Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bov is, or BCG have been identified as major immunodominant antigens, Mass spec trometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revea led a different mannosylation pattern: species containing 7 to 9 sugar resi dues/mole of protein were in the highest proportion, whereas species bearin g a low number of sugar residues were almost absent. The 45/47-kDa recombin ant antigen expressed in E. coil was devoid of sugar residues. The proteins purified from II M.. tuberculosis, M. bovis, or BCG have a high capacity t o elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and t o stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG, The recombinant Apa expressed in Mycobacterium smegmatis was 4 -fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recomb inant protein expressed in Escherichia coil was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro, Thus the observe d biological effects were related to the extent of glycosylation of the ant igen.