Abstract
Earlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I-IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-alpha in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response.
Highlights
A key feature of all members of the genus Mycobacterium is a cell wall of unique and complex structure, which plays an important role in antibiotic resistance and pathogenesis of mycobacteria by modulating the host immune system and phagocytic cell functions [1]
We report the identification of a natural mutant of M. marinum, devoid of LOS-IV production, which allowed the production of large amounts of LOS-III and the determination of the fine structure of all LOSs
The major difference was found in M. marinum 7 (Mma7), a strain originally isolated from the butterfly fish [22], which exhibited an altered LOS profile, characterized by a defect of LOS-IV synthesis and a concomitant accumulation of LOS-III
Summary
Caryophyllose-containing Cell Wall Glycolipids That Inhibit Tumor Necrosis Factor-␣ Secretion in Macrophages*□S. A LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I– IV) are still unknown This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wallassociated glycolipids. LOSs were found and described in Mycobacterium kansasii (6 – 8), Mycobacterium gastri [8, 9], Mycobacterium szulgai [10], Mycobacterium malmoense [11], Mycobacterium gordonae [12], Mycobacterium butyricum [13], Mycobacterium mucogenicum [14], the Canetti variant of Mycobacterium tuberculosis [15] and, more recently in Mycobacterium marinum (Mma) [16] They remain among the less studied mycobacterial glycolipids at a biosynthetic, structural, and functional point of view. These molecules were used in in vitro assays to uncover their potent biological roles
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