Structural characterization and functional properties of a novel lipomannan variant isolated from a Corynebacterium glutamicum pimB′ mutant

Abstract

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by complex lipids and amongst these, lipoglycans are of particular interest. The disruption of NCgl2106 in C. glutamicum resulted in a mutant devoid of monoacylated phosphatidyl-myo-inositol dimannoside (Ac 1 PIM 2 ) resulting in the accumulation of Ac 1 PIM 1 and cessation of phosphatidyl-myo-inositol (PI) based lipomannan (Cg-LM, now also termed 'Cg-LM-A') and lipoarabinomannan (Cg-LAM) biosynthesis. Interestingly, SDS-analysis of the lipoglycan fraction from the mutant revealed the synthesis of a single novel lipoglycan, now termed 'Cg-LM-B'. Further chemical analyses established the lipoglycan possessed an a-D-glucopyranosyluronic acid-(1 ? 3)-glycerol (GlcAGroAc 2 ) based anchor which was then further glycosylated by 8-22 mannose residues, with Man 12-20 GlcAGroAC 2 molecular species being the most abundant, to form a novel lipomannan structure (Cg-LM-B). The deletion of NCgl2106 in C. glutamicum has now provided a useful strain, in addition with a deletion mutant of NCgl0452 in C. glutamicum for the purification of Cg-LM-A and Cg-LM-B. Interestingly, both Cg-LM species induced a similar production of TNF-a by a human macrophage cell line suggesting that the phospho-myo-inositol residue of the PI-anchor does not play a key role in lipoglycan pro-inflammatory activity.

Characterization of theCorynebacterium glutamicumΔpimB′ ΔmgtADouble Deletion Mutant and the Role ofMycobacterium tuberculosisOrthologues Rv2188c and Rv0557 in Glycolipid Biosynthesis

“…4) using a Pro-Q emerald glycoprotein stain according to an established protocol (15,25). Extracts from C. glutamicum showed the presence of LAM Cg as well as LM-A Cg and LM-B Cg (which comigrates with LM-A Cg ), as shown previously (16,25). The lipoglycan extract from C. glutamicum ⌬pimBЈ showed the absence of LAM Cg and LM-A Cg and the presence of Man GlcAGroAc 2 -based LM-B Cg (16), while C. glutamicum ⌬mgtA showed the presence of the PI-based lipoglycans LAM Cg and LM-A Cg and the absence of LM-B Cg (25).…”

“…Using Rv0557 as a query sequence in a BLAST comparison, the next paralog among the six members of M. tuberculosis within the GT4 family was Rv2188c (identity score, 35%), revealing a structural similarity between the two proteins. Both proteins possess orthologs in C. glutamicum, and previous genetic and biochemical studies confirmed that the orthologous proteins have identical functions (13,16,25). When either pimBЈ Cg or mgtA Cg was deleted, no reliable growth defect was observed (data not shown).…”

“…The study was based on a cell-free assay using GDP[ (25). More recently, Rv2188c was also proposed to be involved in the synthesis of Ac 1 PIM 2 as the second ␣-D-mannose-␣-(136)-phosphatidyl-myo-inositol-mannosyl transferase (termed PimBЈ Mt ) (13,16), which has augmented ongoing confusion in the field. Due to the essentiality of M. tuberculosis PIM biosynthesis (3) in this study, we have generated C. glutamicum ⌬pimBЈ ⌬mgtA, deficient in pimBЈ Cg and mgtA Cg (C. glutamicum pimBЈ and mgtA) and subsequently overexpressed Rv2188c and Rv0557 individually to identify their true in vivo and in vitro biochemical activities.…”

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

“…1B). Although both pathways share many common enzymes, LM-A species are based on a glucopyranosyluronic acid diacylglycerol (Gl-A) anchor rather than PI (16,18). On the cytoplasmic side, Gl-A is mannosylated by MgtA (previously named PimB), producing mannosyl-glucuronic acid diacylglycerol (Gl-X), which is extended by MptB (17,29) and other enzymes common to both pathways to produce LM-B, the major LM pool of C. glutamicum (37,38).…”

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria

“…This pathway shares some PPM-dependent enzymes with the PI-based LM pathway including MptB, since an mptB (NCgl1505) mutant of C. glutamicum fails to produce intermediates beyond Gl-X or AcPIM2 [133]. For clarity, the PI-based LM pool has been designated LM-A while this second pathway produces LM-B [121,146], the major LM pool in C. glutamicum [118]. While C. glutamicum produces LAMs via LM-A using a similar pathway to mycobacteria, its LAMs are smaller and structurally distinct, with more extensive mannosylation and singular Araf capping [147].…”