Tuberculous Meningitis Alters the Proteomic Landscape of Brain-Derived Extracellular Vesicles.

Tuberculous meningitis (TB meningitis), the deadliest form of Mycobacterium tuberculosis infection, causes devastating mortality and severe neurological disability despite standard therapy. Brain injury and microglial activation are major determinants of outcome, yet the molecular mechanisms linking infection, inflammation, and neuronal injury remain poorly understood. Extracellular vesicles (EVs), critical mediators of cell-to-cell communication, have been investigated in pulmonary TB, but their role in TB meningitis remains unexplored. We used a young New Zealand White rabbit model of TB meningitis to isolate and characterize brain-derived EVs from infected and uninfected rabbits employing nanoflow cytometry, transmission electron microscopy, proteomic profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and bioinformatic analysis of pathways, cell-type enrichment, and protein-protein interaction networks (DAVID, Enrichr, and STRING databases). EV recovery from flash-frozen brain tissue proved feasible, enabling future studies using biobanked samples. M. tuberculosis infection increased EV release in brain tissue and induced a selective proteomic shift characterized by enrichment of proteins associated with TB host defense, microglial activation, and stress-response pathways, coupled with a reduced representation of neuronal and neuroprotective EV-associated proteins. These findings suggest that infection reprograms the brain EV proteome toward inflammatory and injury-associated pathways. Collectively, this study provides the first comprehensive proteomic characterization of brain-derived EVs in TB meningitis and establishes a foundational atlas of EV-associated proteins altered during infection. These findings identify candidate proteins for future biomarker validation in accessible biofluids (e.g., plasma and CSF) and provide a framework for generating hypotheses about EV-mediated host-pathogen interactions in the CNS during TB meningitis.