Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

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.

Acyltransferases in Bacteria

“…Lipooligosaccharides (LOS), composed of a highly complex glycan backbone and the acyltrehalose part (Fig. 11C), are synthesized by the close relative Mycobacterium marinum and contribute to its virulence (62,(163)(164)(165)(166)(167).…”

“…11C). The LOS biosynthesis cluster strongly resembles the SL-1 and PAT synthesis gene clusters, and the encoded PapA4 was recently identified as crucial for the assembly of the complete, acylated LOS structure (167).…”

The cell envelope glycoconjugates ofMycobacterium tuberculosis

“…Additionally, the cluster encompasses several genes encoding glycosyltransferases, methyltransferases and other glycosyl modifying enzymes likely to be involved in the synthesis and modification of the species-specific oligosaccharidyl unit (Ren et al , 2007; Alibaud et al ., 2014). A M. marinum mutant carrying a transposon insertion in papA4 fails to synthesize LOS, confirming the involvement of this acyltransferase in the pathway (Rombouts et al ., 2011). Interestingly, while a homologous gene cluster is present in the genome of Mtb H37Rv, pks5.1 is missing from this cluster and the H37Rv ortholog of papA4 is predicted to encode a truncated protein of only 165 amino acids instead of the 465 residue protein encoded for instance by papA4 from M. marinum .…”

Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids

“…The MSMEG_4727 ( pks5 ) gene, whose sequence is 65.6 % identical to that of the Mtb Mas-like gene Rv1527c , was involved in the biosynthesis of LOS in M. smegmatis [207]. The genomic surroundings of pks5 from M. smegmatis reminds those described earlier for other acyltrehaloses (see SL, DAT and PAT in sections IV and Fig 9) in that pap - and fadD -like genes likely to be required for the activation and transfer of the acyl groups of LOS [206], and an mmpL gene putatively involved in the translocation of these lipids are found (Fig. 11C).…”

“…The biosynthesis of LOS molecules is still poorly understood with only a few genes experimentally demonstrated to be involved in their elongation and assembly [204–206]. The synthesis of polymethyl-branched fatty acids invariably requires a polyketide synthase (Pks) which uses methylmalonyl-CoA instead of malonyl-CoA as the elongation unit, resulting in the formation of a polymethyl branched aliphatic chain.…”