Understanding Mycobacterial redox response towards endogenous stress produced by peroxynitrite donor

Abstract

Peroxynitrite is a reactive nitrogen species (RNS), biologically generated from the reaction of superoxide radical with nitric oxide. At elevated levels, ONOO causes redox imbalances and disrupt cellular functions which can lead to cell death. Immune cells like macrophages utilises ONOO as cytotoxic effector molecule in response to invading bacteria and parasites. It has been shown that IFN-activated macrophages releases superoxide and nitric oxide that react to form ONOO. Mycobacterium tuberculosis (Mtb), the etiologic agent of Tuberculosis, proliferates in human macrophages and also experiences this oxidative and nitrostative stress. It has been recently reported that Mtb has higher susceptibility to the endogenously produced superoxide. However, there are not many reports on bactericidal activity of peroxynitrite mainly because of its short half-life. Recently a novel molecule (HyPR-1) has been reported that can enhance intracellular ONOOlevel but it has a low potency. In this study, we have synthesized a peroxynitrite donor (CJ067, analogue of HyPR-1) that generates high fluxes of ONOOin buffer as well as inside non-pathogenic Mycobacterium smegmatis (Msm). Using a mycothiol- specific redox biosensor (Mrx1-roGFP2), we have captured the real time oxido-reductive response of mycobacteria in the presence of CJ067 and observed a delayed recovery as compared to other donors. We have also utilised CJ067 to compare the antibacterial activity of ONOO with superoxide and nitric oxide donors in Msm, and a few pathogenic and drug resistant strains of Mtb. We found that a very low concentration (MM) of CJ067 can inhibit the growth of M. smegmatis and Mtb. In sum, our study constitutes synthesizing a potent peroxynitrite donor, CJ067 and understanding the mycobacterial response to ONOO-.