Isotopic tracing in Mycobacterium tuberculosis -infected alveolar epithelial cells indicates metabolic specificity to their virulence and drug-resistance status

Tuberculosis (TB) remains a global health threat aggravated by the emergence of drug-resistant strains. Mycobacterium tuberculosis promotes metabolic remodeling within host cells to support its survival. Type II alveolar epithelial cells have been established as a primary niche for bacterial infection, adherence, and invasion. However, compared to macrophages, metabolic interactions within these cells, particularly the strain-specific exploitation of host metabolism, remain inadequately explored. To address this, we integrated intracellular bacterial growth and host cell viability assays with C-based proteinogenic amino acid kinetics to gain insights into metabolic changes in A549 alveolar epithelial cells infected with laboratory strains (virulent H37Rv, avirulent H37Ra) and clinical isolates (drug-resistant S6 and S11; drug-sensitive S4 and S5). Virulent strain H37Rv showed higher intracellular growth (6-fold increase) but significantly reduced host cell viability relative to avirulent H37Ra. Interestingly, the multidrug-resistant isolate S6 showed the highest intracellular growth (18-fold increase) while maintaining host cell viability. Mass isotopomer distribution analysis revealed that virulent strains showed increased reliance on central carbon metabolism, including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. H37Rv showed greater 13C enrichment in host amino acids, such as alanine (1.6-fold), compared to H37Ra. Drug-resistant isolates displayed distinct 13C enrichment in TCA-derived threonine (0.21–0.25%), methionine (0.18–0.28%), and lysine (0.12–0.27%). We observed 13C enrichment in essential amino acids (lysine, methionine, threonine, valine) across all infected conditions, confirming the pathogen’s dependence on host-derived metabolic intermediates. These findings highlight the need to explore host-to-pathogen amino acid transport as a precise host-directed therapeutic target.