Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid

Katherine Vallejos-Sánchez,Helena Maruenda,Mirko Zimic,Emily Toscano,Daniela E Kirwan,Harry Saavedra,Patricia Sheen,R H Gilman,Juan M Lopez,Ricardo Antiparra,L M Amzel

doi:10.1038/s41598-020-65173-z

Abstract

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported.

Highlights

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens
The present study evaluated the interaction between RpsA and pyrazinoic acid (POA), as well as between RpsA and transfer-messenger RNA (tmRNA), for both the RpsA wild type (RpsA WT) and RpsA ∆A438 proteins, using a range of techniques including Electrophoretic Mobility Shift Assay (EMSA), Isothermal Titration Calorimetry (ITC), Microscale Themophoresis (MST) and Nuclear Magnetic Resonance spectroscopy (NMR)
Free tmRNA showed greater electrophoretic mobility than tmRNA incubated in the presence of RpsA WT or RpsA ∆A438 (Fig. 2), confirming an interaction between both RpsA variants with tmRNA, as described previously[11,12]

Summary

Introduction

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. If the pH of the extracellular environment is acidic, the POA is protonated to POAH, re-enters the cell, and the proton is released in the cytosol This cycle repeats itself, causing an intra-cellular accumulation of POA and a reduction in the pH of the www.nature.com/scientificreports cytoplasm. This leads to a lethal alteration of membrane permeability; the exact mechanism by which this occurs is not yet known[7] It is important to remark, that other recent studies, found that extracellular acid pH is not required to have PZA/POA achieve its lethal effect[8,9,10]. This confirms that the real mechanism of action of PZA is more complicated than though, and significant further research is required

Methods

Results

Conclusion