The overall objectives of the proposed research are to develop a diagnostic method for the early stage of M. avium-intracellulare infection in AIDS patients and also to find an effective chemotherapeutic method which will overcome multiple drug resistance of this group of mycobacteria. The immediate objectives are 1) to determine the genetic relatedness among various strains of M. avium-intracellulare, 2) to identify a species-specific DNA sequence of this group of mycobacteria, 3) to isolate a monospecific antigen of these organisms, and 4) to clarify the role played by chromosomal and plasmid DNAs in drug resistance. The following experimental approach will be undertaken to achieve this goal. Chromosomal DNAs of representative strains of M. avium-intracellulare will be analyzed kinetically using spectrophotometric assays of DNA-DNA hybridization in free solution. Through this method, genome sizes and base ratios of individual strains, and DNA homologies among them will be determined. DNA relatedness will be also examined by the dot hybridization method, using 32p- or biotin-labeled DNA probes. These studies will facilitate the screening process of the following cloning experiments. To determine a species-specific DNA sequence, a colony bank of each representative strains of M. avium-intracellulare will be constructed in Escherichia coli. Small fragments of chromosomal DNA prepared mechanically or enzymatically will be ligated to plasmid vectors such as pBR322 and pMOB45, either directly or using synthetic linkers or homopolymeric tailing. Resultant composite plasmid molecules will be introduced into E. coli through transformation. Colonies harboring plasmids with a species-specific sequence will be selected by repeated colony hybridization using DNA probes derived from closely related species. A species-specific DNA thus prepared will be labeled with either 32P or biotin and used as a probe for identification of M. avium-intracellulare by dot hybridization. E. coli colonies containing a species-specific sequence will be further examined for their ability to produce a monospecific antigen by radioimmunoassay and also by delayed hypersensitivity test in guinea pigs. To study the mechanisms involved in drug resistance, chromosomal and plasmid DNAs isolated from drug-resistant strains will be transferred to a drug-sensitive strain of M. smegmatis using transformation and spheroplast fusion procedures. Resistance due to the permeability barrier will be examined in vitro in the presence of surfactants. Resistance due to an acquisition of the inactivating enzymes will be examined in the presence of various metal compounds which block enzymatic activity.
