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

Abstract

Mycobacterium tuberculosis and related Corynebacterineae synthesize a family of lipomannans (LM) and lipoarabinomannans (LAM) that are abundant components of the multilaminate cell wall and essential virulence factors in pathogenic species. Here we describe a new membrane protein, highly conserved in all Corynebacterineae, that is required for synthesis of full-length LM and LAM. Deletion of the Corynebacterium glutamicum NCgl2760 gene resulted in a complete loss of mature LM/LAM and the appearance of a truncated LM (t-LM). Complementation of the mutant with the NCgl2760 gene fully restored LM/LAM synthesis. Structural studies, including monosaccharide analysis, methylation linkage analysis, and mass spectrometry of native LM species, indicated that the ΔNCgl2760 t-LM comprised a series of short LM species (8-27 residues long) containing an α1-6-linked mannose backbone with greatly reduced α1-2-mannose side chains and no arabinose caps. The structure of the ΔNCgl2760 t-LM was similar to that of the t-LM produced by a C. glutamicum mutant lacking the mptA gene, encoding a membrane α1-6-mannosyltransferase involved in extending the α1-6-mannan backbone of LM intermediates. Interestingly, NCgl2760 lacks any motifs or homology to other proteins of known function. Attempts to delete the NCgl2760 orthologue in Mycobacterium smegmatis were unsuccessful, consistent with previous studies indicating that the M. tuberculosis orthologue, Rv0227c, is an essential gene. Together, these data suggest that NCgl2760/Rv0227c plays a critical role in the elongation of the mannan backbone of mycobacterial and corynebacterial LM, further highlighting the complexity of lipoglycan pathways of Corynebacterineae.

Keywords: Corynebacterium glutamicum; Mycobacterium tuberculosis; cell wall; glycolipid; lipid synthesis; lipoarabinomannan; lipomannan; membrane protein.

FIGURE 1.

Summary of key steps of lipoglycan biosynthesis in C. glutamicum. A, the LM-A/LAM pathway. The early PIM intermediate AcPIM2 is assembled on the cytoplasmic leaflet of the cell membrane by GDP-Man-dependent mannosyltransferases PimA/PimB and acyltransferase PatA before being transported across the membrane for further elongation by polyprenol-phosphate-mannose-dependent mannosyltransferases (LpqW-activated MptB and MptA that form the main mannan chain; MptC and MptD that add mannose side chains) to form Cg-LM-A. Cg-LM-A is further modified by glycosyltransferases to form Cg-LAM. In mycobacteria, additional enzymes (e.g. EmbC, AftC, AftD, and CapA) are involved in the formation of a significantly larger and more complex arabinan structure. B, LM-B pathway. In C. glutamicum, a second family of lipomannans, termed Cg-LM-B, are assembled on a structurally distinct glycolipid anchor that contains the core structure Gl-A. Gl-A is extended with a single mannose residue by the cytoplasmic enzyme, MgtA, forming Gl-X, which is thought to be flipped and then elongated by MptA/B/C/D to form the end product, Cg-LM-B. This abundant species forms the bulk of LM in C. glutamicum. Whether this pathway exists in mycobacteria is unknown.

FIGURE 2.

NCgl2760 is a predicted membrane protein encoded within a conserved genetic locus. A, the tmaT locus of Corynebacterineae. Orthologous genes in the four species are shown using the same color. The focus of the current study, NCgl2760, and its likely mycobacterial orthologues are indicated by a black box. B, Protter prediction for NCgl2760 (48). The protein is predicted to contain an N-terminal signal sequence (red) and a single transmembrane domain.

FIGURE 3.

Disruption strategy and analysis of ΔNCgl2760. A, diagram showing the arrangement of genes in the NCgl2760 region of WT C. glutamicum (top) and ΔNCgl2760 mutant (bottom). NruI and SalI restriction sites, sizes of expected bands on Southern blotting, and position of the probe used are indicated. Small horizontal arrows indicate the binding sites for the four primers used to construct the ΔNCgl2760 mutant (a, NCgl2760-left_F; b, NCgl2760-left_R; c, NCgl2760-right_F; d, NCgl2760-right_R). B, Southern blotting analysis of NruI-digested DNA of C. glutamicum WT (lane 1), a single crossover strain (lane 2), and the ΔNCgl2761 mutant (lane 3). C, Southern blotting analysis of SalI-digested DNA of C. glutamicum WT (lane 1), a single crossover strain (lane 2), and the ΔNCgl2760 mutant (lane 3). Positions of DIG-labeled λ DNA standards digested with HindIII are indicated in kb. Bands showing that the NCgl2760 gene has been deleted (a 6.2-kb NruI fragment in B and a 1.8-kb SalI fragment in C) are indicated by asterisks.

