鼠疫耶尔森氏菌YscW对小鼠巨噬细胞生物效应的研究

发布时间：2018-08-27 16:33

【摘要】： 鼠疫是由鼠疫耶尔森氏菌(以下简称鼠疫菌)引起的一种烈性传染病,其流行给人类带来了巨大的灾难,因此对鼠疫菌致病机制的研究是做好鼠疫防控的重要切入点。鼠疫菌感染宿主引发致病是个复杂的多步骤过程。鼠疫菌在体内致病的关键环节是在巨噬细胞内的生存繁殖,该菌与巨噬细胞相互作用不仅不会被杀死,反而能够生存,并合成大量毒力因子,最终巨噬细胞破裂,释放出的鼠疫菌具备强大的抗吞噬能力,并为分泌毒力因子做好准备。因此,在感染早期,鼠疫菌在宿主巨噬细胞的生存与繁殖是鼠疫菌致病的一个重要环节。鼠疫菌的毒力之一是pCD1质粒编码的Ⅲ型分泌系统(type III secretion system, T3SS),通过六个效应蛋白输入真核细胞从而打断宿主的防御机制。本研究以鼠疫菌T3SS注射小体结构蛋白YscW为研究对象,分析yscW基因缺失后对鼠疫菌毒力的影响;探究yscW突变株与互补株巨噬细胞效应;研究YscW蛋白是否会直接影响巨噬细胞生存及免疫调节功能;探讨YscW蛋白功能的可能机制。 首先利用自杀质粒方法构建yscW突变株,在此基础上导入与鼠疫菌拷贝数接近的pACYC184-YscW的重组质粒构建互补株。LD50实验观察yscW对鼠疫菌致病性的影响,结果表明鼠疫菌缺失yscW基因,其致病性明显减弱;同时体外感染巨噬细胞,显微镜观察表明YscW影响巨噬细胞形态。 进而构建小鼠腹腔感染模型,分析鼠疫菌野生株、yscW突变株与互补株对巨噬细胞生存的影响,体、内外实验证实yscW突变株明显降低巨噬细胞凋亡,而互补株恢复了突变株导致的凋亡改变。通过对凋亡的进一步分析,我们发现不同菌株间导致细胞凋亡的差异主要来源于细胞早期凋亡的改变,而对于晚期凋亡和细胞坏死无差异。此外,细胞周期的分析显示,三株菌无显著差异。同时,凋亡诱导关键蛋白Caspase-3的表达检测结果与三株菌感染后细胞水平凋亡变化相一致,从分子水平证实凋亡差异。然而过表达YscW至RAW264.7细胞后,实验组与对照组细胞凋亡与细胞周期间无显著差异,表明YscW并不是诱导细胞凋亡的直接效应蛋白。YopJ是鼠疫菌Ⅲ型分泌系统六个效应蛋白中唯一与细胞凋亡相关的蛋白,为了研究凋亡差异的机制,对三株菌YopJ分泌进行检测,结果表明yscW突变导致YopJ分泌减少。因此,鼠疫菌yscW突变株YopJ分泌减少是导致该突变株诱导细胞凋亡减少的机制之一。 对于不同鼠疫菌株感染后巨噬细胞的功能改变进行深入研究,结果显示鼠疫菌YscW影响了巨噬细胞的免疫调节功能。吞噬功能、抗原提呈能力和细胞因子分泌是巨噬细胞的三大主要功能。以鸡红细胞作为靶细胞,分析不同菌株感染后巨噬细胞吞噬功能的变化,结果显示无论野生株、突变株还是互补株,感染后巨噬细胞的吞噬功能明显低于正常巨噬细胞,表明鼠疫菌感染后,宿主巨噬细胞吞噬功能降低;而感染鼠疫菌yscW突变株的细胞,与野生株和互补株感染比较,其吞噬功能又明显增强,说明yscW基因直接或间接的参与了病原菌调节宿主细胞的吞噬反应。进而过表达YscW的细胞,其吞噬功能明显低于对照组细胞,更直接的给出了YscW直接调节巨噬细胞吞噬功能的证据。巨噬细胞对OVA(卵白蛋白)抗原小肽的提呈能力通过混合淋巴细胞培养,T细胞增殖程度加以判定。结果显示突变株感染后的巨噬细胞抗原提呈能力明显高于同样条件下野生株感染的细胞,而过表达YscW的细胞其抗原提呈能力又显著低于对照组细胞,这些结果表明YscW直接影响了宿主巨噬细胞的抗原提呈能力。此外,感染后巨噬细胞及过表达细胞表面分子的检测,进一步证实YscW对细胞抗原提呈能力的影响。巨噬细胞体外感染鼠疫菌后,细胞分泌的TNF-α水平明显高于未受到细菌刺激的正常细胞;yscW突变株感染后巨噬细胞TNF-α分泌显著高于其它两株菌,表明yscW基因缺失巨噬细胞促炎症因子释放增多,从影响细胞因子分泌的角度研究了YscW的作用。上述研究结果首次表明,鼠疫菌Ⅲ型分泌系统的结构蛋白YscW直接参与到宿主巨噬细胞免疫调节反应中,干扰了细胞正常的生理机能。 本实验室前期工作发现鼠疫菌YscW蛋白可能与巨噬细胞Rnf149蛋白存在相互作用,这就提示上述观察到的YscW巨噬细胞生物学效应是否与YscW和Rnf149的相互作用相关联。因此对两种蛋白相互作用的验证成为后续实验的基础。对于上述两种蛋白,利用分子克隆技术分别与多个表达载体相连,并通过镍柱和GS-4B柱进行蛋白的纯化,最终获得带有GST和His标签的两种蛋白。进而GST-Pull Down实验体外验证了YscW与巨噬细胞靶蛋白Rnf149结合,为进一步的体内验证奠定基础。 本研究从整体、细胞、分子三水平上,确定鼠疫菌yscW突变株及互补株对原代小鼠巨噬细胞及RAW264.7细胞系生物学功能的影响,并对YscW蛋白本身的巨噬细胞免疫应答状态做出综合评价,特别是感染后或过表达后巨噬细胞的吞噬能力以及抗原提呈能力进行系统研究。在前期实验筛选到鼠疫菌YscW与巨噬细胞Rnf149可能存在相互作用的基础上,进一步验证两者相互作用,以期探究鼠疫菌III型分泌系统结构蛋白YscW的致病机制,对鼠疫菌结构蛋白的新功能研究具有重要的意义。
[Abstract]:Yersinia pestis is a severe infectious disease caused by Yersinia pestis. The epidemic of Yersinia pestis has brought great disaster to human beings. Therefore, the study on the pathogenic mechanism of Yersinia pestis is an important breakthrough point for the prevention and control of plague. The key link is to survive and reproduce in macrophages. The bacterium interacts with the macrophages not only to survive, but also to synthesize a large number of virulence factors. Eventually the macrophages break down, releasing Yersinia pestis with strong anti-phagocytosis ability, and to prepare for the secretion of virulence factors. One of the