Acylation state of the phosphatidylinositol mannosides from Mycobacterium bovis bacillus Calmette Guerin and ability to induce granuloma and recruit natural killer T cells

Previous studies have found that, when injected into mice, glycolipidic fra ctions of mycobacterial cell walls containing phosphatidylinositol mannosid es (PIM) induced a granuloma and recruitment of Natural Killer T cells in t he lesions. The dimannoside (PIM2) and the hexamannoside (PIM6) PIM were is olated from Mycobacterium bovis bacillus Calmette Guerin and shown to act a like, but the activity was found to be dependent on the presence of the lip idic part. The chemical structure of PIM was then re-evaluated, focusing on the characterization of their lipidic part, defining mono- to tetra-acylat ed PIM2. The structure of these acyl forms was elucidated using a sophistic ated combination of chemical degradations and analytical tools including el ectrospray ionization/mass spectrometry, electrospray ionization/mass spect rometry/mass spectrometry, and two-dimensional NMR. Finally, the acyl forms were purified by hydrophobic interaction chromatography and tested for the ir capacity to induce the granuloma and Natural Killer T cell recruitment. We found that there is an absolute requirement for the molecules to possess at least one fatty acyl chain, but the number, location, and size of the a cyl chains was without effect. Moreover, increasing the complexity of the c arbohydrate moiety did not lead to significant differences in the biologica l responses.