GREEN FLUORESCENT PROTEIN-BASED DRUG SCREENING ASSAY AGAINST INTRACELLULAR MYCOBACTERIUM TUBERCULOSIS

Abstract

Novel anti-tuberculosis drugs are urgently needed. In addition to killing extracellular Mycobacterium tuberculosis, an ideal drug needs to be able to eliminate M. tuberculosis residing in macrophages. Determining viability of this intracellular organism is cumbersome, thereby making testing of drugs against macrophage-resident M. tuberculosis laborious. In order to examine macrophage-resident bacteria, M. tuberculosis H37Rv carrying a plasmid expressing a red-shifted green fluorescent protein (GFP) under control of an icl promoter that allows expression only in macrophage was constructed. Recombinant bacteria were allowed to infect U937 cells (a macrophage-like cell line) in a liquid culture for 18 hours prior to drug testing. MIC90 (minimum concentration inhibiting growth by 90%) of isoniazid, rifampin and streptomycin of 0.16, 0.08 and 2.5 µg/ml, respectively were obtained against macrophage-resident recombinant M. tuberculosis, while pyrazinamide was non-inhibitory. This GFP- based macrophage- resident M. tuberculosis drug susceptibility assay (iGFPMA) is rapid, inexpensive and suitable for a high throughput drug screening platform.