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bioRxiv · 10.1101/2020.03.31.018390

EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE

Antiparra, R. A.·Santos, M.·Toledo, A. K.·Gilman, R. H.·Kirwan, D. E.·Sheen, P.·Zimic, M.
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Abstract

Pyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood. It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis. To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility. Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 {micro}g/mL and >7500 {micro}g/mL respectively).

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Antiparra, R. A., Santos, M., Toledo, A. K., Gilman, R. H., Kirwan, D. E., Sheen, P., Zimic, M.. 2020-03-31. EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE. https://doi.org/10.1101/2020.03.31.018390

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