Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune responses, apoptosis, etc., for survival. Mtb protein secretion comprises three different secretion systems: Sec dependent, Sec independent (Tat), and Type VII (T7SS) secretion pathways. CRISPR-based knockdown strains of secA1 and tatA were generated to characterize SecA1 and TatA-mediated secretion systems in Mtb. The absence of both compromised pathogen survival in vitro and ex vivo. The secretome analysis of depletion strains showed a significant decrease in pathway specific secretory proteins compared to Rv. Membrane proteomics suggested substantial changes in the membrane proteome upon depletion. SEM analysis of depleted strains changes in the cellular morphology with cells bulging from the poles upon SecA1 depletion and increasing the length upon TatA depletion. TEM analysis indicates thinning of the cell membrane and cell wall and confirms the changes in the cellular morphology in depletion strains. Together, data suggests that disruption of sec and tat pathway leads to changes in the membrane permeability.