Lipooligosaccharidic antigen containing a novel C4-branched 3,6-dideoxy-alpha-hexopyranose typifies Mycobacterium...

Lipooligosaccharides (LOSs) have recently been proposed as markers of mycobacterial avirulence. They have been characterized in Mycobacterium kansasii cell wall and were investigated in Mycobacterium gastri since the distinction between the two mycobacterial species remains in some question. A set of unknown LOSs was isolated from M. gastri W471. The highly antigenic lipooligosaccharide, LOS-III, was purified and appeared in all the M. gastri strains investigated regardless of their morphology. Moreover, by enzyme-linked immunosorbent assay and chromatographic approaches, it was found that LOS-III unambiguously distinguished M. gastri from the opportunistic pathogen M. kansasii. The LOS-III structure was established from its native form using NMR spectroscopy. This strategy revealed the presence of a supplementary monosaccharide (X) which was not characterized by routine carbohydrate analysis. Its core structure, 3,6-dideoxy-alpha-hexopyranose, was established from the complete assignment of the 1H and 13C spectra by two-dimensional homonuclear (COSY, HOHAHA) and heteronuclear 1H-13C heteronuclear multiple quantum correlation spectroscopy (HMQC) and HMQC-HOHAHA spectroscopy. Due to the absence of a proton at C4, the key data of the C4 side chain structure came from the heteronuclear multiple bond correlation spectroscopy (HMBC) spectrum. It was revealed to be a C-alkyl chain of partial structure 1,3-dimethoxypropyl. From the HMBC spectrum, this novel C-branched monosaccharide was located at the nonreducing end of the LOS, while the putative reducing end was found to consist of a 2',4,6-triacylated alpha-alpha-trehalose. The following structure, based on the evidence presented in this paper, is proposed for LOS-III: Xp alpha(1-->3)[L-Xylp beta(1-->4)](6)3-O-Me-Rhap alpha(1-->3)D- Galp beta(1-->3)-D-Glcp beta(1-->4)2-O-acyl-D-Glcp-alpha(1<==>1)alpha 4,6-di-O- acyl-D-Glcp.