New variants of polar glycopeptidolipids detected in Mycobacterium simiae, including 'habana' strains, as evidenced by electrospray ionization-ion trap-mass spectrometry.

AIMS

To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of 'habana' strains, in a search for specific markers given the immunogenic potential of 'habana' TMC 5135 in experimental tuberculosis.

METHODS AND RESULTS

pGPLs were determined in free lipid extracts using electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS), working in both negative- and positive-ion mode. In the case of TMC 5135, the presence of the previously characterized GPL-II (containing 2,4-di-O-CH(3) glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL-III (containing 4-O-CH(3) glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some 'habana' strains presented variants of GPL-II, designated GPL-II'-A and GPL-II'-B. A di-O-CH(3)-deoxy-hexose (tentatively, 2,3-di-O-CH(3)-fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL-II'-A, whereas in GPL-II'-B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275(T) was identified as tri-O-CH(3)-glucuronic acid (designated GPL-sim(T)-I, with two variants: GPL-sim(T)-I-A and GPL-sim(T)-I-B), O-CH(3)-glucuronic acid (designated GPL-sim(T)-II) and di-O-CH(3)-glucuronic acid (GPL-II'-A and GPL-II'-B). The penultimate sugar of the oligosaccharide chain of GPL-sim(T)-I-A and GPL-sim(T)-II was identified as di-O-CH(3)-deoxy-hexose (tentatively, 2,3-di-O-CH(3) fucose), and that of GPL-sim(T)-I-B as deoxy-hexose (tentatively, fucose). In all strains studied, each M-H and M+Na ion was revealed as a mixture of homologous compounds varying in the number of -O-CH(3) groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide.

CONCLUSIONS

The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL-II'-A, GPL-II'-B, GPL-sim(T)-I-A, GPL-sim(T)-I-B and GPL-sim(T)-II) were defined. Noteworthy was the fact that the 'habana' strains clearly differed from the type strain of Myco. simiae.

SIGNIFICANCE AND IMPACT OF THE STUDY

The data obtained can be used in the delineation of the 'habana' group of Myco. simiae, including the quality control of the immunogenic strain 'habana' TMC 5135.