Manipulating Bacterial and Host Reactive Oxygen Species (ROS)- based mechanisms to potentiate killing of Mycobacterium tuberculosis (Mtb)

Abstract

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how eROS levels are controlled is essential yet remains uncharacterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining non-toxic levels of eROS in Mtb. Integrating redoxosome with a global network of protein-protein interactions and transcriptional regulators revealed a hypothetical protein (rv0158) as a top node managing eROS and redox homeostasis in Mtb. RNA sequencing, seahorse XF flux measurements, and lipid analysis indicate that rv0158 is required to balance the deployment of fatty acid substrates between lipid anabolism and oxidation. Disruption of rv0158 perturbed redox balance in a carbon-source-specific manner, promoted killing in response to anti-TB drugs, reduced survival in macrophages, and lowered persistence in mice. We describe a novel pathogen response to moxifloxacin. Mtb, unlike Escherichia coli, decreases respiration in response to moxifloxacin. Nevertheless, cells were killed, as ROS increased due to NADH-dependent reductive stress. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with a ROS scavenger (thiourea), and an iron chelator (bipyridyl), indicating ROS is part and not a consequence of death processes. Treatment with N-acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Thus, redox and bioenergetic imbalance contribute to the moxifloxacin-mediated killing of Mtb. These results provide a way to make fluoroquinolones more effective anti-tuberculosis agents. We have previously reported that Mtb H37Rv sets up a gradient of mycothiol redox potential: EMSH-oxidized (-240 mV) to EMSH-reduced (-320 mV) inside macrophages, where the EMSH -reduced Mtb subpopulation are significantly more tolerant to anti-TB drugs. Therefore, one of the keys to subverting drug-tolerance is to impede the emergence of EMSH -reduced subpopulation by inducing overwhelming oxidative stress. In this study, we exposed THP1-macrophages infected with Mtb H37Rv expressing Mrx1-roGFP2-biosensor, to a library of FDA-approved drugs (Enzo Life Sciences; BML-2842) and scored for the oxidative shift in the Mtb- EMSH at 24 hours post infection. Based on their activity to trigger oxidative stress inside the bacterium, non-cytotoxicity to host, and inhibition of bacterial growth inside macrophages, C5 molecule emerged as the top hit. Pre-treatment with C5 potentiated killing of Mtb by all tested antibiotics (isoniazid, rifampicin, and moxifloxacin) and reduced drug-tolerance.