Abstract
The dimannoside (PIM2) and hexamannoside (PIM6) phosphatidyl-myo-inositol mannosides are the two most abundant classes of PIM found in Mycobacterium bovis bacillus Calmette Guérin, Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis 607. Recently, these long known molecules received a renewed interest due to the fact that PIM2 constitute the anchor motif of an important constituent of the mycobacterial cell wall, the lipoarabinomannans (LAM), and that both LAM (phosphoinositol-capped LAM) and PIM are agonists of Toll-like receptor 2 (TLR2), a pattern recognition receptor involved in innate immunity. Due to the biological importance of these molecules, the chemical structure of PIM was revisited. The structure of PIM2 was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G. (2001) J. Biol. Chem. 276, 34896-34904). Here we report the purification and molecular characterization of PIM6 in their native form. For the first time, four acyl forms of this molecule have been purified, using hydrophobic interaction chromatography. Mono- to tetra-acylated molecules were identified in M. bovis bacillus Calmette Guérin, M. tuberculosis H37Rv, and M. smegmatis 607 using a sophisticated combination of analytical tools, including matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and two-dimensional homo- and heteronuclear NMR spectroscopy. These experiments revealed that the major acyl forms are similar to the ones described for PIM2. Finally, we show that PIM6, like PIM2, activate primary macrophages to secrete TNF-alpha through TLR2, irrespective of their acylation pattern, and that they signal through the adaptor MyD88.
Highlights
Mycobacterium bovis BCG 1173P2 [1], Mycobacterium smegmatis ATCC-607 [1], and Mycobacterium tuberculosis H37Rv ATCC-27294 were found to mainly contain two phosphatidyl-myo-inositol mannosides (PIM) families, the dimmanosylated (PIM2) and the hexamannosylated (PIM6) ones
M. tuberculosis H37Rv was found to produce as major PIM families PIM2 and PIM6 in the same acyl forms as the ones described for M. bovis BCG
MyD88 is involved in most of the TLR-mediated signals [31], including Tolllike receptor 2 (TLR2) as shown here for the TLR2 agonist BLP and for the best of the response to the TLR4 agonist LPS (Fig. 8D), and we showed that macrophages deficient for MyD88 did not respond to the PIM2 or PIM6 stimulation (Fig. 8, E and F)
Summary
Mycobacterium bovis BCG 1173P2 (the Pasteur strain) [1], Mycobacterium smegmatis ATCC-607 [1], and Mycobacterium tuberculosis H37Rv ATCC-27294 were found to mainly contain two PIM families, the dimmanosylated (PIM2) and the hexamannosylated (PIM6) ones. PIM1, PIM3, PIM4, and PIM5 were observed in very small amounts, suggesting that they are biosynthetic intermediates These experiments revealed that the major acyl forms are similar to the ones described for PIM2. The complete structure of native PIM2 has been achieved [1, 2] These last studies focused on the characterization of their lipidic part and unambiguously established the existence of a tetra-acylated form that was far suggested [4]. Analysis of infected macrophages revealed that PIM, among other mycobacterial lipids, are actively trafficked out of the mycobacterial phagosome [14]. This could be of particular importance relating to the potential
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