Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans.

The physical arrangement of cell envelope components leads to the exposure of selected structural motifs which in turn may influence host-parasite interactions. To gain insight into the exposed epitopes, the present study describes a flow cytometric method designed to probe defined molecules on dispersed mycobacteria. The hydrophobic fluorophore N-hexadecanoyl aminofluorescein inserted in the mycobacterial cell envelope permitted focusing of fluorescence-activated cell sorter analysis on cells that were further labeled with defined monoclonal antibodies and fluorochrome-coupled streptavidin. The use of antibodies directed against the lipooligosaccharide of Mycobacterium tuberculosis demonstrated the specific detection of the antigen on the cell surface of a Canetti-like strain of M. tuberculosis, and not on those of mycobacterial strains that were devoid of the glycolipid. Thus, the method was applied to investigate the relative amounts of surface-exposed mannosylated compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpathogenic species Mycobacterium smegmatis. Both M. tuberculosis and M. smegmatis are endowed with mannosyl and arabinan epitopes on their surfaces, although there are many differences in terms of exposed mannosyl epitopes between the various strains of the tubercle bacillus examined. These differences are correlated with the amounts of terminal mannosyl residues that cap the surface-exposed arabinomannans (A. Ortalo-Magne, A. B. Andersen, and M. Daffe, Microbiology 142:927-935, 1996) but not with the degrees of virulence of the strains. This novel approach could provide new insights into the distribution of defined surface-exposed antigens and thereby into the architecture of the cell envelopes.