Proteins secreted from Mycobacterium tuberculosis during growth are believed to be important for protective immunity against tuberculosis. We have investigated the growth of M. tuberculosis in an enriched liquid medium. The release of isocitrate dehydrogenase from the bacilli served as a marker of autolysis and was observed during the late logarithmic growth phase. The release of proteins during the culture period was investigated by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three major groups of proteins, which differed markedly with respect to profile of release and location in intact bacilli, were defined. A short-term filtrate devoid of autolytic products was defined and found to be composed of 33 major components. Five proteins were identified by monoclonal antibodies. Pronounced superoxide dismutase activity was detected in the filtrate. The enzyme was purified and identified as a dominating component of short-term filtrate.

An infection model of human tuberculosis was established with C57BL/6J mice. The lymphocyte proliferative responses to antigens from Mycobacterium tuberculosis were investigated during the course of infection and compared with results obtained with a group of mice immunized with large amounts of killed bacteria. The two groups responded similarly to a number of mycobacterial antigens, but marked differences in responses against secreted antigens were found; only infected mice responded vigorously to these. The responding lymphocyte subpopulation was made up of L3T4+ T lymphocytes under restriction of the Ia molecule.

Cross-reactions between five proteins actively secreted by Mycobacterium tuberculosis were studied by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting, and ELISA using polyclonal rabbit antisera and mouse monoclonal antibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT51 protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross-react extensively, each of the components containing component-specific as well as cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. N-terminal sequence studies revealed striking homology at the amino acid level between 85A, 85B, 85C and MPT51. MPT64 showed less homology. In addition, striking homology was demonstrated between two different stretches within the 85B sequence and indicated between three stretches within the MPT64 molecule. Thus, a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. MPT64 may also belong to this family.

Protein antigen b (Pab) of Mycobacterium tuberculosis has previously attracted interest because of its immunological and diagnostic relevance. In this study we present evidence that Pab possesses a signal sequence and is secreted from the cytoplasm of M. tuberculosis. The synthesis of Pab is enhanced under phosphate starvation indicating that the protein is involved in phosphate metabolism in M. tuberculosis.

A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals. * Corresponding author. weeks at 43°C, and Mycobacterium marinum was grown for 5 weeks at 33°C. Mycobacterium scrofilaceum was grown 2317 Vol. 60,No. 6 2318 ANDERSEN ET AL.