Mycobacterium tuberculosis Pst/SenX3-RegX3 Regulates Membrane Vesicle Production Independently of ESX-5 Activity

Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, although they may have additional unappreciated functions. MV production appears to be a regulated process, but virR remains the only characterized genetic regulator of vesiculogenesis. Here, we present data supporting a role for the M. tuberculosis Pst/SenX3-RegX3 signal transduction system in regulating MV production. Deletion of pstA1, which encodes a transmembrane component of the phosphate-specific transport (Pst) system, causes constitutive activation of the SenX3-RegX3 two-component system, leading to increased protein secretion via the specialized ESX-5 type VII secretion system. Using proteomic mass spectrometry, we identified several additional proteins hyper-secreted by the ΔpstA1 mutant, including LpqH, an MV-associated lipoprotein. Nanoparticle tracking analysis revealed a 15-fold increase in MV production by the ΔpstA1 mutant. Both hyper-secretion of LpqH and increased MV release required RegX3 but were independent of VirR, suggesting that Pst/SenX3-RegX3 controls MV release by a novel mechanism. Prior proteomic analysis identified ESX-5 substrates associated with MV. We therefore hypothesized that MV release requires ESX-5 activity. We constructed strains that conditionally express eccD(5), which encodes the predicted ESX-5 transmembrane channel. Upon EccD(5) depletion, we observed reduced secretion of the ESX-5 substrates EsxN and PPE41, but MV release was unaffected. Our data suggest that ESX-5 does not affect vesicle production and imply that further characterization of the Pst/SenX3-RegX3 regulon might reveal novel mechanisms of M. tuberculosis vesicle biogenesis.