基于全基因组关联分析的单增李斯特菌克隆复合群87型菌株持留能力形成的分子机制研究

单增李斯特菌(Listeria monocytogenes)是重要的食源性致病菌之一，具有持留能力是某些亚型持久性污染食品生产过程的重要因素。申请人前期研究发现单增李斯特菌克隆复合群（clonal complex，CC）87型菌株是持续污染我国金针菇生产过程的持留菌，但其持留能力形成的分子机制尚未阐明。本项目拟基于分离自金针菇产业链的CC87型菌株，利用全基因组关联分析挖掘可能与CC87型菌株持留能力形成相关的遗传决定因子；以核心基因组多位点序列分型筛选CC87型优势株作为出发株，利用基因同源重组技术和基因功能验证研究，揭示遗传决定因子在持留能力形成中的功能；利用转录组学剖析遗传决定因子可能参与的代谢通路，阐明CC87型菌株在食品生产环境中形成持留能力的分子机制。项目预期研究结果将丰富持留菌持留能力形成的分子调控理论，为防控单增李斯特菌持续性污染食品生产过程提供重要的作用靶点和新策略。

Listeria monocytogenes, an intracellular Gram-positive pathogen, is considered as one of the important food-borne pathogens worldwide. The persistence of some subtype of L. monocytogenes strains is thought to be the leading cause for persist contamination in food production process. Our previous studies revealed that L. monocytogenes clonal complex (CC) 87 strains dominate and persist in Flammulina velutipes plants. However, little information is available about the molecular mechanism of the persistence of L. monocytogenes CC87 strains. In this project, we will collect CC87 isolates from Flammulina velutipes plants and construct a genome dataset of L. monocytogenes strains belonged to different CCs by whole genome sequencing, and then screen the potential determinants of persistence of L. monocytogenes CC87 strains via genome-wide association study. The dominant strain of CC87 is used as original strain screened by core genome multilocus sequence typing, the deletion mutants of determinant genes will be constructed by homologous recombination, and the function of these determinants were analyzed by determining the changes of phenotype characteristics associated with persistence in mutants, further the function of determinants associated with persistence will be also validated by gene complementation. Then we will analyze the differential expression genes regulated by these determinants by RNA-seq approach and explore the potential metabolic pathway associated with these determinants. The aim of this project is to elucidate the molecular mechanism of the persistence of L. monocytogenes CC87 strains. The results of this project will explore the novel knowledge for the molecular mechanism of persistence of L. monocytogenes, and also provide novel targets and strategies for control and precaution of L. monocytogenes contamination in food production process.