IDENTIFICATION OF GENETIC-LOCI IMPLICATED IN THE SURVIVAL OF MYCOBACTERIUM-SMEGMATIS IN HUMAN MONONUCLEAR PHAGOCYTES

A luminescence-based procedure that permits the rapid evaluation of th e survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstra ted impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the g ene interrupted by the transposition event was amplified by ligand-med iated PCR and sequenced, in all cases, the existence of homologous gen es of as yet unknown function were identified in the Mycobacteium tube rculosis genome. Complementation of the mutant mycobacterial strains w ith cosmids containing the homologous loci from M. tuberculosis restor ed normal intracellular growth in three of the four mutants tested, su pporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of di rectly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mono nuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.