C-Terminal Domain of Mycobacterium tuberculosis Glutamate Decarboxylase determines the bacterial stress-adaptive metabolic state that steers macrophage polarisation supporting intracellular persistence

Mycobacterium tuberculosis (Mtb) adapts metabolically to survive hostile conditions within alveolar macrophages, but whether specific enzyme-driven metabolic states of Mtb link stress adaptation to host immunomodulation remains unclear. Using LC-MRM/MS metabolite profiling and metabolic modeling, we previously identified elevated {gamma}-aminobutyric acid (GABA) production via the GABA shunt during adaptation to oxidative and acidic stress. Here, we show that stress-responsive enzymatic efficiency of glutamate decarboxylase (GadB) in producing GABA, not only mitigates acidic and oxidative stress but also drives macrophage polarisation towards the M2 phenotype, extending its role beyond bioenergetics into host-pathogen cross-talk. We identify the C-terminal domain (CTD) of mycobacterial GadB as a key regulator. Enzyme kinetics and fluorescence spectroscopy revealed that CTD deletion abolishes pH-dependent regulation of catalytic efficiency (kcat/Km) and substrate affinity (Ka). Under oxidative stress, the CTD enhances catalytic efficiency without altering substrate affinity, suggesting condition-specific roles. Accordingly, in vitro, Mtb::MtbGadB with higher GABA and [~]40% lower ROS outgrew Mtb::MtbGadB{Delta}Ct under acidic and oxidative conditions. These biochemical differences influenced host immunity, wherein infection with high-GABA-producing Mtb::MtbGadB strain induced M2 polarisation, with decreased CD86, reduced RNS, limited endosomal maturation, as indicated by Rab5/Rab7 ratio and Lysotracker-based confocal microscopy, and decreased TNF-, IL-12p70, and IFN-{gamma} release compared to low-GABA-producing Mtb::MtbGadB{Delta}Ct. Consequently, Mtb::MtbGadB{Delta}Ct showed higher clearance. We conclude that by enabling pH-dependent activity of GadB, the CTD orchestrates proton consumption, ROS reduction, and GABA-mediated immunomodulation. Distinct structural features of CTDs in human versus Mtb GADs highlight the CTD as a selective drug target for pathogen-specific, host-compatible therapies. ImportanceThis work establishes a direct mechanistic link between metabolic state of Mycobacterium tuberculosis (Mtb) actively shaping host immune responses, deciding infection outcome. The study identifies the C-terminal domain (CTD) of MtbGadB in steering enzymatic efficiency, regulating intrabacterial GABA production and ROS quenching, determining the stress-responsive metabolic state of the bacteria. Infection with metabolically distinct strains had differential impacts on host immunity, wherein infection with high-GABA-producing Mtb::MtbGadB induced M2-polarisation, reduced RNS, limited endosomal maturation, and reduced proinflammatory cytokine release, compared to low-GABA-producing Mtb::MtbGadB{Delta}Ct. Consequently, Mtb::MtbGadB{Delta}Ct could be cleared, but Mtb::MtbGadB persisted, deciding the fate of the infection. Structural differences between CTDs of human and Mtb GADs, make it a therapeutically-exploitable determinant for developing pathogen-specific drugs that remain compatible with the host.