TY - JOUR
T1 - Structural and functional evidence that lipoprotein LpqN supports cell envelope biogenesis in Mycobacterium tuberculosis
AU - Melly, Geoff C.
AU - Stokas, Haley
AU - Dunaj, Jennifer L.
AU - Hsu, Fong Fu
AU - Rajavel, Malligarjunan
AU - Su, Chih Chia
AU - Yu, Edward W.
AU - Purdy, Georgiana E.
N1 - Funding Information:
Acknowledgments—Protein mass spectrometric analysis was performed by the Oregon Health & Science University Proteomics Shared Resource with partial support from National Institutes of Health Core Grants P30EY010572, P30CA069533, and S10OD012246. Lipid mass spectrometric analysis was performed by the Washington University Biomedical Mass Spectrometry Research Resource, which is supported by National Institutes of Health Grants P41-GM103422, P60-DK-20579, and P30-DK56341. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
Funding Information:
This work was supported by NIAID, National Institutes of Health Grants R21 AI113074 (to G. E. P.), R01 AI123148 (to G. E. P. and E. W. Y.), and by Grant T32 AI007472 (to G. C. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 Melly et al.
PY - 2019/10/25
Y1 - 2019/10/25
N2 - The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid- containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the δlpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis.
AB - The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid- containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the δlpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis.
UR - http://www.scopus.com/inward/record.url?scp=85074184739&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA119.008781
DO - 10.1074/jbc.RA119.008781
M3 - Article
C2 - 31471317
AN - SCOPUS:85074184739
SN - 0021-9258
VL - 294
SP - 15711
EP - 15723
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -