Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug‐resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug‐resistant phenotype have been identified by evaluating INH‐resistant clinical isolates and in vitro mutants. In this study, site‐directed mutagenesis techniques were utilized to alter the wild‐type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG ‐defective, INH‐resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.

The resurgence of tuberculosis, the emergence of multiple drug resistant tuberculosis, and the increasing prevalence of mycobacterial disease in AIDS patients have increased the importance of defining new mycobacterial antigens that can be utilized in the development of improved diagnostic reagents and more effective vaccines. In this report, a highly immunogenic Mycobacterium kansasii protein (MK35) and the gene encoding this antigen were characterized. MK35 gene probes reacted with genomic DNA from M. avium, M. bovis BCG, M. intracellulare and M. tuberculosis but not with DNA isolated from nine other mycobacterial species. Nucleotide sequence analysis showed that the MK35 gene encodes a 26 kDa protein which contains a consensus bacterial lipoprotein processing sequence. In addition, detergent-phase separation studies strongly suggested that MK35 is a lipoprotein. Skin test assays demonstrated that MK35 elicited a strong response in guinea pigs sensitized with M. kansasii but did not react in M. tuberculosis-sensitized guinea pigs. These results further suggest that mycobacterial lipoproteins are immunogenic antigens that should be considered in the development of new mycobacterial vaccines and diagnostic reagents.

Mycobacterium avium, Mycobacterium intracellulare complex (MAC) bacilli are an important cause of bacteraemia in AIDS patients but treatment is complicated by their resistance to the usual antimycobacterial agents. In this study of 20 strains of MAC none was found to have the mutations associated with resistance to rifampicin and streptomycin in M. tuberculosis suggesting that MAC have unique mechanisms for resistance to these agents.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.

Genetic and biochemical studies have suggested a link between reduced catalase activity and resistance to isoniazid in Mycobacterium tuberculosis. In this study, we examined the molecular mechanisms of resistance to isoniazid with six in vitro mutants of the M. tuberculosis complex (Mycobacterium bovis and M. tuberculosis). Five of six mutants resistant to isoniazid were negative by catalase assays. Immunoblot analyses using a polyclonal antibody against the katG gene product (catalase-peroxidase) demonstrated that the enzyme is not produced in four of these isoniazid-resistant strains. A complete deletion of the katG gene was detected in only one of these isoniazid-resistant M. tuberculosis complex strains by Southern blot analyses. In two other resistant strains, partial deletions of the katG gene were identified. A point mutation which resulted in the insertion of a termination codon in the katG coding sequence caused a catalase-negative phenotype in a fourth strain. Of the two resistant strains which produce the enzyme, one was shown to be negative by a catalase assay. Single-stranded conformational polymorphism and DNA sequence analyses identified a mutation in the katG gene of this strain which may contribute to reduced enzymatic activity and subsequent isoniazid resistance. These data demonstrate that genetic alterations to the katG gene other than complete deletions are prevalent and may contribute significantly to the number of cases of isoniazid-resistant tuberculosis.

Mycobacterial disease remains a serious international public health concern. Improved methods to rapidly and specifically detect mycobacterial infections would greatly enhance clinical management of these diseases. To define species-specific T cell epitopes that may be useful for the immunodiagnosis of mycobacterial infections, polymerized synthetic peptides from the 19-kD Mycobacterium tuberculosis and Myco. intracellulare protein homologues were tested in guinea pig DTH assays. Five Myco. tuberculosis and eight Myco. intracellulare peptides evoked skin test responses. Although all of the active Myco. tuberculosis and seven of the Myco. intracellulare peptides elicited non-specific DTH reactions, the peptide IN13 induced a Myco. intracellulare-specific skin test reaction, and thus represents a specific Myco. intracellulare T cell DTH epitope. This result suggests that the development of monospecific peptide-based immunodiagnostic reagents may be feasible for future clinical use.

Disseminated mycobacteremia resulting from Mycobacterium avium-Mycobacterium intracellulare complex (MAC) infections frequently contribute to the morbidity and mortality seen in AIDS patients. To better understand the immunopathology of MAC disease and to identify molecules that may have potential diagnostic and vaccine utility, an immunoreactive M. intracellulare protein (MI43) and the gene encoding this antigen were characterized. Southern blot hybridizations demonstrated that MI43 gene probes reacted only with genomic DNA from M. intracellulare, M. avium, and Mycobacterium asiaticum and not with DNA isolated from 11 other mycobacterial species. Nucleotide sequence analysis showed that the MI43 gene encodes a 27-kDa protein which contains a consensus bacterial lipoprotein processing sequence. Detergent-phase separations and metabolic labeling with [3H]palmitate also suggested that MI43 is a lipoprotein. Serological assays demonstrated that recombinant MI43 fusion proteins react with sera from M. avium-infected mice, sera from patients with MAC disease, and sera from patients with active tuberculosis. These results further suggest that mycobacterial lipoproteins are important immunogens that should be considered in the development of improved mycobacterial vaccines and diagnostic reagents.

The recent emergence of indolent and rapidly fatal drug-resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal S12 protein (rpsL gene) from streptomycin-resistant strains and their streptomycin-sensitive parental strains. We have demonstrated that five singly SmR M. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug-resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug-sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.

