Integrative Transcriptomic and Metabolomic Analysis of Mycobacterium tuberculosis Drug-Tolerant Persisters: Redox Disequilibrium, Respiratory Suppression, and ATP Depletion in Phenotypic Drug Tolerance
DOI:
https://doi.org/10.59675/P421Keywords:
Mycobacterium tuberculosis; drug-tolerant persisters; phenotypic drug tolerance; redox homeostasis; oxidative phosphorylationAbstract
Background: The treatment of tuberculosis is impacted by drug-tolerant Mycobacterium tuberculosis (Mtb) persisters that endure prolonged antibiotic exposure without developing permanent resistance. Objective: - This study investigated the connections between transcriptional changes, redox homeostasis, respiration, and energy during the initial enrichment of persisters using an integrated approach of RNA sequencing, metabolomics, and biochemical assays.
Methods: - Samples were collected at three distinct experimental timepoints: Exponential Growth Stage (T0), Early Stationary Stage (T1), Post-Antibiotic Treatment (T2): Evaluated after 48 hours of isoniazid/rifampicin treatment. Exploratory Comparison: Assessed a ΔmshA mutant for susceptibility to isoniazid and reactive oxygen species (ROS).
Results: - Transcriptional Changes: Marked downregulation of ventilation and ATP-producing genes occurred in T2 cells, including ndh (Rv1854c), atpB, and atpE, along with the activation of stress-related regulators (relA, dosR, sigF, mshA, and ahpC). Energetics & Redox Balance: ATP levels dropped to 0.232 times the baseline at T0, the 〖"NADH/NAD" 〗^+ ratio increased, and oxidized mycothiol accumulated. Gene-metabolite association analysis revealed an atpB-related module connecting respiration suppression to central carbon metabolism changes. Respiration: Oxygen consumption measurements indicated a continuous decline in both basal and maximal respiration levels over time. Mutant Susceptibility: The ΔmshA strain showed heightened susceptibility to isoniazid and ROS, indicating a likely mechanistic role for mycothiol-associated buffering, though serving as supportive rather than conclusive evidence of genetic validation. Conclusion: - The data support a working hypothesis where redox imbalances and respiration interference correlate with progressive ATP depletion during persister selection. However, because T2 sampling occurred post-antibiotic treatment and NADPH was not directly measured, the findings do not prove a causative link between redox status and ATP depletion.
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