Abstract
Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.
Highlights
Mycolic acids are essential building blocks of the outer membrane of all mycobacteria
We resorted to the E. coli adenylate cyclase two-hybrid (BACTH) system as our primary approach to identify M. tuberculosis proteins interacting with MmpL3
This system exploits the fact that the catalytic domain of the adenylate cyclase (CyaA) from Bordetella pertussis consists of two complementary fragments, T25 and T18, that are not active when physically separated but whose functional complementation when fused to interacting polypeptides results in cyclic AMP synthesis in an E. coli cya mutant30. cAMP produced by the reconstituted chimeric enzyme activates a β-galactosidase reporter allowing positive interactions to be identified on indicator agar media
Summary
Mycolic acids are essential building blocks of the outer membrane of all mycobacteria. Bacilli division and elongation without compromising the integrity of their existing multilayered cell wall implies spatiotemporal coordination of biosynthetic and export activities at the sites of septal and polar elongation, a process apparently facilitated in mycobacteria by the uneven distribution of the peptidoglycan, arabinogalactan, mycolic acid and phospho(glyco)lipid synthetic complexes in the inner membrane, and their enrichment in the polar and subpolar regions of actively elongating cells[20,21,22,23,24] Despite these advances, much remains to be done in understanding the molecular mechanisms coordinating new cell wall deposition with cell septation/elongation, and characterizing the molecular interactions and dynamic features of the individual cell envelope biosynthetic pathways involved. The central involvement of MmpL3 in mycolic acid translocation, the localization of this transporter at the pole and septum of actively-growing cells[20,25] where mycolic acid transfer to their outer membrane acceptors has been shown to occur[26,27,28,29], and its ability to directly or indirectly interact with the elongation regulator Wag[31] (aka DivIVA)[20] led us to hypothesize that MmpL3 may be at the center of a protein scaffold coordinating new mycolic acid and, perhaps, other cell envelope constituents deposition during cell elongation and division
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