Characterization of the MurT/GatD complex in Mycobacterium tuberculosis towards validating a novel anti-tubercular drug target.

Arundhati Maitra,Sanjib Bhakta,Nicholas H Keep,Liam T Martin,Antima Gupta,Kristine B Arnvig,Alethea B Tabor,Syamasundari Nukala,Rachael Dickman,Adrian J Shepherd,Tulika Munshi

doi:10.1093/jacamr/dlab028

Abstract

ObjectivesIdentification and validation of novel therapeutic targets is imperative to tackle the rise of drug resistance in tuberculosis. An essential Mur ligase-like gene (Rv3712), expected to be involved in cell-wall peptidoglycan (PG) biogenesis and conserved across mycobacteria, including the genetically depleted Mycobacterium leprae, was the primary focus of this study.MethodsBiochemical analysis of Rv3712 was performed using inorganic phosphate release assays. The operon structure was identified using reverse-transcriptase PCR and a transcription/translation fusion vector. In vivo mycobacterial protein fragment complementation assays helped generate the interactome.ResultsRv3712 was found to be an ATPase. Characterization of its operon revealed a mycobacteria-specific promoter driving the co-transcription of Rv3712 and Rv3713. The two gene products were found to interact with each other in vivo. Sequence-based functional assignments reveal that Rv3712 and Rv3713 are likely to be the mycobacterial PG precursor-modifying enzymes MurT and GatD, respectively. An in vivo network involving Mtb-MurT, regulatory proteins and cell division proteins was also identified.ConclusionsUnderstanding the role of the enzyme complex in the context of PG metabolism and cell division, and the implications for antimicrobial resistance and host immune responses will facilitate the design of therapeutics that are targeted specifically to M. tuberculosis.

Highlights

TB is an airborne, infectious disease responsible for 10 million new cases of infection and 1.2 million deaths in 2019 alone.[1]
Mycobacterium smegmatis mc2155 was used with pUAB100 and pUAB200 plasmids for in vivo protein–protein interaction studies. pYUB76 constructs were used with both M. smegmatis mc2155 and E. coli for promoter analysis
Rv3712 comprises two domains that span residues 56–264 and 303–402 (Figure 1c) as identified by InterPro[18] and Phyre 2.0.19 The first domain is homologous to the central domain of Mur ligases, whereas the second is classified as a domain of unknown function (DUF1727)

Summary

Introduction

TB is an airborne, infectious disease responsible for 10 million new cases of infection and 1.2 million deaths in 2019 alone.[1]. As the PG-synthesizing Mur ligase enzymes have emerged as potential drug targets,[5,6,7] a genome-wide search of M. tuberculosis led us to Rv3712—a ‘possible ligase’ expected to be involved in PG metabolism. Rv3712 is essential for the survival of M. tuberculosis, it is conserved across all mycobacteria and has survived the genomic decay in Mycobacterium leprae, making it an attractive candidate for investigation as a novel drug target.[8,9,10] its role in PG metabolism was unknown. Based on its predicted ligase activity, Rv3712 could perform one of three PG-associated roles in M. tuberculosis: (a) that of an ATP-dependent Mur ligase, involved in de novo PG synthesis; (b) that of a murein peptide ligase (Mpl), a PG-recycling enzyme found in Gram-negative bacteria; or (c) that

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Conclusion