BPS2026 – Mycobacterial extracellular vesicles disrupt phagosomal maturation by altering membrane tension and suppressing transcription of fusion machinery

The success of mycobacteria as a pathogen is largely attributed to its ability to evade lysosomal degradation, notably by modulating phagosomal maturation and preventing phagosome-lysosome fusion. Among the various virulence strategies employed, the release of mycobacterial extracellular vesicles (MEVs) has emerged as a critical mechanism for host immune modulation. However, the biophysical and molecular basis of MEV-mediated immune interference remains poorly defined. Here, we show that MEVs fuse with host plasma and phagosomal membranes, inducing substantial changes in membrane fluidity, tension, and stiffness as revealed by live-cell imaging and fluorescence lifetime imaging microscopy (FLIM). These biophysical alterations impair the recruitment of early endosomal markers, such as Rab5, delay phagosome maturation, and disrupt lysosomal integrity. Using in vitro reconstitution and micropipette aspiration, we demonstrate that MEV lipids alter membrane mechanical properties, specifically increasing membrane order, line tension, and bending modulus, which collectively hinder phagosome-lysosome fusion. Lipidomic profiling identifies distinct mycobacterial lipid species enriched in MEVs that correlate with these effects. Notably, artificial vesicles composed solely of MEV lipids reproduce the observed lysosomal defects, confirming lipid sufficiency in driving host membrane dysfunction. Furthermore, transcriptomic profiling reveals widespread reprogramming of host gene expression, particularly affecting phagosome maturation, trafficking, and immune regulatory pathways. Together, our findings establish that MEV-derived lipids function as stealth effectors that remodel host membrane architecture, inhibit phagolysosomal progression, and reprogram innate immune responses.