All mycobacterial species, including pathogenic Mycobacterium tuberculosis, synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for normal growth and viability. These glycolipids are important cell-wall and/or plasma-membrane components in their own right and can also be hyperglycosylated to form other wall components, such as lipomannan and lipoarabinomannan. We have investigated the steps involved in the biosynthesis of the major PIM species in a new M. smegmatis cell-free system. A number of apolar and polar PIM intermediates were labelled when this system was continuously labelled or pulse–chase-labelled with GDP-[3H]Man, and the glycan head groups and the acylation states of these species were determined by chemical and enzymic treatments and octyl-Sepharose chromatography respectively. These analyses showed that (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least two pathways that differ in the timing of the first acylation step, (2) early PIM intermediates containing a single mannose residue can be modified with two fatty acid residues, (3) formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicating that polyprenol phosphate-Man, rather than GDP-Man, is the donor for these reactions, (4) modification of acylated PIM4 with α1-2- or α1-6-linked mannose residues is probably the branch point in the biosyntheses of polar PIM and lipoarabinomannan respectively and (5) GDP strongly inhibits the synthesis of early PIM intermediates and increases the turnover of polyprenol phosphate-Man. These findings are incorporated into a revised pathway for mycobacterial PIM biosynthesis.
- cell wall
- phosphatidylinositol mannoside
- polyprenol phosphate
Abbreviations used: ASAM, Aspergillus saitoi α1-2-mannosidase; PIM, phosphatidylinositol mannoside; AcxPIMy, PIM species with x (1 or 2) fatty acyl chains, linked to either the core α1-2-linked Man or the myo-inositol head group, and y Man residues; ESI–MS, electrospray ionization MS; HPTLC, high-performance TLC; JBAM, Jack-bean α-mannosidase; LM, lipomannan; LAM, lipoarabinomannan; MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; PPM, polyprenol (C35/C50) phosphate-Man; TFA, trifluoroacetic acid.
- The Biochemical Society, London ©2004