Single-Cell Tracking Reveals Antibiotic-Induced Changes in Mycobacterial Energy Metabolism

Abstract

ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening.

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

“…In the light of the fact that many of the antimycobacterial drugs are linked to energy metabolism of Mtb (Black et al, 2014; Koul et al, 2014; Maglica et al, 2015), measuring the levels of the NADH:NAD + ratio becomes clinically important. Thus, we treated the Mtb Peredox with sub-lethal concentrations of antituberculosis agents for 6 and 24 h and calculated the NADH:NAD + ratio though CLSM analysis.…”

Imaging Adenosine Triphosphate (ATP)

“…In unicellular organisms, Maglica et al (2015) used ATeam sensors to monitor antibiotic induced cell death by single cell tracking of ATP levels in Mycobacterium smegmatis , demonstrating its potential as an important technology in drug discovery for antibiotic screening and mechanism of action imaging assays. In multicellular organisms, Ozawa et al (2015) found that glycolysis dependent ATP production was necessary for lamellipodia formation in podocytes.…”

Antibiotic efficacy — context matters

“…Targeted metabolomic studies have revealed significant changes in intracellular energy metabolites following antibiotic treatment [29-31], corroborated by live-cell imaging experiments directly measuring transient changes in ATP [32]. Experiments using the Seahorse XF Analyzer have captured real-time increases to cellular respiration by bactericidal antibiotics [33-35], complementing real-time measurements of overflow ROS production using electrochemical [36] or genetically encoded biosensors [33].…”