单核细胞增生性李斯特菌一新基因组岛功能解析

Listeria monocytogenes (LM) is a zoonotic pathogen that causes listeriosis, which has higher mortality comparing with the infection of other foodborne pathogen. High pathogenic L.monocytogenes XYSN is a serotype 4b strain isolated from cerebral parenchyma in an outbreak of sheep listeriosis. Based on the complete genome sequence and comparative genomics analysis, one 25kbp genomic island encoding virulence-asscociated genes was found in the genome of LM XYSN. In this study, the structural feature of this genomic island is analyzed and identified. Additionally, the genomic island and virulence-asscociated genes are knocked out with homologous recombination technology. Furthermore, the biological characteristics and interaction with cell lines and animal hosts are studied with the mutants strains and wild type strain. In vitro infection model, the interaction between listerial strains and brain endothelial cell line, epithelial cell line is observed and determined with microscope examination and immunological technology. In vivo infection model, the cross-talking between listerial strains and hosts including the kinetics of infection, the localization and pathological changes are evaluated by deterimnation bacterial load in organs, immunohischemical technique, etc. Totally, structural feature and biological function analysis of the new genomic island in Listeria monocytogenes can reveal the role in this high pathogenic strain, our research work is helpful to elucidate the pathogenic mechanism of high virulent LM and laws of genetic evloution.

单核细胞增生性李斯特菌(LM)是人兽共患李斯特菌病的病原菌，所引发的感染具有较高死亡率。血清型4b的李斯特菌LM XYSN是本实验室从暴发李斯特菌病的绵羊脑中分离的高致病力菌株，经前期全基因组测序和比较基因组学分析，发现该菌株存在一个由潜在毒力相关基因构成的25kbp基因组岛。本研究拟在对该基因组岛结构特征分析的基础上，构建基因组岛、潜在毒力相关基因缺失的突变株和回复株，通过生物学特性的测定及体内外感染实验，对其功能特性进行研究。以脑内皮细胞系、肠上皮细胞系为体外感染模型，借助显微观察及免疫学测定等技术，对其与细胞的互作效应进行测定。以小鼠和绵羊为体内感染模型，借助脏器细菌载量、免疫组化测定等技术，对细菌在体内地消长规律、组织定位及病理变化进行测定。基因组岛功能的解析，能有效地揭示其在高致病力李斯特菌中的作用，有助于阐明高毒力菌株的致病机制及遗传演化规律。

李斯特菌属由19个种构成，但只有单核细胞增生李斯特菌和伊氏李斯特菌具有致病性，其中单核细胞增生李斯特菌是重要的食源性人兽共患李斯特菌病的致病菌。本项目旨在对引起山羊李斯特菌病暴发的分离株的一新基因组岛的功能进行研究。经比较基因组学分析，发现该基因组岛与伊氏李斯特菌的毒力岛2（LIPI-2）高度同源, 推测伊氏李斯特菌LIPI-2的一部分，即GI9通过水平基因转移的方式插入XYSN基因组中，进而推动XYSN菌株的进化。在此基础上，本研究构建了基因组岛敲除突变株以及该基因组岛中smcL缺失株和重组菌株，以Caco-2BBE细胞系和C57BL/6小鼠进行了体内外感染试验，结果表明，smcL基因能增强谱系Ⅰ菌株NTSN和谱系Ⅱ菌株EGDe侵袭肠道的能力，具有诱导宿主细胞早期凋亡的功能；该基因组岛的敲除显著降低了XYSN的体内感染能力。本研究揭示了XYSN新获得的基因组岛为致病岛，在突破肠道屏障、体内定殖中和增强其毒力中发挥重要作用。