Title : Investigating the role of Mycobacterium tuberculosis transcriptional regulator VirS in acidic responses and identification of inhibitors against it
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 an AraC/XylS type transcriptional regulator, VirS, is induced under 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, by 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 and 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 identify inhibitors against VirS. We identified a few hit compounds that inhibited VirS DNA binding activity as well as the growth of M. tuberculosis in vitro broth culture. 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. These studies also pave way to design novel M. tb inhibitors targeting VirS.
