Ectopic Expression of Rv0023 Mediates Isoniazid/Ethionamide Tolerance via Altering NADH/NAD+ Levels in Mycobacterium smegmatis.

Shailesh Kumar Gupta,Rajendra Kumar Angara,Suhail Yousuf,Akash Ranjan,Chilakala Gangi Reddy

doi:10.3389/fmicb.2020.00003

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) accounts for nearly 1.2 million deaths per annum worldwide. Due to the emergence of multidrug-resistant (MDR) Mtb strains, TB, a curable and avertable disease, remains one of the leading causes of morbidity and mortality. Isoniazid (INH) is a first-line anti-TB drug while ethionamide (ETH) is used as a second-line anti-TB drug. INH and ETH resistance develop through a network of genes involved in various biosynthetic pathways. In this study, we identified Rv0023, an Mtb protein belonging to the xenobiotic response element (XRE) family of transcription regulators, which has a role in generating higher tolerance toward INH and ETH in Mycobacterium smegmatis (Msmeg). Overexpression of Rv0023 in Msmeg leads to the development of INH- and ETH-tolerant strains. The strains expressing Rv0023 have a higher ratio of NADH/NAD+, and this physiological event is known to play a crucial role in the development of INH/ETH co-resistance in Msmeg. Gene expression analysis of some target genes revealed reduction in the expression of the ndh gene, but no direct interaction was observed between Rv0023 and the ndh promoter region. Rv0023 is divergently expressed to Rv0022c (whiB5) and we observed a direct interaction between the recombinant Rv0023 protein with the upstream region of Rv0022c, confirmed using reporter constructs of Msmeg. However, we found no indication that this interaction might play a role in the development of INH/ETH drug tolerance.

Highlights

Tuberculosis (TB) remains a major cause of death worldwide and the leading cause by a single infectious agent (World Health Organisation, 2018)
Rv0023 is a non-essential gene in Mycobacterium tuberculosis (Mtb), yet it regulates a high number of genes in the genome (Sassetti et al, 2003; Rustad et al, 2014)
Mycobacterium smegmatis (Msmeg) genome does not harbor the ortholog of Rv0023 and serves as a good model to study the function of Rv0023

Summary

Introduction

Tuberculosis (TB) remains a major cause of death worldwide and the leading cause by a single infectious agent (World Health Organisation, 2018). MDR TB constitutes 3.5% of new TB cases and 18% of previously treated cases (World Health Organisation, 2018) To overcome these challenges and to better counter resistance in Mycobacterium tuberculosis (Mtb), understanding the mechanisms and deciphering the pathways majorly responsible for generating resistance are greatly required. The role of the ndh gene mutations in INH and ETH co-resistance in Mycobacterium smegmatis (Msmeg) and Mycobacterium bovis (Mbovis) has been shown (Vilcheze et al, 2005), while the role of ndh in conferring resistance in Mtb is yet to be determined. In Msmeg, mutations in ndh lead to an increase in NADH cellular concentration and inhibition of INH-NAD and ETH-NAD adducts formation (Vilcheze et al, 2005)

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Conclusion