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 expo sure of selected structural motifs which in turn may influence host-pa rasite interactions. To gain insight into the exposed epitopes, the pr esent study describes a flow cytometric method designed to probe defin ed molecules on dispersed mycobacteria. The hydrophobic fluorophore N- hexadecanoyl aminofluorescein inserted in the mycobacterial cell envel ope 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 demonstr ated the specific detection of the antigen on the cell surface of a Ca netti-like strain of M. tuberculosis, and not on those of mycobacteria l strains that were devoid of the glycolipid. Thus, the method was app lied to investigate the relative amounts of surface-exposed mannosylat ed compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpatho genic species Mycobacterium smegmatis. Both M. tuberculosis and M. sme gmatis are endowed with mannosyl and arabinan epitopes on their surfac es, although there are many differences in terms of exposed mannosyl e pitopes between the various strains of the tubercle bacillus examined. These differences are correlated with the amounts of terminal mannosy l residues that cap the surface-exposed arabinomannans (A. Ortalo-Magn e, A. B. Andersen, and M. Daffe, Microbiology 142:927-935, 1996) but n ot with the degrees of virulence of the strains. This novel approach c ould provide new insights into the distribution of defined surface-exp osed antigens and thereby into the architecture of the cell envelopes.