A guardian role of TagA in protecting <i>Mycobacterium tuberculosis</i> from nitrosative killing.

Abstract

Upon activation, macrophages generate substantial levels of reactive nitrogen species, which can induce alkylating damage in the DNA of intracellular <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) and thereby restrict bacterial replication. However, the molecular mechanisms by which <i>Mtb</i> repairs such DNA lesions remain poorly understood. Here, we identified genes required for <i>Mtb</i> survival in distinct macrophage subsets using transposon insertion sequencing. Among these, <i>tagA</i> displayed a specialized role in <i>Mtb</i> survival in M1-polarized macrophages, as well as in mice at 4 wk postinfection, a stage when macrophages are biased toward an M1-polarized state. Mechanistically, TagA conferred resistance to the DNA alkylating agent methyl methanesulfonate through its 3-methyladenine (3-MA) excision activity with Glu48 serving as a key catalytic residue for substrate binding. Critically, TagA was found to protect the <i>Mtb</i> genome from alkylation damage caused by nitrosative stress-a hallmark of the M1-polarized macrophage microenvironment. Furthermore, pharmacological inhibition of inducible nitric oxide synthase (iNOS) with S-methylisothiourea sulfate in mice or genetic deletion of <i>nos2a</i> in zebrafish markedly rescued the survival defect of Δ<i>tagA</i>. Together, these findings reveal a previously unappreciated mechanism by which the DNA repair enzyme TagA protects <i>Mtb</i> against 3-MA DNA damage under nitrosative stress, thereby promoting bacterial survival in M1-polarized macrophages and during in vivo infection.