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.

Structure–Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis

“…42 TMM acetylation would follow the transfer of mycolic acids onto trehalose by Pks13 43 and, perhaps, serve as a signal for export by MmpL3 or another component of the mycolic acid translocation machinery. Whether acetylation is required for TMM export in mycobacteria was not known.…”

Identification of a Membrane Protein Required for Lipomannan Maturation and Lipoarabinomannan Synthesis in Corynebacterineae

“…We have recently shown that the C. glutamicum acetyltransferase TmaT (TMCM mycolyl acetyltransferase; NCgl2759) catalyzes the acetylation of the major cell wall glycolipid TMCM and that this modification is important for periplasmic transport of this glycolipid (41). A subsequent study showed that the mycobacterial orthologue has the same function (44).…”

“…Amino acid sequence alignments were produced using Clustal Omega. Construction of C. glutamicum ⌬NCgl2760 and Complementation Strains-The NCgl2760 gene was deleted using a twostep allelic replacement strategy previously used in our laboratory to create other cell wall mutants (11,16,29,41). A 1.0-kb fragment containing sequence from the left side of the NCgl2760 gene was amplified using ProofStart DNA polymerase (Qiagen) and the primers NCgl2760-left_F (5Ј-GTCACC-CGGGTAAATAAAGGCAACC) and NCgl2760-left_R (5Ј-GATAGGATCCCAATAATGAGGACCG) and cloned into the XmaI/BamHI sites (underlined) of pUC19 (49), creating plasmid pUC-NCgl2760left.…”

“…Free lipids, including PIMs and trehalose mycolates, were extracted and purified as described previously (41). The delipidated cell pellet was then subjected to ethanol reflux by suspending in 5 ml of 50% (v/v) ethanol and incubating at 100°C for 2 h, with occasional vortex mixing.…”

“…Gene, Rv0227c-We have recently shown that the C. glutamicum gene NCgl2759 (orthologue of the essential M. tuberculosis gene, Rv0228) encodes an acetyltransferase that converts trehalose monocorynomycolate (TMCM) to an acetylated form (AcTMCM) and is essential for efficient transport of TMCM across the inner membrane for conversion to trehalose dicorynomycolate (TDCM) (41). To determine whether other genes in this locus are involved in trehalose corynomycolate biosynthesis, we investigated the function of NCgl2760, the next gene in the genome of C. glutamicum ATCC 13032.…”