MtrA regulation of essential peptidoglycan cleavage in Mycobacterium tuberculosis during infection [mtrA_knockdown]

The success of Mycobacterium tuberculosis (Mtb) is largely due to its ability to withstand numerous stresses imposed by host immunity. Here, we present a data-driven model that captures these adaptive mechanisms and reveals the dynamic interplay of host-derived stresses and genome-encoded regulatory programs in Mtb. The model captures the genome-wide distribution of cis-acting gene regulatory elements and the conditional influences of transcription factors at those elements to elicit environment-specific responses. Analysis of transcriptional responses that may be essential for Mtb’s survival in acidic conditions identified regulatory control by the MtrAB two-component signal system. Using genome-wide transcriptomics as well as imaging studies, we have characterized the MtrAB circuit by tunable CRISPRi knockdown in both Mtb and the non-pathogenic organism, M. smegmatis (Msm). These experiments validated the essentiality of MtrA in Mtb, but not Msm. We identified that MtrA regulates multiple enzymes that cleave cell wall peptidoglycan and is required for efficient cell division. Moreover, our results suggest that peptidoglycan cleavage, regulated by MtrA, is required for Mtb to survive intracellular stress. Further, we present MtrA as an attractive drug target, as even weak repression of mtrA results in loss of Mtb viability and completely clears the bacteria with low-dose isoniazid or rifampicin treatment. Cultures were grown to mid-log phase in 7H9-rich media with Kanamycin (50 μg/ml) and then diluted back in the presence or absence of ATc to induce CRISPRi-mediated mtrA knockdown. Knockdown was allowed to proceed for 14 hours (M. smegmatis) or 4 days (M. tuberculosis). Samples, in biological quadruplicate, were collected by centrifugation at high speed for 5 min, discarding supernatant and immediately flash freezing the cell pellet in liquid nitrogen. Cell pellets were stored at -80° C until RNA extraction was performed. The sequence reads that passed quality filters were aligned with Bowtie2 and processed using the R package DuffyNGS.

结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的致病与存活能力，很大程度上源于其耐受宿主免疫系统施加的多种胁迫的能力。本研究构建了数据驱动模型，用以阐释该菌的适应性调控机制，并揭示宿主来源胁迫与结核分枝杆菌基因组编码的调控程序之间的动态互作关系。该模型可捕捉全基因组范围内顺式作用基因调控元件（cis-acting gene regulatory elements）的分布特征，以及转录因子在这些元件上的条件性调控影响，从而触发环境特异性的基因应答反应。针对结核分枝杆菌在酸性条件下生存所必需的转录应答分析，本研究鉴定出MtrAB双组分信号系统的调控作用。本研究通过全基因组转录组学及成像研究，在结核分枝杆菌与非致病性的耻垢分枝杆菌（M. smegmatis, Msm）中，采用可诱导调控的CRISPRi敲低技术对MtrAB通路进行了系统表征。实验结果证实，MtrA在结核分枝杆菌中具有必需性，但在耻垢分枝杆菌中并非必需。研究发现，MtrA可调控多种裂解细胞壁肽聚糖的酶的表达，且对于细菌高效完成细胞分裂不可或缺。此外，本研究结果表明，由MtrA调控的肽聚糖裂解过程，是结核分枝杆菌耐受胞内胁迫的必要条件。进一步研究显示，MtrA是极具潜力的药物靶点：即使仅对mtrA进行轻度抑制，也会导致结核分枝杆菌的生存能力完全丧失，且联合低剂量异烟肼（isoniazid）或利福平（rifampicin）治疗，可彻底清除体内的结核分枝杆菌。实验流程如下：将培养物在含卡那霉素（Kanamycin，50 μg/ml）的7H9完全培养基中培养至对数中期，随后分别在添加或不添加ATc的条件下进行稀释，以诱导CRISPRi介导的mtrA基因敲低。敲低过程分别持续14小时（耻垢分枝杆菌）或4天（结核分枝杆菌）。以生物学重复四次的方式收集样本：高速离心5分钟后弃去上清液，立即将细胞沉淀置于液氮中快速冷冻。细胞沉淀保存于-80℃环境中，直至开展RNA提取实验。通过质量过滤的测序reads经Bowtie2进行序列比对，并使用R包DuffyNGS完成后续数据处理。