Biochemical studies on some aspects of nitrogen metabolism in mycobacterium sp

Abstract

The inexorable rise in cases of tuberculosis worldwide fuelled by the HIV epidemic highlights the need for new drugs and particularly those that can shorten the duration of treatment. This thesis describes three approaches for the discovery of new novel therapeutic agents against the disease. First approach is the evaluation and characterization of the biochemical role of enzymes related to nitrate metabolism of Mycobacteria during survival in its dormant or latent stage. The second approach is development of a simple screening assay, which could be used to pick up dormant stage specific inhibitors at large scale. The third approach is screening of diverse chemical library to search biologically active molecules against dormant stage of the bacilli. A respiratory type of nitrate reductase, NarGHJI was identified as a first report in Mycobacterium smegmatis during this study. This enzyme’s activity was found to be induced during hypoxic shiftdown of the culture to dormant stage in Wayne’s in vitro model. More significantly blockage of the enzyme’s function by specific inhibitors led to a steep reduction in viability of the bacilli during hypoxic stage survival in Wayne’s in vitro model, which indicated this enzyme as a potential drug target for latent stage. Furthermore, a complete pathway of the assimilation of nitrate was found to be present in M. smegmatis, M. bovis BCG and M. tuberculosis, which also played significant role during its hypoxic stage persistence. Nitrate reductase, NarGHJI also played a significant role during survival of M. tuberculosis within the THP- macrophage environment which indicated the generation of microaerobic environment in the host cells which harbors the pathogen, similar to Wayne’s hypoxic model. Interestingly the essentiality of nitrate reductase, NarGHJI was found irrespective of presence or absence of nitrate in the medium during this intracellular survival of the organism, which indicated the production of nitrate and/ or nitrite in the activated macrophages. We could also use the whole cell activity of nitrate reductase as a reporter system of dormant stage to develop a simple, rapid, and robust cell based high throughput assay for facilitating dormant stage antitubercular screening.