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

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 remains the only characterized genetic regulator of vesiculogenesis. Here, we present data supporting a role for the Pst/SenX3-RegX3 signal transduction system in regulating MV production. Deletion of , 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 Δ mutant, including LpqH, an MV-associated lipoprotein. Nanoparticle tracking analysis revealed a 15-fold increase in MV production by the Δ 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 , which encodes the predicted ESX-5 transmembrane channel. Upon EccD 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 vesicle biogenesis. In Gram-negative bacteria, MV derived from the outer membrane have diverse functions in bacterial physiology and pathogenesis, and several factors regulating their production have been identified. Though Gram-positive bacteria and mycobacteria that lack an outer membrane also produce vesicles with described roles in pathogenesis, the mechanisms of MV biogenesis in these organisms remain poorly characterized. Defining mechanisms of MV biogenesis might yield significant insights into the importance of MV production during infection. In , only a single genetic element, , is known to regulate MV production. Our work reveals that the Pst/SenX3-RegX3 signal transduction system is a novel regulator of MV biogenesis that controls MV production by a mechanism that is independent of both VirR and activation of the specialized ESX-5 protein secretion system. Understanding which genes in the RegX3 regulon cause increased MV production might reveal novel molecular mechanisms of MV release.