Quantification of the Metabolic Heterogeneity in Mycobacterial Cells Through the Measurement of the NADH/NAD+ Ratio Using a Genetically Encoded Sensor
NADH/NAD+ levels are an indicator of the bacterial metabolic state. NAD(H) levels are maintained through coordination of pathways involved in NAD(H) synthesis and its catabolic utilization. Conventional methods of estimating NADH/NAD+ require cell disruption and suffer from low specificity and sensitivity and are inadequate in providing spatiotemporal resolution. Recently, genetically encoded biosensors of the NADH/NAD+ ratio have been developed. One of these sensors, Peredox-mCherry, was adapted for the measurement of cellular levels of NADH/NAD+ in the slow-growing Mycobacterium tuberculosis (Mtb) and the fast-growing Mycobacterium smegmatis. Importantly, the use of the engineered reporter strains of Mtb demonstrated a significantly higher heterogeneity among the bacteria residing in macrophages compared to the bacteria grown in synthetic media. Previous estimations of NADH/NAD+ levels have missed this important aspect of the biology of Mtb, which may contribute to the variable response of intracellular Mtb to different antimycobacterial agents. In this chapter, we describe the details of a method used in the generation of reporter strains for the measurement of the NADH/NAD+ ratio in mycobacteria. Importantly, once the reporter strains are created, they can be exploited with fluorescence spectroscopy, FACS, and confocal microscopy to access the dynamic changes in the NADH/NAD+ levels in intact individual bacterial cells. Although we have only described the method for the creation of reporter strains capable of measuring NADH/NAD+ in mycobacteria in this chapter, a similar method can be used for generating reporter strains for other bacterial species, as well. We believe that such reporter stains can be used in novel screens for small molecules that could alter the metabolism of bacterial cells and thus aid in the development of new class of therapeutic agents.
KeywordsBacterial metabolic state Metabolic heterogeneity Peredox Tuberculosis pathogenesis
This work was supported by funding from CSIR (OLP070) and Department of Biotechnology (BT/PR/5086/GBD/27/307/2011). We are thankful to Mr. Deepak Bhat for his help with confocal microscopy. We are thankful to Dr. Hariom Kushwaha for managing the AK laboratory requirements. S.A.B. and I.K.I. are grateful to the CSIR for JRF and SRF. A.K. is supported by DST, India (DST/INT/AUS/GCP-7/13 and SR/SO/BB-0037/2013), and DBT, India (BT/PR15097/MED/29/237/2011), and CSIR through Supra Institutional Projects—BSC0210G (INFECT), BSC0211E (Bugs to drugs), and Network project BSC0119F (human microbiome).
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