virulence of Yersinia pestis is the type III secretion system (T3SS) encoded by pCD1 plasmid, which is transfected into eukaryotic cells via six effector proteins to interrupt the host defense mechanism. In this study, the structure of mice injected with Yersinia pestis T3SS was studied. YscW protein was used as the research object to analyze the effect of yscW gene deletion on the virulence of Yersinia pestis; to explore the macrophage effect of yscW mutant and complementary strains; to study whether YscW protein directly affects the survival and immune regulation of macrophages; and to explore the possible mechanism of YscW protein function.
Firstly, yscW mutant was constructed by suicide plasmid method, and then a complementary strain was constructed by introducing the recombinant plasmid pACYC184-YscW which was close to the copy number of Y. pestis. The effect of yscW on the pathogenicity of Y. pestis was observed by LD50 assay. The results showed that Y. pestis lacked yscW gene and its pathogenicity was significantly weakened. Observation indicates that YscW affects macrophage morphology.
Then the mouse abdominal infection model was constructed to analyze the effect of wild Yersinia pestis strain, yscW mutant and complementary strain on the survival of macrophages. In vivo, in vitro and in vivo experiments showed that yscW mutant significantly reduced the apoptosis of macrophages, while complementary strain recovered the apoptotic changes caused by the mutant. In addition, cell cycle analysis showed that there was no significant difference among the three strains. Meanwhile, the expression of Caspase-3 was consistent with the changes of cell apoptosis at the cellular level after infection. However, after overexpression of YscW to RAW264.7 cells, there was no significant difference in cell apoptosis and cell cycle between the experimental group and the control group, suggesting that YscW was not a direct effector protein inducing cell apoptosis. YopJ was the only one of the six effector proteins in the secretory system of Yersinia pestis type III related to cell apoptosis. The results showed that YscW mutation resulted in the decrease of YopJ secretion. Therefore, the decrease of YopJ secretion of Y. pestis YscW mutant was one of the mechanisms leading to the decrease of YopJ secretion.
The function changes of macrophages infected by different strains of Y. pestis were studied. The results showed that Y. pestis YscW affected the immune regulation function of macrophages. Phagocytosis, antigen presenting ability and cytokine secretion were the three main functions of macrophages. The phagocytic function of macrophages in wild strains, mutants and complementary strains was significantly lower than that in normal macrophages, indicating that the phagocytic function of host macrophages decreased after infection by