【作者】 陈泽良；

【导师】 杨瑞馥；

【作者基本信息】 中国人民解放军军事医学科学院 ， 军事预防医学， 2005， 博士

【摘要】 背景:鼠疫是由鼠疫耶尔森氏菌(简称鼠疫菌)引起的自然疫源性烈性传染病。鼠疫菌独特的致病能力,一直是鼠疫研究工作者关注的焦点。密度感应系统(quorum sensing, QS)是一种细胞密度依赖性的细菌胞间信号传递系统,通过这种机制细菌可以协调基因的表达,从而使细菌可以适应环境、产生毒力因子等,使细胞的活动具有组织性,成为类似于多细胞生物的群体性活动。本文报道了鼠疫菌密度感应系统在鼠疫菌毒力中的作用。 方法:结合基因突变技术、高效液相分离、质潜鉴定和细胞凋亡分析实验,从鼠疫菌野生株和QS突变株分离鉴定QS系统的信号分子及其对巨噬细胞的作用;利用全基因组芯片,比较分析QS突变株与野生株转录谱差异,鉴定QS系统调控元;利用鼠疫菌毒力相关蛋白的蛋白芯片,分析QS系统在体内对毒力相关蛋白表达的影响;通过一系列表型实验,比较QS突变株在体外、细胞和动物毒力表型的差异。 结果:从鼠疫菌的培养上清中,分离鉴定了两种信号分子,C6-HSL和C8-HSL,并证实了两种信号分子的合成基因。巨噬细胞毒性实验证实,这两种信号分子对小鼠巨噬细胞均具有促凋亡作用。QS系统调控了大量与细菌的基础生理功能和毒力相关的基因,并在体内影响了大量毒力蛋白的表达。细胞和动物实验结果显示,QS突变株在巨噬细胞内的生存能力下降,对小鼠的毒力减弱。 结论:鼠疫菌QS系统合成了两个信号分子,C6-HSL和C8-HSL。信号分子直接作为毒力因子发挥作用。QS系统调控大量基因的表达,包括一些与鼠疫菌基本生理功能和鼠疫菌毒力相关的基因,QS突变株中这些基因的表达变化,导致各毒力表型改变,动物毒力减弱。更多还原

【Abstract】 Backgroud: Yersinia pestis (Y. pestis)is the etiologic agent of bubonic and pneumonic plague, which has claimed millions of lives in human history. The unique pathogenicity is the focus of the recent researches on this pathogen. Quorum sensing is a cell-density dependent global regulation system, which is used for cell to cell communication mediated by the signal molecule, N-acyl-homoserine lactone (AHL). Here, we report the virulence role of quorum sensing system in the pathogenicity of Y. pestis. Methods: By combining the gene mutation technique, HPLC, MS and macrophage apoptosis assays, we isolated and identified the signal molecules of Y. pestis and tested their virulence to macrophage. Using the DNA microarray containing all ORFs of Y. pestis, we compared the transcriptome of the QS mutant to that of its parent strain. With the protein microarray containing the virulence-associated proteins from Y. pestis, the antibody profile of the QS mutant was compared with its parent strain. And then the phenotype differences were analyzed between the QS mutant and its parent. Results: From the supernatant of the wild type strain, two QS signal molecules were identified, C6-HSL and C8-HSL. Both molecules are virulent to macrophage J774A.1. Quorum sensing system controlled the transcription of a large number of genes, including those with basic cellular functions and those encoding proven virulence determinants. The results from antibody profiling showed that many antibodies to the virulence proteins were not detected in the serum from the QS mutant, suggesting that QS influence the expression of these proteins in vivo. The QS mutant is less virulent to cells and mice, as showed by the survival rate in macrophage and LD50 to mice. Conclusion: The quorum sensing system produce two signal molecules, C6-HSL and C8-HSL, which could act as virulence determinants directly. The quorum sensing system controls directly or indirectly a large number of virulence genes, which contribute to the pathogenicity of Y. pestis.更多还原