Biosynthesis of mycobacterial lipoarabinomannan.

Gurdyal S. Besra,Charles J. Waechter,Caroline B. Morehouse,Christian M. Rittner,Patrick J. Brennan

doi:10.1074/jbc.272.29.18460

Abstract

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are potent immunomodulators in tuberculosis and leprosy. Little is known of their biosynthesis, other than being based on phosphatidylinositol (PI), and they probably originate in the phosphatidylinositol mannosides (PIMs; PIMans). A novel form of cell-free incubation involving in vitro and in situ labeling with GDP-[14C]Man of the polyprenyl-P-mannoses (C35/C50-P-Man) and the simpler PIMs of mycobacterial membranes, reisolation of the [14C]Man-labeled membranes, and in situ chase demonstrated the synthesis of a novel alpha(1-->6)-linked linear form of LM at the expense of the C35/C50-P-Man. There was little or no synthesis under these conditions of PIMan5 with its terminal alpha(1-->2)Man unit or the mature LM or LAM with copious alpha(1-->2)Man branching. Synthesis of the linear LM, but not of the simpler PIMan2, was susceptible to amphomycin, a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases. A mixture of P[3H]I and P[3H]IMan2 was incorporated into the linear LM, supporting other evidence that, like the PIMs, LM and LAM, it is a lipid-linked mannooligosaccharide and a new member of the mycobacterial glycosylphosphatidylinositol lipoglycan/glycolipid class. Hence, the simpler PIMs originate in PI and GDP-Man, but further growth of the linear backbone emanates from C35-/C50-P-Man and is amphomycin-sensitive. The origin of the alpha(1-->2)Man branches of mature PIMan5, LM, and LAM is not known at this time but is probably GDP-Man.

Highlights

The cell wall of Mycobacterium spp. consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan which, in turn, is attached to the mycolic acids [1]
CoA to yield a mixture of PIMan2, the monoacyl (Ac1PIMan2) and the diacyl (Ac2PIMan2) derivatives
It was later recognized that LAM, in its various forms, i.e. ManLAM and AraLAM [22], and LM contain a mannan core linked to PI similar to that in the family of phosphatidylinositol mannosides (PIMs) (PIMan1–6) (6 – 8)

Summary

Biosynthesis of Mycobacterial Lipoarabinomannan*

(Received for publication, February 28, 1997, and in revised form, May 12, 1997). Gurdyal S. The biological importance of LAM dictates an understanding of its biosynthesis Both LM and LAM are based on phosphatidylinositol (PI) [6, 7], on monoacyl phosphatidylinositol dimannoside (Ac1PIMan2) [8, 9], a member of the PIM family with a characteristic myoinositol (Ino) 2,6-dimannosyl unit [10, 11]. The structural progression from PI to PIMan to Ac1PIMan to LM and LAM has suggested a similar biosynthetic order [8]; to date, this was mere speculation In this present study, we have defined the origins of the Man units of the PIMs and LM and, in the course of the work, identified an ␣136linked linear form of LM, the apparent precursor of mature LM and LAM

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Biosynthesis of LM and LAM

Nature of products