Sec and Tat Mediated Secretion Safeguards Mycobacterium tuberculosis Membrane Homeostasis

Protein secretion is essential for the growth and virulence of Mycobacterium tuberculosis, yet the organization and function of its secretion pathways remain poorly understood. We reviewed the existing literature, combined it with systematic queries, and finalized annotations based on experimental data and computational predictions to compile a curated list of 92 secretory components and 198 reactions involved in Sec, twin-arginine translocation (Tat), and ESX pathways. Using CRISPRi, targeted depletion of SecA1 or TatAC impaired both in vitro growth and ex vivo survival. Label-free quantitative secretome analysis revealed decreased export of substrates dependent on SecA1 and TatAC, with enrichment of cytosolic proteins in culture filtrates, indicating increased membrane dysbiosis. Membrane proteomics showed elevated levels of proteins engaged in intermediary and lipid metabolism, while proteins associated with the cell wall and cell processes decreased, suggesting weakened membrane integrity. Loss of SecA1 or TatAC increased membrane permeability, with the effect being more pronounced in the case of TatAC, and caused structural abnormalities seen under electron microscopy. Overall, our integrated multi-omics and functional genetics studies demonstrate that the SecA1 and Tat pathways are essential for maintaining membrane homeostasis in Mycobacterium tuberculosis. These results suggest that essential secretory proteins may be promising targets for therapeutic intervention.