SummaryDisseminated Mycobacterium avium/Mycobacterium intracellulare complex (MAC) disease is a frequent complication in patients with the acquired immune deficiency syndrome (AIDS). In this report, we present the nucleotide sequence of the M. intracellulare MI22 gene. Computer sequence comparisons reveal that the MI22 gene, which encodes a serologically active protein, has 78% DNA sequence identity and 77% protein sequence identity with the seroreactive 19 kDa Mycobacterium tuberculosis lipoprotein antigen. Southern blot hybridizations indicate that an MI22 gene probe binds similar—‐sized restriction fragments in M. tuberculosis and M. intracellulare genomic DNA. In addition, immunoblot analyses demonstrate that MI22 is recognized by sera from tuberculosis patients. These data further support the existence of 19 kDa MAC and M. tuberculosis protein homologues. Phase partitioning experiments and the presence of a consensus lipid modification site in the deduced MI22 protein sequence strongly suggest that MI22 is also a lipoprotein. Comparative analyses of these mycobacterial antigenic homologues may provide the basis for the design of species‐specific diagnostic reagents.

The immunoreactivity of four recombinant Mycobacterium intracellulare beta-galactosidase fusion proteins, which correspond to 22, 40, 43 and 85 kDa M. intracellulare antigens, was assessed. Lymphoproliferative assays demonstrated that Escherichia coli lysates containing each of the fusion proteins stimulated T cells in vitro. Purified preparations of three of these recombinant M. intracellulare antigens (22, 43 and 85 kDa) also induced delayed-type hypersensitivity (DTH) reactions in sensitized guinea pigs. However, the skin test responses evoked by each of these antigens was not species-specific. Given these results, the potential utility as skin test reagents of the purified antigens or peptides derived from these proteins is discussed.

The activation of catalase genes in response to oxidative stress may contribute to the intracellular survival of mycobacteria. In this report, the nucleotide sequence of a mycobacterial catalase gene is described. The deduced protein sequence of this Mycobacterium intracellulare gene (MI85) was 60% identical to the Escherichia coli hydroperoxidase I (HPI) protein, 59% identical to the Salmonella typhimurium (HPI) catalase, and 47% identical to a Bacillus stearothermophilus peroxidase. The MI85 protein, expressed in E. coli, has also been shown to have peroxidase and catalase activities. Furthermore, Southern blot hybridizations, which demonstrated that a MI85 gene probe hybridizes with chromosomal DNA from thirteen different strains of mycobacteria, suggest that this catalase-peroxidase gene is prevalent in the mycobacterial genus. The availability of catalase gene probes should permit an evaluation, at the molecular level, of the role of catalase in mycobacterial pathogenesis.

Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection of M. avium infection is difficult, and treatment regimens are often ineffective. Much needs to be learned about antigens and factors responsible for immunity to and pathogenesis of the disease. Specific antigens and diagnostic procedures for infection need to be developed. To address some of these problems, we have generated 25 different monoclonal antibodies against a serovar 4 strain of M. avium isolated from a patient with AIDS. Protease sensitivity studies have demonstrated that each of these antibodies recognizes a protein-associated epitope. Immunoblot analyses suggest that seven of these monoclonal antibodies react specifically with M. avium and M. intracellular epitopes. Immunoreactive bacteriophages were identified from an M. avium lambda gt11 expression library with two of these monoclonal antibodies (3808 C3 and 3954 B12). Lambda lysogens, generated from the immunoreactive bacteriophages, overproduced beta-galactosidase fusion proteins which were reactive with the two monoclonal antibodies in immunoblot assays. The purified fusion proteins were shown to elicit skin test reactions in sensitized guinea pigs.

The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in recent years primarily because a significant proportion of acquired immunodeficiency syndrome patients develop disseminated M. avium complex disease. In an effort to develop new tools to study these infections, we have produced eight monoclonal antibodies directed against M. avium. Western blot (immunoblot) specificity analysis and protease sensitivity assays indicate that four of these antibodies recognize M. avium-specific protein epitopes and two react with M. avium complex-specific peptide determinants. These monoclonal antibodies may be useful clinically in the diagnosis of M. avium complex disease and in the laboratory for isolation and characterization of native and recombinant M. avium complex antigens.

Four bacteriophages expressing different immunoreactive recombinant Mycobacterium intracellulare antigens were isolated from a lambda gt11 library with monoclonal antibodies to M. intracellulare. These four antibodies reacted with native M. intracellulare proteins of 54, 43, 40/38, and 22 kilodaltons. Southern blot hybridizations with DNA probes prepared from insert fragments of these bacteriophages confirmed the M. intracellulare derivation of the inserts. The physical maps of the immunoreactive phages were deduced by restriction enzyme digestions. The molecular weights of the expressed recombinant antigens were determined by Western (immuno-) blotting.

The plasmid profiles of 12 Mycobacterium avium strains isolated from 12 different patients with acquired immunodeficiency syndrome were analysed. Plasmids were identified in 9 of these strains. Plasmids were isolated from all 7 serovars 4 and 8 strains, a serovar 20a strain and an untypeable strain, but were not detected in either of 2 serovar 3b strains or an untypeable isolate. Southern blot hybridisations revealed that extracts derived from all of the plasmid-containing strains hybridised to a DNA probe prepared from known mycobacterial plasmid sequences. However, restriction analyses suggest that native plasmids which hybridised to the DNA probe and were similar in mass were not identical.

Disseminated Mycobacterium avium-Mycobacterium intracellulare (M. avium complex) disease is a prevalent opportunistic infection in patients with acquired immune deficiency syndrome. Because of the increasing importance of this disease, an M. avium complex lambda gt11 expression library was prepared. We screened the library with an absorbed anti-M. intracellulare serum and identified a recombinant phage which expressed a 190-kilodalton beta-galactosidase-M. intracellulare fusion protein. Lysates containing the 190-kilodalton fusion protein evoked strong humoral and cell-mediated responses. The immunoreactivity of the M. intracellulare recombinant protein suggests that antigens isolated from the expression library may be useful as skin test, serodiagnostic, or immunoprophylactic reagents for M. avium complex disease.