单增李斯特菌（Listeria monocytogenes, LM）是重要的食源性致病菌之一，较强的生物膜形成能力是其持久性污染食品生产的重要因素。我们发现：LuxS/AI-2群体感应系统中luxS基因缺失株生物膜形成能力显著增强，基于定量蛋白质组学结果推测显著上调表达的LadR蛋白在生物膜形成中起重要作用，但其作用机制仍未有研究。本项目拟通过同源重组技术构建LM GIM798的ladR基因缺失株和回复株，运用RNA-seq、RT-qPCR技术分析LadR转录调控的基因表达谱；以EMSA和DNase I足迹技术综合分析LadR的转录调控机制，通过表型分析、功能验证探讨LadR在LM生物膜形成中的作用机制。本项目旨在阐明LadR在LM中的调控机制及其在生物膜形成中的功能，进一步揭示LuxS/AI-2群体感应系统调控生物膜形成的可能通路，为开发基于阻断群体感应调控通路的防控新技术提供新思路。

Listeria monocytogenes, an intracellular Gram positive pathogen, is considered as one of the important food-borne pathogens by WHO. Biofilm formation is thought to be the leading cause for the persistence of L. monocytogenes contamination in food production. Our previous study found that the ability of biofilm formation was significantly increased luxS mutant, and the expression of LadR protein were also significantly increased based on quantatitive proteomics analysis. Previous studies demonstrated AphA, which secondary structure is highly similar to LadR, plays an important role in biofilm formation via regulating the c-di-GMP production. So we infer that LadR plays an important role in biofilm formation. However, little information is available on the effect of LadR protein on biofilm formation in L. monocytogenes. In this project, the dominant food-borne L. monocytogenes GIM798 will use as an original strain, which isolated from Flammulina velutipes product in our previous national investigations of foodborne pathogens contamination in China. First, ΔladR mutant and complement stain will be constructed by homologous recombination, and then explore the differential expression genes regulated by LadR protein using RNA-seq analysis and verifying the exact DNA-binding sites of potential genes regulated by LadR protein using EMSA and DNase I footprinting. Meanwhile, the biofilm formation of wild type strain and mutant are measured for the functional analysis of differential expression genes regulated by LadR. The aim of this project is to elucidate the effect of LadR protein on biofilm formation, which may further explore the regulation circuit of LuxS/AI-2 based quorum sensing in L. monocytogenes. The results of this project will provide new idea for exploring the novel techniques to control L. monocytogene contamination based on the disruption of quorum sensing signal transduction.