【作者】 董浩；

【导师】 吴清民；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 布鲁氏菌病(Brucellosis)是一种由布鲁氏菌(Brucella spp)引起的严重的人兽共患病,给人类的健康和畜牧业的发展带来巨大的危害。转录调控因子MucR是布鲁氏菌的一个重要的毒力因子,但mucR基因缺失引起布鲁氏菌毒力降低的原因以及MucR蛋白调控的靶点基因尚未研究清楚。本文试图通过转录组测序分析及染色质免疫共沉淀探讨上述问题。为了确定转录组测序和染色质免疫共沉淀的最佳条件,通过lacZ报告基因和Western blot检测了MucR蛋白在不同生长阶段和应激环境中的转录和翻译水平,结果显示在生长迟缓期和对数期早期,MucR蛋白随着细菌密度的增加而增加,当细菌到达对数期后期,MucR蛋白的表达水平达到最高并且基本保持不变。这种细菌表达水平随细菌密度而变化的特点使我们怀疑MucR蛋白的表达可能与细菌的群体感应系统存在一定的联系。研究发现,在群体感应相关基因vjbR的缺失株中mucR基因的转录水平与亲本菌株16M无差异,说明MucR蛋白的表达不依赖于VjbR的调控。当布鲁氏菌处于不同的应激环境中,MucR蛋白会在pH4.5的条件下迅速减少。为了研究MucR蛋白水平降低是由于蛋白合成水平降低还是蛋白降解增强,采用氯霉素抑制细菌蛋白的合成后检测MucR蛋白降解的特性,结果表明MucR蛋白水平降低主要是由于在pH4.5的环境中蛋白降解作用增强引起的。根据上述结果,确定转录组测序和染色质免疫共沉淀的最佳细菌培养条件应该是将布鲁氏菌在正常的TSB培养基中培养至对数期后期。为了研究MucR蛋白的毒力相关机制,通过转录组测序技术检测了mucR基因缺失后引起布鲁氏菌转录谱的变化,结果显示442个基因的表达水平显著改变,其中310个基因表达水平上调,132个基因表达水平下调。通过COG聚类分析发现这些差异基因主要与布鲁氏菌的代谢途径、生物外膜合成途径、DNA转录翻译以及修复等生物过程密切相关。这些差异表达的基因中,一些重要的毒力因子,如群体感应系统、Ⅳ型分泌系统、反硝化途径、DNA修复途径、耐受缺铁环境、耐受酸性环境、生物膜合成相关基因的异常表达可能是mucR缺失株毒力降低的重要原因。环境应激实验的结果显示,mucR基因缺失后会影响细菌对酸性环境、缺铁环境、阳离子多肽的耐受能力,这说明nucR基因缺失后,可能无法适应宿主细胞内的上述应激环境而导致生存和增殖能力的降低。为了进一步研究MucR的调控靶点,通过染色质免疫共沉淀技术验证了77个差异表达的基因是否被MucR蛋白直接调控,最终筛选到14个MucR蛋白的直接结合位点,包括7个转录调控相关基因,2个细菌外膜合成相关基因,2个金属离子转运相关基因,1个抵御酸性环境相关基因,1个反硝化途径相关基因以及1个编码碱性磷酸酶的基因。上述结果解释了mucR基因缺失后布鲁氏菌对酸性环境和缺铁环境的敏感,细胞外膜特性的改变以及反硝化途径相关基因的差异表达是由于MucR蛋白调控靶点的异常表达引起的。本研究揭示了布鲁氏菌mucR基因缺失后毒力降低的原因和MucR蛋白的调控靶点,为布鲁氏菌的调控网络和胞内生存机制提供了重要的参考依据。更多还原

【Abstract】 Brucellosis is a serious zoonotic disease caused by Brucella spp,which threats human health and animal husbandry. Transcriptional regulator MucR is a virulence factor of Brucella spp., while the related mechanism of Brucella virulence of MucR and its target genes is still unknown. In this study, we used RNA-seq and chromatin Immunoprecipitation to solve these questions.To determine the optimum condition of RNA-seq and chromatin Immunoprecipitation, lacZ report gene assay and Western blot were used to detect the expression level of mucR gene at different growth stages and stress conditions. It showed that the transcriptional level and translational level of the mucR gene were increased at lag phase and early logarithmic phase, and achieved the highest level during the late logarithmic phase. This feature made us consider that the expression of MucR might be affected by quorum sensing system. Our date showed that the expression of MucR was almost the same in△vibR and16M, and it suggested that the expression of MucR was not affected by quorum sensing related protein VjbR. When Brucella melitensis16M was cultured at different stress mediums, the expression levels of MucR were also detected, and this protein was rapidly decreased at acidic medium (pH4.5). To detect if the decrease of MucR was caused by proteolysis, bacterial protein synthesis was inhibited by chloromycetin, and it demonstrated that the decrease of MucR was mainly caused by the enhancement of proteolysis. According to above results, the optimum condition of RNA-seq and chromatin Immunoprecipitation is culturing bacteria to late logarithmic phase in TSB medium.To study the MucR related mechanism of bacterial virulence, we performed RNA-seq analysis using Brucella melitensis RNA obtained from B. melitensis16M and the mucR mutant. In total, the expression levels of442genes were significantly changed in the mucR mutant, including310up-regulated genes and132down-regulated genes. According to the result of COG analysis, these differently expressed genes were involved in metabolism, cell wall/envelope biogenesis, and transcriptional regulation. Results of stress assays demonstrated that MucR was involved in tolerance to acid, iron-limitation, and cationic polypeptides. These results mentioned above should be the possible reasons of the attenuation of the mucR mutant.The target genes regulated by MucR were also detected in our study.77differently expressed genes were verified by chromatin immunoprecipitation, and14target genes were found. These target genes contained7genes involved in transcriptional regulation,2genes encoding outer membrane proteins,2metal ion transport genes,1gene involved in resistance of acidic stress,1gene related to denitrification, and1alkaline phosphatase. These results suggested that the sensitive to acidic and iron-limiting environment, changes of bacterial outer membrane properties, and different expression of genes involved in denitrification pathway were caused by different expression of MucR target genes in mucR mutant.Our study demonstrated MucR related mechanism of bacterial virulence and the target genes regulated by MucR, which provided insights for further studies of Brucella regulation network and intracellular survival mechanisms.更多还原