Yersinia pestis, and the phagocytic function of cells infected with Y. pestis yscW mutant was lower than that of wild strains and complementary strains. The phagocytic function of YscW gene was also significantly enhanced, indicating that yscW gene directly or indirectly participated in the phagocytic response of host cells regulated by pathogenic bacteria. The results showed that the antigen presenting ability of macrophages infected by the mutant strain was significantly higher than that of the cells infected by the wild strain under the same conditions, while the antigen presenting ability of the cells overexpressing YscW was significantly lower than that of the control group. These results indicated that YscW was direct. In addition, the detection of macrophages and over-expressed cell surface molecules after infection further confirmed the effect of YscW on cell antigen presenting ability. TNF-a secretion of macrophages after infection was significantly higher than that of the other two strains, suggesting that yscW gene-deleted macrophages increased the release of pro-inflammatory factors. The effect of YscW on the secretion of cytokines was studied from the point of view of influencing the secretion of cytokines. The regulation of reaction interferes with the normal physiological functions of cells.
Previous work in our laboratory has found that Y. pestis YscW protein may interact with macrophage Rnf149 protein, which indicates whether the biological effects of YscW macrophages observed above are related to the interaction between YscW and Rnf149. Therefore, the validation of the interaction between the two proteins is the basis for subsequent experiments. The protein was purified by nickel column and GS-4B column. The binding of YscW to macrophage target protein Rnf149 was confirmed by GST-Pull Down assay in vitro.
In this study, we determined the effects of Y. pestis yscW mutants and complementary strains on the biological functions of primary mouse macrophages and RAW264.7 cell lines from the overall, cellular and molecular levels, and made a comprehensive evaluation of the macrophage immune response status of YscW protein itself, especially the phagocytosis ability of macrophages after infection or overexpression. The antigen presenting ability of Y. pestis YscW was systematically studied. On the basis of the possible interaction between Y. pestis YscW and macrophage Rnf149, the interaction between Y. pestis YscW and macrophage Rnf149 was further verified in order to explore the pathogenic mechanism of Y. pestis type III secretory system structural protein YscW. Righteousness.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R378

【引证文献】

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1 江凌晓;江凌云;;S系统及时序布尔网络模型构建鼠疫耶尔森菌基因调控网络的初步研究[J];热带医学杂志;2011年09期

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