TolC调控猪肠外致病性大肠杆菌生物膜形成的分子机制

As a new zoonotic and foodborne pathogenic bacterium, extraintestinal pathogenic Escherichia coli (ExPEC) has posed serious threats to animal husbandry, food safety and public health. Biofilm formation can enhance the bacterial anti-adversity ability, thus making it more difficulty to prevent and control the bacterial infection. The outer membrane channel TolC protein, an efficient target against the multidrug resistance, has been shown to regulate the bacterial biofilm formation, but the mechanism remains unknown. The previous study by our research group showed that TolC protein of a porcine-source ExPEC strain might be involved in the bacterial resistance against the NaCl and sugar-induced hypertonic stress and the adjustment of curli fimbriae production, thus regulating the bacterial biofilm formation. Using such techniques as RNA-seq, comparative membrane proteinomics, real time RT-PCR and western-blot, this project will screen and identify the factors involved in the osmotic responsiveness and curli fimbriae production that regulated by TolC protein through gene transcription and protein expression or phosphorylation. Then the function of these factors will be investigated by performing the genetic deletion or complement. In combination with the functional analysis of TolC-involved efflux pumps, this project will try to disclose the molecular mechanism how TolC protein adjusts the transmembrane transportation of sodium or sugar to induce the bacterial resistance against the hypertonic stress, and regulates the curli fimbriae production and biofilm formation. This study will also lay the foundation for identifying the drug targets to control the bacterial biofilm formation.

肠外致病性大肠杆菌（ExPEC）是一种新的人兽共患性和食源性致病菌，对畜禽养殖、食品安全和人类健康具有严重威胁。生物膜形成可提高该菌抗逆境能力，增大防控难度。外膜通道蛋白TolC是控制细菌多重耐药性的有效靶标之一，对生物膜形成也有重要作用，但其机制尚不清楚。申请人课题组前期研究发现，TolC调控猪ExPEC生物膜形成与其参与高渗胁迫抗性和Curli菌毛合成调节有关。本项目拟采用RNA-seq、膜蛋白质组学、real time RT-PCR、Western-blot等技术，筛选和鉴定受TolC表达调控的ExPEC渗透压响应和Curli菌毛合成相关因子，然后通过基因缺失或补偿解析其功能，结合分析TolC相关外排泵的作用，系统阐明TolC调节ExPEC 钠离子和糖跨膜转运以参与高渗胁迫抗性、进而调控Curli菌毛合成和生物膜形成的分子机制，也为鉴定有效药物靶标控制细菌生物膜的形成提供理论基础。

肠外致病性大肠杆菌（ExPEC）是一种新的食源性致病菌，对畜禽养殖、食品安全和人类健康具有严重威胁。生物被膜形成可提高细菌抗逆境能力，增大防控难度。项目组前期证实，外排泵蛋白TolC缺失可降低ExPEC高渗胁迫抗性，进而降低ExEPC在高渗条件下Curli菌毛合成和生物被膜形成能力。本项目利用比较外膜蛋白组学、RNA-seq基因转录组学、RT-qPCR和扫描电镜等技术，筛选到受TolC调控的渗透压响应和Curli菌毛合成相关因子。进一步研究发现：在高盐和高蔗糖环境下，TolC缺失可造成不同外排泵系统受损，从而降低ExPEC对高NaCl和蔗糖环境的耐受性，但外膜蛋白OmpX过表达可代偿TolC缺失的影响。OmpW过表达可降低ExEPC对高盐环境的耐受性；通过基因缺失突变和磷酸化位点突变菌株的构建，证实CpxA/R双组分系统响应TolC缺失后形成的周质高渗信号，调控csgD基因转录影响curli菌毛的合成，最终影响生物被膜的形成。本研究结果提示TolC、OmpX和OmpW可作为药物靶标，研发添加剂以增强高渗环境控制食品及环境中病原菌及其生物被膜形成的效果。