Characterization of Membrane Vesicles Released by the Opportunistic Human Pathogen Mycobacterium avium subsp. hominissuis (MAH) in Response to an in vitro System Mimicking the Phagosomal Environment
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Mycobacterium avium subsp. hominissuis (MAH) belongs to the most-clinically significant non-tuberculous mycobacterial (NTM) pathogens with constant increase in disease prevalence, mainly in several industrialized western countries where tuberculosis is less prevalent. Upon entry into the alveolar space, MAH is engulfed by resident-macrophages, where the pathogen adapts to the hostile phagosomal environment and proliferates. Mycobacteria bypass host immune defenses by secreting virulence factors. Several intracellular pathogens including mycobacteria are known to form membrane vesicles (MVs) in response to intracellular stress, helping bacteria to
deliver virulence effectors in the host cell. MVs are bacterial membrane-derived spheres filled with the cargo of biologically active materials such as proteins, lipids and nucleic acids. MVs play important role in bacterial pathogenesis, nutrient acquisition, biofilm formation and immunomodulation essential for bacterial survival within the host. Macrophage phagosomal environment, partly consisting of elemental mixture and low pH, activates several virulence mechanisms of MAH. Using the in vitro model mimicking the phagosomal environment of MAH (metal mix), we characterized cargo of MVs and investigated whether MV-associated bacterial virulence effectors are delivered to the cytosol of the host macrophages. Scanning electron microscopy of MAH exposed to the in vitro phagosomal model revealed formation of MVs on bacterial surface. Further, transmission electron microscopy confirmed presence of MVs in the purified samples of isolated vesicles. Using mass spectrometric analysis, 202 proteins were identified in MVs of minimal media of starvation model and 263 proteins were found in the in vitro model mimicking phagosome environment. Out of 263 cargo proteins, 211 are unique to the metal mix and are enriched in several virulence factors including enzymes involved in lipid and fatty acid metabolism and cell wall-associated processes. Non-canonical amino acid metabolic labeling and click chemistry-based enrichment assay confirmed that at least 5 proteins found in the in vitro phagosomal model are also present in the cytosol of THP1 macrophages during MAH infection at 24h. Lipidomic analysis showed presence of outer cell wall lipids with no compositional differences between MVs of minimal media and metal mix. Using PicoGreen assay, we demonstrate that MVs of both minimal media and metal mix harbor dsDNA; however, the concentration was found to be significantly lower in
the in vitro phagosomal model. Similar results are observed by laser confocal microscopy of MVs stained with lipophilic nucleic acid dye SYTO-61. Our study suggests that MAH produces MVs in phagosomes and carry important virulence factors with potential roles in bacterial intracellular survival and in the immunomodulation of host cellular defenses.