Abstract
We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR-2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR-2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR-2343 gene. Disruption of MMAR-2343 resulted in a severe LOS breakdown, indicating that MMAR-2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly.