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

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. To investigate gene expression changes of mtrA overexpression, we used M. tuberculosis strain containing an ATc-inducible vector as described previously in Minch et al (PMID: 25581030). For each sample, cultures of M. tuberculosis were cultured in standard 7H9-rich media containing 50 μg/ml hygromycin B to mid-log phase, washed three times in 1x PBS and diluted back to OD600 = 0.05 into either neutral or acidic pH media with 50 μg/ml hygromycin B and the presence or absence of ATc inducer. Samples, in biological triplicate from two separate experiments, were collected after 18 hours of ATc induction, centrifuged at high speed for 5 min, supernatant was discarded and the cell pellet was immediately flash frozen 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.