Abstract
The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry 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 revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to 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 recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.
Highlights
The immune protection against tuberculosis can be achieved only by prior vaccination with a living vaccine [1,2,3]
The development of recombinant DNA systems for efficient expression of mycobacterial genes in Escherichia coli appeared as an attractive alternative for obtaining larger amounts of mycobacterial antigens important for immune responses
We describe the purification and biochemical characterization of Apa known as the 45/47-kDa complex, a major immunodominant antigen secreted in vitro by the bacteria of the M. tuberculosis complex (i.e. M. tuberculosis, Mycobacterium bovis, and BCG)
Summary
The immune protection against tuberculosis can be achieved only by prior vaccination with a living vaccine [1,2,3] An explanation of this phenomenon could be that living bacilli release protective antigens, which are not present in sufficient amounts in dead bacteria or which are not appropriately presented to T lymphocytes. The development of recombinant DNA systems for efficient expression of mycobacterial genes in Escherichia coli appeared as an attractive alternative for obtaining larger amounts of mycobacterial antigens important for immune responses. Recent reports on post-translational modifications of mycobacterial antigens such as acylation and glycosylation [7, 8] emphasized the importance of comparing structure and biological properties of native bacterial products to those obtained by recombinant DNA technology
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