A host-vector system for molecular study of the intracellular growth ofMycobacterium tuberculosisin phagocytic cells

Abstract

The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc(2)155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli- mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes.