Clofazimine is a member of the phenazine pigment family which has been successfully used in chemotherapy against a variety of mycobacteria including M. avium. The presence of clofazimine in growth medium resulted in higher carotenoid pigmentation in M. avium cells. Carotenoid pigments have been shown to quench superoxide radicals supporting the hypothesis that pigmentation possibly protected cells against superoxide. Clofazimine caused the generation of superoxide radicals in M. intracellulare strain LR163 represented by cyanide-resistant oxygen consumption. The amount of oxygen consumed was dependant upon the clofazimine concentration. This supports the hypothesis that clofazimine is antibiotic via its ability to generate toxic oxygen metabolites. Higher catalase activity was found in extracts of cells grown in the presence of a low concentration of clofazimine. At a higher concentration, the amount of catalase and superoxide dismutase activities were lower than the basal level. This finding did not agree with the hypothesis. At this point the reason for the drop in the activities (i.e. lower than basal level) is not known. Clofazimine was mildly synergistic with rifampicin. This result supports hypothesis that the defense mechanism of M. intracellulare to clofazimine was enzymatic. Clofazimine-resistant derivatives of M. intracellulare strain LR163 have been isolated. Their characterization will provide a direct approach towards determining the mode of action of clofazimine in cells. / Master of Science

Proteins of the cytoplasmic membrane of Mycobacterium avium were investigated to identify those which were: (1)intrinsic or extrinsic, (2) attached to the cell wall, (3)surface accessible and (4) excreted. In addition sera containing anti-cytoplasmic membrane proteins were obtained and preliminary purification of the cytoplasmic membrane protein was attempted. The predominating cytoplasmic membrane protein of 31,000 daltons (MCMP) was found to be intrinsic, attached to the cell wall and possibly surface accessible. The MCMP was not excreted, even in media in which the MCMP is not found in the cytoplasmic membrane. Other cytoplasmic membrane proteins were also found to be intrinsic; a few were likely to be extrinsic based upon their separation from the membrane in sucrose gradients. Cytoplasmic membrane proteins of 66, 000, 115, 000 and 129 dalton were surface accessible as judged by I 125-Iodobead labeling. Antisera against the HCMP and other cytoplasmic membrane proteins was obtained and will be useful in further cytoplasmic membrane protein characterization. Acetone precipitation of a cytoplasmic membrane preparation was performed to partially purify the MCMP. The data from this study can be used for the development of serodiagnostic reagents for detecting mycobacterial infection. / Master of Science