title: Acetylation of trehalose mycolates is required for efficient MmpL-mediated membrane transport in Corynebacterineae.
abstract: Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum NCgl2759 resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The in vitro synthesis of AcTMCM was dependent on acetyl-CoA, whereas in vivo [(14)C]-acetate pulse-chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the ΔNCgl2759 mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel TMCM mycolyl acetyltransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaryo-Botte
authors.forename: Arek K
authors.initials: AK
authors.lastname: Rainczuk
authors.forename: David J
authors.initials: DJ
authors.lastname: Lea-Smith
authors.forename: Rajini
authors.initials: R
authors.lastname: Brammananth
authors.forename: Phillip L
authors.initials: PL
authors.lastname: van der Peet
authors.affiliation: #Metabolomics Laboratory, Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Meikle
authors.forename: Julie E
authors.initials: JE
authors.lastname: Ralton
authors.forename: Thusita W T
authors.initials: TW
authors.lastname: Rupasinghe
authors.forename: Spencer J
authors.initials: SJ
authors.lastname: Williams
authors.forename: Ross L
authors.initials: RL
authors.lastname: Coppel
authors.forename: Paul K
authors.initials: PK
authors.lastname: Crellin
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: McConville
mesh_terms: D000107:Acetylation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D002473:Cell Wall / Q000201:enzymology / Q000235:genetics; D003311:Cord Factors / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D009171:Mycolic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014199:Trehalose / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003311:Cord Factors; D026901:Membrane Transport Proteins; D009171:Mycolic Acids; D011994:Recombinant Proteins; C021099:trehalose monomycolate; D014199:Trehalose