Structural study of the LipoMannans from Mycobacterium bovis BCG: Characterisation of multiacylated forms of the phosphatidyl-myo-inositol anchor

A biosynthetic filiation is postulated between the mycobacterial phosphatid yl-myo-inositol mannosides (PIMs), the lipomannans (LMs) and the lipoarabin omannans (LAMs), the major antigens of the envelopes. Moreover, as the PI a nchor is thought to play a role in the biological functions of the LAMs, we characterized the lipid moiety of the PI anchor from Mycobacterium bovis B CG cellular LMs. Their structure was investigated along with that of a puri fied tetra-acylated form of PLM, (Ac4PIM2). A two-dimensional H-1-P-31 hete ronuclear multiple quantum correlation homonuclear Hartmann-Hahn spectrosco py study of Ac4PIM2 unambiguously localised a fourth fatty acid on the C3 o f the myo-Ins beside the fatty acids already described on the C1 and C2 pos ition of the glycerol and on the C6 position of the mannose. This analytica l strategy was extended to the structural study of the cellular LM anchor. Using an appropriate solvent system, the one dimensional P-31 NMR spectrum exhibited four major resonances typifying the LM populations. These populat ions differed in number and location of the fatty acids. For one of these p opulations, we established the presence of an extra fatty acid on the C3 of the myo-Ins of the LM anchor. The fact that both types of mol ecules have an elaborated anchor in common, indicates that cellular LMs are multimannos ylated forms of PIMs. In addition, the LM mannan core structure was analyse d by two-dimensional NMR, pointing to a high level of branching by single a lpha 1 --> 2 Manp side-chains. (C) 1999 Academic Press.