Imaging the NADH:NAD+ Homeostasis for Understanding the Metabolic Response of Mycobacterium to Physiologically Relevant Stresses

Abstract

The NADH:NAD+ ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD+ ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD+ ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD+ levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD+ ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD+ ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further studies on mycobacterial physiology and will create new avenues of research for anti-tuberculosis drug discovery.

The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets

“…Given our observation that MetT affects eukaryotic mitochondrial ROS production, we reasoned MetT may also affect mycobacterial energetics. To visualise disruption of cellular respiration in M. marinum , we created a transgenic M. marinum Δesx1 strain carrying pMV762-Peredox-mCherry (Addgene 90217) to visualise build up of NADH:NAD ratio [25, 26]. Analysis of constitutive mCherry expression in a late log culture revealed a small but statistically significant decrease in bacteria number following 1 mM MetT treatment and a larger decrease following 5 mM MetT treatment that was correlated with CFU counts (Fig 6B).…”

Protein–protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis

“…Rv0148 is a NAD oxidoreductase involved in the function of intermediary metabolism and respiration. The exposure of Δ0148 to components inhibiting ETC complexes I and II [27] revealed that Δ0148 responds to changes in the electron transport system, thus predicting its role in cell homeostasis. M. tuberculosis adapts to various stress conditions to survive as a successful pathogen inside the host [28].…”

“…To understand the role of oxidative and nitrogen stress in H 37 Rv, Δ0148 and CΔ0148, the strains were grown to mid-log phase in 7H9-OADC-T medium and exposed to various stress components such as electron transport chain (ETC) inhibitors, peroxides, sulphoxides, thiol stress with slight modification to the procedure by [27] and OD values were measured at 600 nm (OD600) for 4, 24 and 48 h time points. The graphs were plotted and significance was reported considering P < 0.01 and P < 0.001.…”

Evolutionary Views of Tuberculosis: Indoleamine 2,3‐Dioxygenase Catalyzed Nicotinamide Synthesis Reflects Shifts in Macrophage Metabolism

“…[ 50 ] Isoniazid and pyrazinamide induce changes to the NADH/NAD + ratio of the mycobacterium. [ 77 ] Isoniazid and pyrazinamide affect host, as well as mycobacterial, nicotinamide metabolism. Counterintuitively, isoniazid (INH) treatment is a known cause of pellagra, a host niacin (Vitamin B3) deficiency, possibly through competition between INH and niacin.…”

“…[ 114 ] Other relevant strategies have included using inhibitors targeting mycobacterial tryptophan synthesis [ 40,115 ] or the mycobacterial NAD + synthesis or recycling pathways. [ 50,72,77 ]…”