Comparative Proteomic Analysis of Hypoxic Adaptation Reveals Conserved and Species-Specific Responses in Mycobacterium tuberculosis and Mycobacterium bovis

Abstract Tuberculosis, caused by members of the Mycobacterium tuberculosis complex, remains a major global health concern. While M. tuberculosis and M. bovis share similar pathogenic mechanisms, important differences in disease progression and adaptive responses have been observed. Hypoxia is a critical stressor during latent infection, however, the comparative molecular adaptations of these two species under oxygen-limited conditions remain incompletely understood. In this study, we used the Wayne gradual oxygen depletion model to investigate the cellular proteomes of the reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv under hypoxic and aerobic conditions. Our analysis revealed that pathways involved in acyl-CoA generation constitute a conserved response to hypoxia in both species. Proteins associated with lipid metabolism and stress responses, particularly members of the DosRST regulon, emerged as hallmarks of hypoxic adaptation in both strains. Notably, a greater number of DosRST- and EHR-regulated proteins showed an increase in abundance in M. bovis than in M. tuberculosis . Lipid metabolism and cell envelope remodeling were central features of the hypoxic response in both species, consistent with observed changes in PDIM A and PDIM B levels. Both species also showed accumulation of proteins involved in molybdenum metabolism, whereas remodeling of nitrogen metabolism was a prominent feature specific to the M. bovis response. These findings support the notion that pathogenic mycobacteria rely on lipid utilization to maintain energy homeostasis and redox balance under oxygen-limited conditions, and that cell envelope remodeling may contribute to long-term survival. The identified species-specific differences in metabolic adaptation and PDIM modulation provide new insights into the distinct physiological responses of M. tuberculosis and M. bovis during hypoxic stress.