Uncovering asparagine utilization pathway, a critical requirement for mycobacterial pH-driven adaptation at the host-pathogen crossroads

M. tuberculosis, an intracellular pathogen, thrives in a specialized membrane-bound vacuole, the phagosome with acidic pH and limited access to nutrients. To survive and replicate within human host, M. tuberculosis must adapt and fulfil its nutritional requirements. To understand how the bacillus captures nutrients from its host, we studied mycobacterial acquisition of host-derived amino acids, the preferred nitrogen sources. We discovered that PhoP, a key determinant of mycobacterial adaptation to phagosomal acidification, controls expression of AnsP1 and AnsP2 to facilitate acquisition of host aspartate and asparagine, respectively. Thus, macrophage-infected WT-H37Rv showed a significantly higher level of intra-bacterial Asn compared to the phoP-KO mutant and a complemented mutant could restore Asn level to that of WT-H37Rv. Under acidic conditions, elevated DNA binding of PhoP within the promoters lead to direct activation of ansP1 and ansP2. Consistently, phoP-KO is unable to utilize Asn under acidic condition, and over-expression of ansP1 or ansP2 restored intracellular growth defect of the mutant. These findings uncover the regulatory network allowing utilization of organic nitrogen sources by the pathogen during infection. Author summaryHuman Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global public health burden claiming [~]1.25 million deaths in 2023. To investigate how the bacillus captures nitrogen sources from its host, we studied acquisition of host amino acids aspartate (Asp) and asparagine (Asn) by the tubercle bacilli. We show that PhoP, a key determinant of mycobacterial adaptation to phagosomal acidification, controls expression of Asp and Asn transporters to facilitate acquisition of host amino acids by the pathogen. A mutant lacking phoP is unable to utilize Asn under acidic conditions, whereas over-expression of ansP1 and/or ansP2 restored growth defect of the phoP-KO in vitro and in macrophages. Together, these findings uncover the transcriptional landscape allowing utilization of organic nitrogen sources by the tubercle bacilli during infection.