Molecular characterization of the eis promoter of Mycobacterium tuberculosis and expression of eis in culture and during macrophage infection

Abstract

Previous work in the laboratory of Dr. Richard Friedman identified the eis gene as capable of enhancing the survival of avirulent Mycobacterium smegmatis within U-937 macrophage-like cells. Studies were undertaken to identify the promoter for eis in both M. smegmatis and M. tuberculosis H37Ra. A 412 base pair region upstream of the eis coding sequence, as well as a DinR-like cis element, was necessary for the full expression of eis in both mycobacteria. To study the expression of eis further, a destabilized luciferase-based reporter system was constructed for fusion of the 412 base pair eis promoter. The expression of eis was monitored throughout culture growth of both M. smegmatis and M. tuberculosis H37Ra using this novel reporter and quantitative real-time PCR. In M. smegmatis, eis was induced upon transition from logarithmic to stationary phase as detected by both luciferase activity and real-time PCR. In M. tuberculosis H37Ra, expression from the eis promoter driven luciferase construct could not be detected, although real-time PCR showed that eis was constitutively expressed. Analysis of groEL2 expression by real-time PCR in comparison with phsp60 driven luciferase production showed that this novel luciferase system could also detect expression changes in M. tuberculosis H37Ra. The lack of luciferase expression from the eis promoter suggested that the utility of this system was dependent on the promoter. eis and groEL2 expression were then monitored in both mycobacteria during the infection of U-937 cells. In M. smegmatis, analysis of eis expression using luciferase suggested that eis was not induced during initial infection, but showed that eis is repressed between 3 h and 24 h of infection. Luciferase production from the eis promoter did not change upon the infection of macrophages with M. tuberculosis H37Ra, suggesting that the eis promoter construct was non-functional. Real-time PCR analysis of eis expression during infection of U-937 cells with M. tuberculosis H37Ra showed that eis is constitutively expressed during macrophage infection. A comparison of groEL2 expression and luciferase activity from phsp60 showed high variability in heat-shock expression and suggested that the luciferase system was not suitable for detecting groEL2 expression changes during macrophage infection.