LadR蛋白在单增李斯特菌LuxS/AI-2群体感应系统调控生物膜形成中的功能研究

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.

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。

单核细胞增生李斯特菌（Listeria monocytogenes，简称单增李斯特菌）是一种重要的食源性致病菌，致死率高达20~30%。它能在食品及其加工环境表面形成生物膜，是造成消费者持续性污染的重要原因之一。因此，研究单增李斯特菌生物膜的形成机制对防控该病原菌污染具有重大意义。.本项目以我国食品源优势菌株（血清组I.1，ST8）GIM798-1（WT）为出发株，利用同源重组技术和pKSV7温度敏感型质粒构建ladR缺失株（ΔladR），研究发现ΔladR的生长能力和运动性（p <0.05）增强；溶血性（p <0.0001）和早期生物膜形成能力（12-36 h，p <0.001）显著减弱，表明LadR正调控单增李斯特菌早期的生物膜形成能力；利用MTT还原法测定生物膜细胞活性发现生物膜对苯扎氯铵的耐受性与生物膜的生物量相关。利用液相色谱-质谱联用仪测定发现ΔladR胞内环二鸟苷酸（cyclic diguanylate，c-di-GMP）的水平显著减弱（p <0.05），表明LadR可能正调控单增李斯特菌胞内c-di-GMP的水平。.与WT相比，RNA-seq分析ΔladR缺失株共有40个上调基因和17个下调基因存在表达水平差异，RT-qPCR验证RNA-seq数据准确。上调基因主要参与单增李斯特菌碳水化合物磷酸转移酶系统，分别参与单增李斯特菌D-阿洛糖、D-纤维二糖、麦芽糖/麦芽糖糊精、D-阿拉伯糖醇/D-木糖醇和甘露糖的代谢调控。下调基因中有13个基因与单增李斯特菌的毒力相关，其中有11个基因受PrfA毒力激活因子调控。碳水化合物利用试验结果表明ΔladR在葡萄糖和/或麦芽糖为碳源时的生长能力显著高于WT，但生物膜形成能力显著低于WT，表明LadR可能通过负调控lmo2121-lmo2126的表达提高单增李斯特菌对葡萄糖和/或麦芽糖的摄取能力，进而影响生物膜的形成。.另外，利用两种不同亲和纯化标签的原核表达载体进行ladR基因原核表达，结果发现pGEX-4T-1-ladR表达载体的蛋白可溶性表达高于pET-28a-ladR，且两种原核表达载体表达的蛋白通过凝胶阻滞试验表明均具有活性，为后续研究LadR的功能和转录调控机理奠定了良好基础。.综上所述，LadR可能通过调控单增李斯特菌碳水化合物PTS和PrfA及其调控的毒力基因的表达来影响生物膜的形成。