Mycobacterium tuberculosis Pst/SenX3-RegX3 regulates membrane vesicle production independently of ESX-5 activity

Dylan W. White, Sarah R. Elliott, Evan Odean, Lynne T Bemis, Anna Tischler

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

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. IMPORTANCE 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 M. tuberculosis, only a single genetic element, virR, 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.

Original languageEnglish (US)
Article numbere00778-18
JournalmBio
Volume9
Issue number3
DOIs
StatePublished - May 1 2018

Bibliographical note

Funding Information:
We thank Dirk Schnappinger for providing the pDO23A, pDE43-MCH, pEN41A-T38S38, and pEN12A-P750 plasmids used to construct the eccD5 Tet-OFF plasmid, Carl Nathan for providing the pvirR overexpression plasmid and VirR primary antibody, Anthony Baughn for providing plasmids pTIC10a and pJT6a, Ryan Hunter for assistance with electron microscopy, Shannon Kordus for assistance with FPLC, Kelsey Binder for assistance with pTIC-eccD5 plasmid construction, and Alyssa Brokaw for constructing the pJGΔeccD5 plasmid. The following reagents were obtained through BEI Resources, NIAID, NIH: monoclonal anti-Mycobacterium tuberculosis GroEL2 (Rv0440), clone IT-70 (DCA4) (produced in vitro), catalog no. NR-13657; polyclonal anti-Mycobacterium tuberculosis Mpt32 (Rv1860) (antiserum, rabbit), catalog no. NR-13807; polyclonal anti-Mycobacterium tuberculosis antigen 85 complex (FbpA/Fbp/FbpC; Rv3804c, Rv1886c, and Rv0129c) (antiserum, rabbit), catalog no. NR-13800; monoclonal anti-Mycobacterium tuberculosis KatG (Rv1908c), clone IT-42 (HBT1) (produced in vitro), catalog no. NR-13791; monoclonal anti-Mycobacterium tuberculosis LpqH (Rv3763), clone IT-54 (produced in vitro), catalog no. NR-13792; and monoclonal anti-Mycobacterium tuberculosis PhoS1/PstS1 (Rv0934) clone IT-23 (TB71) (produced in vitro), catalog no. NR-13657. This work was supported by an NIH Director’s New Innovator Award (1DP2AI112245 to A.D.T.) and institutional startup funds from the University of Minnesota (A.D.T. and L.T.B.). The purchase of the NanoSight NS300 was partially supported by an equipment grant from the University of Minnesota Foundation (L.T.B.). Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC program.

Funding Information:
We thank Dirk Schnappinger for providing the pDO23A, pDE43-MCH, pEN41AT38S38, and pEN12A-P750 plasmids used to construct the eccD5 Tet-OFF plasmid, Carl Nathan for providing the pvirR overexpression plasmid and VirR primary antibody, Anthony Baughn for providing plasmids pTIC10a and pJT6a, Ryan Hunter for assistance with electron microscopy, Shannon Kordus for assistance with FPLC, Kelsey Binder for assistance with pTIC-eccD5 plasmid construction, and Alyssa Brokaw for constructing the pJGΔeccD5 plasmid. The following reagents were obtained through BEI Resources, NIAID, NIH: monoclonal anti-Mycobacterium tuberculosis GroEL2 (Rv0440), clone IT-70 (DCA4) (produced in vitro), catalog no. NR-13657; polyclonal anti-Mycobacterium tuberculosis Mpt32 (Rv1860) (antiserum, rabbit), catalog no. NR-13807; polyclonal anti-Mycobacterium tuberculosis antigen 85 complex (FbpA/Fbp/FbpC; Rv3804c, Rv1886c, and Rv0129c) (antiserum, rabbit), catalog no. NR-13800; monoclonal anti-Mycobacterium tuberculosis KatG (Rv1908c), clone IT-42 (HBT1) (produced in vitro), catalog no. NR-13791; monoclonal anti-Mycobacterium tuberculosis LpqH (Rv3763), clone IT-54 (produced in vitro), catalog no. NR-13792; and monoclonal anti-Mycobacterium tuberculosis PhoS1/PstS1 (Rv0934) clone IT-23 (TB71) (produced in vitro), catalog no. NR-13657. This work was supported by an NIH Director’s New Innovator Award (1DP2AI112245 to A.D.T.) and institutional startup funds from the University of Minnesota (A.D.T. and L.T.B.). The purchase of the NanoSight NS300 was partially supported by an equipment grant from the University of Minnesota Foundation (L.T.B.). Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC program.

Publisher Copyright:
© 2018 White et al.

Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.

Keywords

  • ESX secretion
  • Lipoproteins
  • Tuberculosis
  • Type VII secretion
  • Vesicle

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