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.
Highlights
A highly flexible and robust metabolic system ensures survival of Mycobacterium tuberculosis (Mtb) inside the host (Boshoff and Barry, 2005; Lee et al, 2013; Gouzy et al, 2014)
The salvage pathway is upregulated during hypoxia and in the lungs of mice infected with Mtb, where it plays a crucial role in Mtb survival (Boshoff et al, 2008; Vilchèze et al, 2010)
To the best of our knowledge, the data presented here are first to quantify the NADH:NAD+ redox state of fast and slow-growing Mycobacterium at single cell levels under various physiologically relevant stresses such as oxidative and nitrosative stress and stress generated through the use of antimycobacterial drugs
Summary
A highly flexible and robust metabolic system ensures survival of Mycobacterium tuberculosis (Mtb) inside the host (Boshoff and Barry, 2005; Lee et al, 2013; Gouzy et al, 2014). In order to normalize the sensor response, the Peredox sensor was fused to mCherry protein that does not change the fluorescence upon binding to NADH.This protein engineering has resulted in an efficient biosensor that does not require cell disruption and can measure metabolically relevant free NADH levels inside the mammalian cells. Such a sensor has not yet been employed for measurement of NADH:NAD+ levels in bacterial cells. These sensors could pave the way for acquiring new insights into the mechanisms of action of drugs and will aid in the development of new methods for discovering antimicrobials
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