Direct contact of the bacterial surface induces phase separation in the host phagosome membrane

Mycobacterium tuberculosis (Mtb) establishes intracellular niches by remodeling host membranes into either spacious or compact phagosomes, yet how direct bacterial contact within these distinct compartments facilitates bacterial egress remains unknown. Using fixed cell imaging, in vitro reconstitution of phagosome-like vesicles, and numerical simulations, we uncover that mycobacterial load and its direct contact determines phagosome fate by driving membrane bending, lipid wrapping and phase separation. Low-to-moderate load induces membrane vesiculation, generating compact phagosome-like structures. High bacterial load drives a scaffold-like architecture that primes compartments for rupture via complete lipid phase separation and changes in the membranes bending and area stretch moduli, rendering it more deformable. Notably, physical contact synergizes with the virulence factor ESAT-6, amplifying its membrane-deforming activity to disrupt phagosomal integrity. We propose that mycobacteria actively switch spacious to compact phagosomal states by modulating host membrane mechanics--a process governed by the membrane-to-contact area ratio and proximity to lipid demixing point. These findings reveal a biophysical switch in pathogen-driven membrane remodeling, with broad implications for understanding how intracellular pathogens manipulate host membranes to survive.