FIGURE 4.

C. glutamicum ΔNCgl2760 synthesizes a novel truncated LM species. A, SDS-PAGE analysis of LM/LAM fractions from WT (lane 1), ΔmptA mutant (lane 2), ΔNCgl2760 mutant (lane 3), ΔNCgl2760 mutant complemented with a functional copy of NCgl2760 (lane 4), or plasmid alone (lane 5). The LM/LAM phenotypes of the ΔmptA and ΔNCgl2760 mutant lines are very similar. B, SDS-PAGE analysis of lipoglycans purified from a ΔmgtA mutant (lane 1).

FIGURE 5.

Linkage analyses of C. glutamicum WT, ΔmptA, and ΔNCgl2760 mutant LM/LAM. LM/LAM samples were methylated, hydrolyzed, and reduced, and the resultant PMAA sugars were analyzed by GC-MS. A–C, PMAA of LM/LAM preparations from C. glutamicum WT (A), ΔmptA (B), and ΔNCgl2760 (C) bacteria. The mutant LM/LAM lacked PMAA corresponding to terminal Ara_f and had altered abundance of PMAA corresponding to branched (2,6-disubstituted Man) and unbranched (6- and 2-monosubstituted Man).

FIGURE 6.

Q-TOF-MS profiling of LM samples from WT and ΔmptA and ΔNCgl2760 mutant lines. Purified LM/LAM samples from WT (A), ΔmptA (B), ΔNCgl2760 (C), and ΔmgtA (D) were analyzed by direct infusion Q-TOF-MS in negative ion mode. Individual LM species were identified based on their exact mass after deconvolution and mass difference (by m/z 162 or 324) from related LM species containing the same lipid moieties. The plots show the mass (m/z) of all detected species and predicted mannan chain length (number of mannose residues). C. glutamicum WT bacteria contained 117 clearly resolved LM species with masses between 2712 and 9190 Da (10–50 mannose residues). In contrast, LM species of the ΔmptA mutant (112 LM species) and ΔmgtA mutant (78 LM species) had masses between 2200 and 4900 Da (with a minor cluster at higher molecular mass) and between 3222 and 8859 Da (13–48 mannose residues), respectively. ΔNCgl2760 LM (91 lipoglycan species detected) had a size distribution between 2626 and 5520 Da (8–27 mannose residues), and no higher mass lipoglycan species were detected at all. ΔNCgl2760 shared 66 lipoglycan species with the ΔmptA mutant and 32 LM species with the ΔmgtA mutant, indicating that both LM pathways (LM-A and LM-B) are still active in the ΔNCgl2760 knock-out mutant.

FIGURE 7.

The M. smegmatis orthologue of NCgl2760, MSMEG_0317, is an essential gene. A, the MSMEG_0317 locus. Primer binding sites are shown by small horizontal arrows, and expected PCR product sizes for the MSMEG_0317 gene and ΔMSMEG_0317 allele are indicated in kb. B, potential DCO clones derived from MSMEG_0317 SCO containing an integrated pMV361:MSMEG_0317 plasmid were analyzed by PCR using primers MSMEG_0319 and MSMEG_0317R. Lane 1, λHindIII/EcoRI molecular weight DNA markers (sizes shown in kb); lane 2, 100-bp DNA ladder; lane 3, wild-type M. smegmatis genomic DNA as template; lanes 4–20, potential DCO strain genomic DNA as templates. Clones in lanes 4, 5, 10, 16, and 20 gave the expected size band (0.6 kb) for a DCO, whereas the remaining 12 clones were WT revertents (1.7 kb). The reaction shown in lane 12 failed to give a detectable product.

FIGURE 8.

NCgl2760 is required for elongation of LM-A/LAM lipoglycans in C. glutamicum. Only the LM-A-based lipoglycan pathway (assembly on PI) is shown for clarity, although the LM-B pathway (assembly on GlcA-DAG) is also defective in the mutant.