Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Department of Host Defense, and Virology, Osaka City University Graduate School of Medicine, Osaka 545-8585, Japan, Department of Microbiology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan, Japan BCG Laboratory, Tokyo 204-0022, Japan, Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan, Department of Microbiology, Immunology and Pathology, Colorado State University, Colorado 80523, USA, Department of Immunology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan, Molecular Epidemiology Division, Mycobacterium Reference Center, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo 204-8533, Japan

^* To whom correspondence should be addressed. Email: fujiwara{at}med.osaka-cu.ac.jp.

	Abstract

Mycobacterium avium-intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria, and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine, and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and NMR analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy--hexose-(13)-4-O-methyl--L-rhamnose-(13)--L-rhamnose-(13)--L-rhamnose-(12)-6-deoxy--L-talose. Next, the 22.9 kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide, was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include 3 glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. The M. avium serotype 1 strain was transformed with cosmid clone #253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Taken together, this serotype 16-specific gene cluster is responsible for the biosynthesis of serotype 16 GPL.