LcrQ调控假结核耶尔森氏菌III型分泌系统的分子机制研究

Type III secretion system (T3SS) in Yersinia is elegantly regulated in apparatus assembly as well as in the secretion of virulent effector proteins. As an important negative regulatory protein to T3SS in Yersinia, LcrQ plays vital feedback inhibition roles at the levels of both gene regulation and effector protein secretion. However, it is largely unclear how LcrQ exerts its negative regulation role and how it secrets outside bacterial cells under T3SS inducible conditions. This project will focus on analyzing the inhibition functions of LcrQ to T3SS at transcriptional, post-transcriptional and protein expression levels, and the mechanism underline the secretion of LcrQ in T3SS inducible conditions will also be investigated. On the basis of the negative regulatory roles of LcrQ to T3SS is based on blocking the role of the transcriptional activator LcrF, we will screen proteins interacting with LcrQ and LcrF, with the emphasis of the secretion apparatus proteins and chaperones which form complex with LcrQ on its regulatory and secretory roles, together with the crystal structure of LcrQ. Eventually, we will dissect the molecular mechanism of the negative feedback regulation of LcrQ at levels of gene expression and protein secretion. Our project will systematically explain the regulation of T3SS under inducible conditions, which will provide information for further dissecting the modulation of the T3SS machinery in pathogenic bacteria.

耶尔森氏菌III型分泌系统(Type III secretion system, T3SS)受到复杂精细的调控。作为重要的负调控蛋白，LcrQ全局性调控T3SS基因的表达和效应蛋白的分泌，但LcrQ如何在胞内发挥负调控T3SS的功能，以及LcrQ如何分泌到胞外解除其负调控的机制一直是未解之谜。本项目围绕LcrQ在胞内如何发挥负调控功能，以及LcrQ在T3SS诱导条件下分泌到胞外解除其抑制T3SS的机制两方面开展研究。在前期LcrQ转录调控T3SS通过阻遏转录激活因子LcrF功能的基础上，系统筛选与LcrQ及LcrF相互作用的蛋白，重点研究装置蛋白及伴侣蛋白与LcrQ互作及形成复合体对调控和分泌的影响，并结合LcrQ结构解析，从基因表达和蛋白分泌两个层面揭示LcrQ负调控T3SS的分子机制。项目的完成将系统阐释耶尔森氏菌T3SS诱导表达的调控细节，为更深入研究T3SS的运转机制奠定基础。

耶尔森氏菌III型分泌系统(T3SS)受到复杂精细的调控。作为T3SS重要的负调控蛋白，LcrQ全局性调控T3SS相关基因的表达和毒力效应蛋白的分泌，但LcrQ如何在胞内发挥负调控T3SS的功能以及LcrQ如何分泌到胞外解除其负调控的机制一直是未解之谜。本项目通过对LcrQ在胞内发挥抑制T3SS作用的作用机理及LcrQ分泌到胞外具体过程的探讨，从基因表达和蛋白分泌两个层面全面揭示了LcrQ负反馈调控耶尔森氏菌T3SS的分子机制。重要结果及主要进展包括：1).筛选了与LcrQ相互作用的蛋白；2).LcrQ在mRNA和蛋白水平抑制T3SS转录激活因子LcrF的表达；3).LcrQ调控LcrF的表达需要YopD/LcrH的参与；4).LcrQ以及YopD的负调功能需要RNaseE参与；5).LcrQ抑制YopD的分泌，导致滞留在胞内的YopD抑制LcrF以及T3SS效应蛋白的表达。本研究首次将LcrQ,YopD以及LcrF三种T3SS调控蛋白的功能联系起来，系统阐释了LcrQ参与耶尔森氏菌T3SS诱导表达的调控细节，揭示了LcrQ负调控T3SS的分子机制，为更深入研究病原细菌T3SS的调控机制奠定了基础。