CLONING, SEQUENCING, AND EXPRESSION OF THE APA GENE CODING FOR THE MYCOBACTERIUM-TUBERCULOSIS 45 47-KILODALTON SECRETED ANTIGEN COMPLEX/

Effective protection against a virulent challenge with Mycobacterium t uberculosis is induced mainly by previous immunization with living att enuated mycobacteria, and it has been hypothesized that secreted prote ins serve as major targets in the specific immune response. To identif y and purify molecules present in culture medium filtrate which are do minant antigens during effective vaccination, a two-step selection pro cedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effecters induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kD a antigen complex, present in BCG culture filtrate, has been previousl y identified and isolated (F. Remain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodi es recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to done, express, and sequ ence the corresponding gene of M. tuberculosis. An M. tuberculosis shu ttle cosmid library was transferred in Mycobacterium smegmatis and scr eened with a competitive enzyme-linked immunosorbent assay to detect t he clones expressing the proteins. A clone containing a 40-kb DNA inse rt was selected, and by means of subcloning in Escherichia coli, a 2-k b fragment that coded for the molecules was identified. An open readin g frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted mole cular mass of 28,779 Da. The gene was referred to as apa because of th e high percentages of proline (21.7%) and alanine (19%) in the purifie d protein. Southern hybridization analysis of digested total genomic D NA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-te rminal identity or homology of the M. tuberculosis and M. bovis BCG pu rified molecules and their similar global and deduced amino acid compo sitions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (2 1.7%) was confirmed and explained the apparent higher molecular mass ( 45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel ele ctrophoresis resulting from the increased rigidity of molecules due to proline residues. Extensive homology with the 43L Mycobacterium lepra e antigen (B. Wieles, M. van Agterveld, A. Janson, J. Clark-Curtiss, T . Rinke de Wit, M. Harboe, and J. Thole, Infect. Immun. 62:252-258, 19 94) and the capacity to induce a potent T- and-dependent response prom pted us to analyze the role of the protein(s) for the bacteria and its effect on the immune response during M. tuberculosis infection.