Unraveling the Role of the Transcriptional Regulator VirS in Low pH-Induced Responses of Mycobacterium Tuberculosis and Identification of VirS Inhibitors

Abstract

The ability of M. tuberculosis to respond to intramacrophage stresses such as oxygen/nitrogen radicals and low pH is important for its persistence. It has been reported earlier that a AraC/XylS type transcriptional regulator, VirS, is induced in response to low pH and regulates cell envelope architecture. However, a comprehensive understanding of how VirS mediates its influence on gene expression to coordinate pH response remains uncharacterized. Here, using multiple approaches, we investigated the contribution of VirS in maintaining intramycobacterial pH homeostasis. Using a genetic biosensor of cytoplasmic pH, we demonstrated that VirS is required to maintain intramycobacterial pH in response to acid stress. Furthermore, loss of VirS reduced M.tuberculosis’s ability to block phagosomal-lysosomal fusion, indicating that VirS regulates phagosomal maturation. Transcriptomics data indicate that VirS affects the expression of genes involved in cell wall synthesis, efflux pumps, ion transporters, metabolic enzymes, transcription regulation and growth under acid stress. Furthermore, we performed EMSA, DNA footprinting, 3-D structure generation. Structure guided mutational studies revealed key residues required for its interaction with DNA. Importantly, we performed structure based virtual screening to target VirS:DNA interaction and identified possible inhibitors. Lastly, the top hit compounds inhibited VirS DNA binding and reduced the growth of M.tuberculosis in vitro. Taken together, our findings establish an empirical role of VirS in mediating M.tuberculosis’s response to acidic stress and suggest that targeting of VirS can be effective anti-mycobacterial strategy.

Keywords: Mycobacterium tuberculosis, gene microarray, site-directed mutagenesis, transcription factor, tuberculosis, virtual screening, inhibitor, VirS, AraC/XylS type transcription factor