The Genes Coding for the Antigen 85 Complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG Are Members of a Gene Family: Cloning, Sequence Determination, and Genomic Organization of the Gene Coding for Antigen 85-C of M. tuberculosis

and genomic organization of the gene coding gene family: cloning, sequence determination, Mycobacterium bovis BCG are members of a of Mycobacterium tuberculosis and The genes coding for the antigen 85 complexes A gene encoding the 33-kDa secreted protein of Mycobacterium tuberculosis (antigen 85-C) was isolated and sequenced. The corresponding DNA sequence contains a 1,020-bp coding region. The deduced amino acid sequence corresponds to a 340-residue protein consisting of a 46-amino-acid signal peptide and a 294-amino-acid mature protein. Comparison with previously described genes for the 30-kDa antigen (the a antigen of M. bovis BCG, also called antigen 85-B) and the 32-kDa antigens from M. bovis BCG and M. tuberculosis (antigens 85-A) indicates that the three genes share considerable sequence homology (70.8 to 77.5%) but may also code for distinctive epitopes. Strong differences among the three sequences are clearly visible upstream and downstream from the region coding for the mature proteins. The three genes have been detected in the genome of M. bovis BCG by Southern blot hybridization with three type-specific probes. Furthermore, hybridization of large DNA fragments (100 to 1,000 kbp) from M. tuberculosis separated by pulsed-field gel electrophoresis showed that the three genes coding for the antigen 85 complex are not clustered within the bacterial genome. Three antigenic proteins (85-A, 85-B, and 85-C) have been identified by using counterimmunoelectrophoresis of myco-bacterial culture filtrates (5, 27). Isoelectric focusing of the purified complex antigen also indicates that the complex consists of several proteins (5a). We have previously purified a protein with a molecular mass of 32 kDa from zinc-deficient Mycobacterium bovis BCG culture filtrate. This protein was identified as antigen 85-A (7). Its NH2-terminal amino acid sequence (Phe-Ser-Arg-Pro-Gly-Leu) is identical to that reported for the a antigen (antigen 85-B) protein purified from M. bovis BCG substrain Tokyo (28). The antigen 85 complex is present among different strains of mycobacteria (6). It is secreted by living bacilli as a predominant protein in normal Sauton culture filtrate and could be useful in the serodiagnosis of tuberculosis (24) and leprosy (19). Furthermore, the 32-kDa protein induces specific lymphoproliferation and gamma interferon production in peripheral blood leukocytes from patients with tuberculosis (11) and those with leprosy and from purified protein derivative-and lepromin-positive healthy subjects (13a). Recent findings indicate that the amount of 32-kDa protein-induced gamma interferon in BCG-sensitized mouse spleen cells is under probable H-2 control (10). Finally, the high affinity of mycobacteria for fibronectin is …