The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis.
The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis.
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DOI：
10.1371/journal.ppat.1004183
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发表时间：
2014-05
期刊：
影响因子：
6.7
通讯作者：
Cole ST
中科院分区：
文献类型：
--
作者：
Solans L;Gonzalo-Asensio J;Sala C;Benjak A;Uplekar S;Rougemont J;Guilhot C;Malaga W;Martín C;Cole ST
PhoPR双组分系统对结核分枝杆菌的毒力至关重要，它控制约2%的基因表达，包括主要毒力决定因素ESX-1分泌装置的基因。phoP的突变导致病原体特异性细胞壁成分的产生受损，以及体内和体外的衰减。在ChIP-seq实验（染色质免疫沉淀后进行高通量测序）中使用针对天然蛋白的抗体，我们证明PhoP与结核分枝杆菌基因组上至少35个位点结合。PhoP调控子包括几个转录调控子以及多酮合成酶和PE/PPE蛋白的基因。ChIP-seq结果与高分辨率转录组学分析（RNA-seq）的整合显示，PhoP直接控制30个基因，而调控级联则通过EspR等蛋白质负责信号放大和下游效应，EspR通过调控espACD操纵子控制Esx1功能。PhoP调控最突出的位点位于rv2395和PE_PGRS41之间的基因间区，其中mcr7基因编码小的非编码RNA （ncRNA）。Northern blot实验证实Mcr7在结核分枝杆菌phoP突变体中缺失，并且在结核分枝杆菌复合体成员中低水平表达ncRNA。通过遗传和蛋白质组学分析，我们证明Mcr7调节tatC mRNA的翻译，从而影响双精氨酸易位（Tat）蛋白分泌装置的活性。结果，免疫优势的Ag85复合物和β -内酰胺酶BlaC的分泌受到影响。因此，Mcr7作为结核分枝杆菌中第一个功能已被确定的ncRNA，代表了PhoPR双组分系统与成功感染宿主所必需的下游功能之间缺失的一环。PhoPR对是结核分枝杆菌中最具特征的双组分系统之一，PhoR是跨膜传感器激酶，PhoP在控制毒力相关基因（如编码ESX-1分泌装置的基因）的表达中起重要作用。先前的研究表明，phoP突变导致小鼠感染模型的衰减，从而为开发一种携带phoP缺失的新型减毒活结核分枝杆菌疫苗提供了基础，该疫苗目前已进入临床试验。为了深入研究PhoP在结核分枝杆菌中的作用，我们采用了一种系统生物学方法，包括ChIP-seq和RNA-seq技术。我们证实PhoP与结核分枝杆菌染色体上至少35个靶点结合，并直接影响30个基因的表达，而信号的进一步扩增是由下游的调节因子提供的。最强的结合位点位于rv2395和PE_PGRS41之间，在这里显示了非编码RNA (ncRNA) Mcr7的转录。Mcr7的表达仅限于结核分枝杆菌，在phoP突变体中完全沉默。遗传学和蛋白质组学方法证明，Mcr7控制双精氨酸（Tat）分泌系统的活性，从而调节免疫优势抗原Ag85复合物和BlaC β -内酰胺酶的分泌。
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. One of the best characterized two-component systems in Mycobacterium tuberculosis is represented by the PhoPR pair, with PhoR being the transmembrane sensor kinase and PhoP playing an essential part in controlling expression of virulence-associated genes, such as those encoding the ESX-1 secretion apparatus. Previous studies showed that mutations in phoP resulted in attenuation in the mouse model of infection, thus providing the basis for the development of a novel live attenuated Mycobacterium tuberculosis vaccine carrying a deletion in phoP which is today in clinical trials. To thoroughly investigate the role of PhoP in M. tuberculosis, we undertook a systems biology approach comprising ChIP-seq and RNA-seq technologies. We demonstrated binding of PhoP to at least 35 targets on the M. tuberculosis chromosome and direct impact on expression of 30 genes, while further amplification of the signal is provided by regulators acting downstream. The strongest binding site was located between rv2395 and PE_PGRS41, where transcription of the non-coding RNA (ncRNA) Mcr7 was demonstrated. Expression of Mcr7 was found to be restricted to M. tuberculosis species and totally silenced in a phoP mutant. Genetics and proteomics approaches proved that Mcr7 controls activity of the Twin Arginine (Tat) secretion system, thus modulating secretion of the immunodominant antigen Ag85 complex and the BlaC beta-lactamase.
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