗DnaJ抗血清预处理MLE12后野生菌对该细胞的黏附能力明显下降。 3、明确S.pn dnaJ基因缺陷后宿主或免疫细胞对该菌天然免疫反应的变化，再从分子水平筛选宿主识别DnaJ诱导先天性免疫的模式识别受体和下游信号通路。建立小鼠S.pn的肺炎模型，取感染肺组织切片染色观察炎症反应，定量PCR和ELISA检测促炎因子的表达；小鼠巨噬细胞RAW264.7感染S.pn，比较该细胞对野生菌和缺陷菌的吞噬能力及炎症因子分泌水平；原核表达并纯化DnaJ全长蛋白刺激RAW264.7，检测促炎因子的水平，定量PCR筛选识别DnaJ蛋白的TLRs，并用抗TLRs单抗验证，蛋白激酶抑制剂筛选DnaJ蛋白诱导促炎因子的相关信号通路，Western blot进行验证。体内实验显示S.pn的dnaJ缺陷后其感染小鼠的肺部炎症反应强度下降、促炎因子分泌峰值降低且达峰时间延迟；体外实验发现dnaJ缺陷后RAW264.7对S.pn的吞噬增加，促炎因子分泌下降。DnaJ蛋白单独作用于RAW264.7后可活化PI3K、JNK信号通路，刺激促炎因子IL-6的分泌，此过程不依赖TLR2和TLR4。
[Abstract]:Streptococcus pneumoniae (S.pn) is a common gram-positive pathogenic bacteria, which causes pneumonia, meningitis and septicemia in the world with high morbidity and mortality. In recent 20 years, many proteins of S.pn have been found to be inflammatory medials or directly damage the host tissues and encode these proteins. The virulence of S.pn decreased significantly after gene mutation, so the virulence protein of S.pn played an important role in the pathogenesis of Streptococcus pneumoniae infection.
When bacteria enter the host environment, they inevitably face changes in survival conditions. They adjust gene expression to adapt to the selection pressure of the environment. The heat shock protein (HeatShock Protein, HSP) of bacteria is a group of stress induced proteins related to the bacteria itself. The expression of HSP is often a potential virulence factor of bacteria. Therefore, a comprehensive understanding of the effect of Streptococcus pneumoniae HSP on its own virulence and its interaction with the host immune cells will contribute to the development of a new therapeutic agent against pneumococcal infection.
DnaJ/Hsp40 protein is a highly conserved and widely existing heat shock protein. As a co molecular chaperone of DnaK, the DnaJ protein that activates DnaK's ATPase activity.S.pn is a surface protein. Studies have shown that the protein has the potential of the vaccine. Peritoneal or mucosal inoculation causes the host to produce acquired immunity against multiple serotype S.pn infections. However, it is not clear whether DnaJ proteins are involved in bacterial pathogenesis and the interaction between DnaJ proteins and host innate immune cells.
In this study, the effects of dnaJ defects on the virulence of Streptococcus pneumoniae and the role of DnaJ in inducing the natural immune response of the host - the secretion of pro-inflammatory factors, the recognition of the pattern recognition receptor (PRR) to the DnaJ protein and the activation of the signal pathway were observed in two aspects, including the following three parts:
1, the dnaJ gene deletion mutant of S.pn was constructed to observe the growth of temperature stress in vitro, providing experimental strains and technical platform for subsequent study of the role of DnaJ in the interaction of bacteria and host.
Using the long arm homologous polymerase chain reaction (LFH-PCR) method, the erythromycin resistance gene (ERM) was prepared in the middle, and both sides were dnaJ genes, the downstream homologous sequences were linked, the Streptococcus pneumoniae D39 or R6 bacteria were transformed, and the defective strains were screened on the erythromycin containing blood plate and used to identify the defective strains by PCR and sequencing. The results showed the dnaJ base of the defective strains. Because of the substitution of ERM gene completely, the morphology of Gram stain was not changed, but its colony was obviously smaller, and the growth curve in vitro was roughly the same as that of wild bacteria at 30 and 37 C, but the growth was obviously inhibited at 20 and 40 C, almost unable to grow.
2, the influence of dnaJ deficiency on the toxicity of pneumococcus pneumoniae was observed from two aspects in vivo and in vitro.
The survival of the mice infected with wild bacteria or defective bacteria was observed directly after the intraperitoneal attack. The nasal infection S.pn established the mice pneumonia model, counted the bacterial load of the susceptible tissue, and determined the effect of dnaJ defects on the self colonization ability of the bacteria. S.pn infected the lung epithelial cells of mice, and compared the adhesion and invasion ability of the wild and defective strains. Differences. The toxicity test showed that the mice in the dnaJ deficient bacteria group could tolerate the lethal dose of S.pn and the long-term survival. The bacterial load in the nasopharynx and lungs of the defective bacteria group was significantly lower than that of the wild bacteria group, and the defective bacteria in the blood were cleared quickly, and the dnaJ defect made the adhesion and invasion ability of MLE12 cells weakened significantly. After pretreatment with DnaJ antiserum, the adhesion ability of wild bacteria to MLE12 cells decreased significantly.
3, the changes in the natural immune response of the host or immune cells to the bacteria after the S.pn dnaJ gene defect were identified, and then the host identification and the downstream signaling pathway were screened at the molecular level by the host to identify the DnaJ induced innate immunity.
The pneumonia model of S.pn in mice was established. The inflammatory reaction was observed in the infected lung tissue section and the expression of proinflammatory factors was detected by quantitative PCR and ELISA. The macrophage macrophage RAW264.7 infected S.pn, compared the phagocytic ability of the cells to the wild and defective bacteria and the secretion level of the inflammatory factors, and the prokaryotic expression and purified DnaJ full length protein stimulated RAW264.. 7, detection of the level of proinflammatory factors, quantitative PCR screening and identification of DnaJ protein TLRs, and using anti TLRs monoclonal antibody test, protein kinase inhibitor screening DnaJ protein induced proinflammatory factor related signal pathway, Western blot to verify. In vivo experiment showed that S.pn dnaJ deficiency after the infection of mice lung inflammation reaction intensity decreased, proinflammatory factor score The peak time was reduced and the peak time was delayed. In vitro, it was found that the phagocytosis of RAW264.7 to S.pn increased after dnaJ defect, and the secretion of proinflammatory cytokines secreted.DnaJ protein to activate PI3K, JNK signaling pathway and stimulate the secretion of pro-inflammatory factor IL-6, which did not depend on TLR2 and TLR4..
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【相似文献】