INDUCTION OF MYCOBACTERIUM-AVIUM GENE-EXPRESSION FOLLOWING PHAGOCYTOSIS BY HUMAN MACROPHAGES

Little is known about the bacterial factors that enable pathogenic myc obacteria to survive and multiply within the macrophages of the infect ed host. By preparing cDNA from Mycobacterium avium bacilli grown in h uman-derived macrophages and in broth culture and using subtractive hy bridization to remove commonly expressed genes, a procedure was develo ped to identify genes of M. avium that are specifically expressed when the bacilli are growing within macrophages. Total RNA was isolated fr om M. avium recovered 5 days after infection of human macrophages and from bacilli grown in vitro in broth. Mycobacterial mRNAs were convert ed to cDNA by reverse transcription. Biotin-modified cDNAs prepared fr om M. avium grown in broth culture were used to subtract the housekeep ing genes from the cDNAs of the macrophage derived M. avium. After eac h round of subtraction, a sample of the unsubtracted cDNA was amplifie d, labeled, and hybridized to cosmid clones of M. avium DNA. After thr ee rounds of subtraction, the amplified DNA hybridized to approximatel y 1% of the cosmid clones under stringent conditions. Although the maj ority of the genes that are induced in phagocytized M. avium cells are expressed in the broth-grown bacilli, one DNA fragment that was ident ified coded for an mRNA that is highly specific for M. avium in phagos omes. This procedure will be especially useful for identifying genes t hat are expressed in response to growth in specific environments from organisms with genetic systems that are not well characterized.