Over the past decade there has been a resurgence of tuberculosis in the United States accompanied by increasing problems with multidrug-resistant strains. This has stimulated a critical reappraisal of our knowledge about the host-pathogen interaction with the hope of understanding factors contributing to virulence and generating new and improved vaccines. For several intracellular bacterial pathogens survival within activated phagocytes is facilitated by the production of superoxide dismutase (SOD) that counters reactive oxygen intermediates (ROI) generated by the phagocytes. The role of superoxide dismutase produced by Mycobacterium tuberculosis in the pathogenesis of disease remains uncertain. Questions regarding the contribution of SOD to the intracellular survival of M. tuberculosis within human macrophages can be best answered by the construction and evaluation of isogeneic strains of M. tuberculosis that differ only in whether or not SOD is produced. Although genetic manipulation of slow-growing mycobacteria has proven to be difficult, new approaches offer hope that the construction of isogeneic SOD-negative and positive mycobacterial strains can be accomplished. We used PCR to amplify a sodA-containing DNA fragment from the chromosomal DNA of M. tuberculosis strain H37Rv. The fragment has been cloned into an E. coli plasmid and a selectable marker inserted into a unique pstI site of sodA. Currently, portions of this recombinant, inactivated sodA comprising about 100 base pairs on either side of and including the selectable marker have been PCR-amplified, cloned into a non-mycobacterial vector, and are being introduced into slow-growing mycobacteria in an attempt to achieve homologous recombination and gene inactivation. The isogeneic strains will then be studied in vitro with human macrophages to determine whether they stimulate the production of superoxide and whether they can be killed by macrophages alone or with the addition of various cytokines. In summary, The primary goal of this project is to determine whether SOD contributes to the intracellular survival of M. tuberculosis within human macrophages.
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