Real-time measurements of ATP dynamics via ATeams inPlasmodium falciparumreveal drug-class-specific response patterns

Abstract

AbstractMalaria tropica, caused by the parasitePlasmodium falciparum(P. falciparum) remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism ofP. falciparumis an interesting target for drug design. To this end, analysis of the central metabolite ATP is of great interest. So far, only cell disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation, and partly inconsistent results. To address this, we have established fluorescent probes, based on FRET and known as ATeam, for use in blood stage parasites. ATeams are capable of measuring MgATP2-levels in a ratiometric manner, thereby facilitatingin cellulomeasurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection, and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) inP. falciparumto monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin proteinin vitroand stably integrated the sensors into the genome of theP. falciparumNF54attBcell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox-cyclers decreased ATP, doxycycline caused first-cycle cytosol alkalization, and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs’ mode of action with possible implications for target identification and drug development.