Gilbert A. Diaz

Serological, Taxonomic, and Kinetic Studies of the T and M Classes of Mycobacterial Catalase

Two types of catalase may be found in extracts of mycobacteria, the heat-labile T class and the heat-stable M class. The T-catalase is resistant to 3-amino-1,2,4-triazole and has a Michaelis constant in the range of 3.1 to 6.8 mM H2O2, whereas the M-catalase is inhibited by 3-amino-1,2,4-triazole and has a Michaelis constant in the range of 143 to 156 mM. Some species of mycobacteria produce only one class of catalase, and others produce both. Of the species studied, only Mycobacterium terrae, M. triviale, and M. nonchromogenicum failed to exhibit T-catalase, although all three of these species had M-catalase. Conversely, M. tuberculosis, M. bovis, M. intracellulare, M. avium, M. gastri, M. marinum, and M. xenopi yielded T-catalase but not M-catalase. Six species, M. szulgai, M. simiae, M. kansasii, M. gordonae, M. scrofulaceum, and M. asiaticum, produced both classes. The differences in resistance to heat and 3-amino-1,2,4-triazole were exploited in the development of methods for quantitative serological characterization of one class of catalase in the presence of the other. These techniques were used with three reference sera to produce a branching diagram of divergence of the T-catalases from 13 species of mycobacteria based on measurements of immunological distance. No T-catalase could be demonstrated in another three species. A first-stage study was also carried out with a single reference antiserum to M-catalase from M. kansasii. Representatives of nine mycobacterial species, including the three that produced no T-catalase, were characterized with this reference system, which tends to yield larger immunological distance values than the T-catalase system.

Reciprocal Immunological Distances of Catalase Derived from Strains of Mycobacterium avium, Mycobacterium tuberculosis, and Closely Related Species

The structural divergence of catalase derived from different mycobacteria has previously been studied in terms of immunological distances as determined by titration with antiserum against catalase extracted from Mycobacterium tuberculosis. This study has been extended by addition of a second reference antiserum: One raised against catalase from M. avium. The two systems show a high reciprocality of immunological distance against one another. The catalase from M. intracellulare is serologically very similar to that of M. avium, and the enzyme from M. bovis is very similar to that of M. tuberculosis. Catalase extracted from BCG, a pathovar of M. bovis, appears to exhibit structural modification which diverges from the parent M. bovis and converges on M. avium. Two classes of catalase have been separated from M. scrofulaceum: One is nonreactive with either reference antiserum, and the other exhibits immunological distances that suggest that M. avium is in a position between M. scrofulaceum and M. tuberculosis.

Immunoprecipitation Studies of Mycobacterial Catalase

Serological techniques have been used to detect small changes in amino acid sequences of specific proteins in closely related biological systems. These changes are believed to reflect evolutionary divergence. In the present study, the differences in binding capacities of catalases from different mycobacteria for a reference antiserum were measured very specifically by assaying the unbound functional enzyme after exposure to antibody. Catalase derived from Mycobacterium tuberculosis has been used to immunize rabbits. The antibody so produced precipitated the enzyme but did not inactivate it. This antibody also precipitated catalase from sonic lysates of other mycobacterial species. The binding capacity of catalase derived from a number of heterologous species for the M. tuberculosis antibody was always lower than that of homologous enzyme. Some species produced catalase that failed to react at all with the reference serum. In other cases, there was evidence of at least two serologically unrelated catalases in a single strain. There was also a limited correlation between rate of inactivation of catalase by heat and the relative antibody-binding capacity of the enzyme. The serological study of catalases offers promise of providing a useful tool for clarifying evolutionary relationships among mycobacterial species.

Differentiation between T-Catalases Derived from Mycobacterium avium and Mycobacterium intracellular by a Solid-Phase Immunosorbent Assay

Antibody to the T-catalase derived from a culture of Mycobacterium avium serovar 8 was purified by immunoaffinity absorption and elution and tested in a solid-phase immunosorbent assay against T-catalases from 31 strains in the M. avium complex. The immunologic distances from the homologous antigen exhibited a bimodal distribution. Strains of serovars 1 through 6 and 8, which correspond to the M. avium deoxyribonucleic acid (DNA) homology group of Baess, yielded immunologic distance scores against the reference serovar 8 preparation ranging from 2 to 16 U. Strains of serovars 7, 12, 14, 16, 18, and 19, which correspond to the Mycobacterium intracellulare DNA homology group, yielded immunologic distance scores ranging from 20 to 30 U. Among serovars for which DNA homology data were not available, strains of serovar 10 fell into the M. avium catalase serogroup, and serovars 13, 15, 17, 20, 23, and 25 fell into the M. intracellulare group. The status of serovar 9 was ambiguous. When the reference antibody was cross-absorbed with T-catalase from a serovar 14 strain, many of the antibodies to common epitopes were removed; the remaining antibodies reacted strongly with catalases from all strains belonging to serovars of the M. avium DNA homology group, but only minimally with catalases from strains of serovars in the M. intracellulare DNA homology group. Our results provide support for continued recognition of M. avium and M. intracellulare as separate species and for reassignment of some serovars from M. intracellulare to M. avium.