全基因组水平筛选和鉴定布鲁氏菌毒力相关效应因子基因

Brucellosis is an important zoonotic disease caused by Brucella, which is widely prevalent in the world and one of the major diseases considering of its serious harm to the public health and food safety. Brucella spp. are facultative intracellular pathogen, which can invade the host through digestive tract, respiratory tract, and skin or mucous membrane, and then resides and replicates in macrophages for long. Because of lacking the classical virulent factors, it was an urgent demand for systematical screening and identification of Brucella virulence-relative genes, particularly the direct effector genes, to support the interpretating the molecular mechanism of persistent intracellular infection and developing the novel technical products against brucellosis. In this study, we will screen and identify the essential genes for brucella intracellular survival in the macrophage cultures infected by Brucella mutants array,which included the mutants with the definite disrupted genes almostly covered the whole genomes of Brucella. On the basis of the function prediction and clustering analysis of newly identified essential genes, the candidate virulence-relative effector genes required for brucella intracellular survival will be selected to determine the role in Brucella intracellular multiplication ex vivo and persistent infection in vivo. Finally, we will appraise the functional properties and evolutionary traits of the novel virulence-relative effector genes by prediction of their protein structures and subcellular localization, as well as homologous comparison of the genes and/or proteins with that in other intracellular pathogens.The data originated from this study will improve on understanding the molecular mechanism of persistent intracellular infection and pathogenesis of Brucella.

布鲁氏菌病是由布鲁氏菌引起的一种重要人畜共患病，在全球各地广泛流行并危害公共卫生和食品安全，已成为全球重点防控的重要疾病之一。布鲁氏菌可通过消化道、呼吸道、皮肤黏膜等途径侵入机体并能在巨噬细胞内长期寄生和繁殖。由于缺乏经典毒力因子，在全基因组水平上全面鉴定布鲁氏菌毒力相关基因，尤其是效应因子基因，成为该病原体持续性感染和致病机制研究以及布病新型防控产品开发的关键。本项目利用布鲁氏菌全基因组已知基因突变菌株阵列，首先通过细胞感染全面筛选鉴定该菌在胞内生存必需基因，在新发现的基因功能预测和聚类分析基础上，选择验证新发现效应因子基因突变对布鲁氏菌在细胞内和小鼠体内生存能力的影响，最后通过其编码产物结构预测、亚细胞定位以及相关胞内寄生菌同源序列比对，评价新发现毒力相关效应因子基因的功能特性和进化特征，为布鲁氏菌持续性感染和致病分子机制的深入研究奠定基础。

布鲁氏菌病是一种重要的动物源性人畜共患病，全球各国广泛流行并严重危害公共卫生和食品安全。布鲁氏菌经消化道、呼吸道、皮肤黏膜侵入机体并在胞内长期寄生，但其逃逸宿主免疫系统并在体内持续感染的分子机制尚不清楚。为此，本研究测序分析转座子突变株4万个，获得有效插入位点信息1.35万条，数据量覆盖布鲁氏菌全基因组4倍。在此基础上，选择6782个突变菌株（涵盖2281/3301个基因内插入突变菌株和1048个基因间隔区插入的突变菌株。用上述96孔板阵列的突变菌株感染小鼠巨噬细胞，初筛出1090个毒力相关的突变株，然后将其接种最低营养培养基（MM），排除营养缺陷型菌株后经细菌计数验证577个弱毒突变株，其中涉及到139个新发现的基因和120个已鉴定的布鲁氏菌毒力相关基因。将毒力相关基因与近年鉴定的布鲁氏菌分泌性蛋白基因进行比对，优选BMEII0580、BMEII0581、BMEII0607、BMNI_I0006、BMNI_I0007、BMNI_I0008、BMNI_I0009、BMNI_I0010构建了基因缺失株及其相应的互补菌株，通过胞内存活试验和小鼠感染试验，发现BMEII0580、BMEII0581、BMEII0607缺失后对细菌毒力影响程度较小，而BMNI_I0006-BMNI_I0010缺失对布鲁氏菌毒力影响显著。羊种布鲁氏菌NI株BMNI_I0006-BMNI_I0010构成YejABEF操纵子。该操纵子缺失导致布鲁氏菌对怀孕小鼠的致病性显著降低，测定各基因缺失株体外生长能力、抗菌肽刺激后存活率及基因表达情况、感染细胞和小鼠模型发现，在酸性环境中除NIΔyejA1缺失株外其他基因缺失株均表现出更高的敏感性，生存能力显著降低；yejE基因缺失株NIΔyejE和全操纵子基因缺失株NIΔyejABEF在抗菌肽处理后表现出更高的敏感性，即在抗菌肽存在下，生存能力降低；通过扫描电镜发现，yejE基因缺失株和全操纵子缺失株经抗菌肽处理后菌体细胞裂解死亡，表明yejE基因对布鲁氏菌抵抗抗菌肽的裂解具有极为重要的作用。因此本研究解读了布鲁氏菌逃逸宿主先天性免疫的分子机制1种。本研究发表SCI论文14篇（第一、二标注各7篇），对照项目约定的研究任务书，完成了任务书规定的研究内容、目标和预期的指标。