The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in Mycobacterium tuberculosis

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.PhoPR双组分系统（PhoPR two-component system）是结核分枝杆菌（Mycobacterium tuberculosis）毒力调控的必需通路，该系统可调控约2%的基因表达，其中包括ESX-1分泌装置（ESX-1 secretion apparatus）——一类核心毒力决定因子。phoP基因突变会导致病原体特异性细胞壁成分合成受损，并使结核分枝杆菌在体外（ex vivo）与体内（in vivo）均出现减毒表型。本研究通过针对天然蛋白的抗体开展染色质免疫共沉淀测序（chromatin immunoprecipitation followed by high-throughput sequencing, ChIP-seq）实验，证实PhoP可结合结核分枝杆菌基因组上至少35个基因座。PhoP调控子包含多种转录调控因子，以及聚酮合酶与PE/PPE蛋白编码基因。将ChIP-seq结果与高分辨率转录组分析（RNA-seq）相结合后发现，PhoP可直接调控30个基因，而调控级联则通过EspR等蛋白实现信号放大与下游效应调控：EspR可通过调控espACD操纵子，进而影响Esx1功能。PhoP调控最显著的位点位于rv2395与PE_PGRS41之间的基因间区，其中mcr7基因编码一种小型非编码RNA（non-coding RNA, ncRNA）。Northern印迹实验证实，结核分枝杆菌phoP突变体中不存在Mcr7，且除结核分枝杆菌外的结核分枝杆菌复合群成员中，该ncRNA的表达水平较低。通过遗传学与蛋白质组学分析，本研究证实Mcr7可调控tatC mRNA的翻译过程，进而影响双精氨酸转运（Twin Arginine Translocation, Tat）蛋白分泌装置的活性。受此影响的蛋白包括免疫显性抗原Ag85复合物以及β-内酰胺酶BlaC等的分泌。Mcr7是首个功能得到阐明的结核分枝杆菌非编码RNA，因此它填补了PhoPR双组分系统与宿主成功感染所需下游功能之间的缺失环节。