Abstract
ABSTRACTMycobacterium 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 eccD5, which encodes the predicted ESX-5 transmembrane channel. Upon EccD5 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.
Highlights
Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, they may have additional unappreciated functions
We demonstrate that the ΔpstA1 mutant hyper-secretes MV containing the lipoproteins LpqH and PstS1 in a RegX3-dependent manner
Because the ΔpstA1 mutant exhibits a RegX3dependent increase in the activity of the ESX-5 secretion system and ESX-5 substrates had previously been identified within MV, we initially hypothesized that MV release and ESX-5 activity were connected
Summary
Mycobacterium tuberculosis releases membrane vesicles (MV) that modulate host immune responses and aid in iron acquisition, they may have additional unappreciated functions. 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. 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. Though Gram-positive bacteria and mycobacteria that lack an outer membrane produce vesicles with described roles in pathogenesis, the mechanisms of MV biogenesis in these organisms remain poorly characterized. 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. M. tuberculosis MV-null mutants might be used to conclusively determine the role of either MV-induced inflammation or MV-mediated inhibition of T cell function in pathogenesis, but such mutants have yet to be described
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