Third party funded individual grant
Start date : 01.01.2016
End date : 31.12.2017
Scientific goals of this project are:
(1) to perform confocal, multiphoton and plate reader live cell imaging in P. falciparum-infected erythrocytes, erythrocyte ghosts, isolated parasites, isolated digestive vacuoles and non-infected erythrocytes
(2) to record Fluo-4 fluorescence as measure for drug/solute export from the digestive food vacuole using recently established 'reverse Ca2+ imaging'
(3) to assess PfMDR1 pump rates in different drug resistant and sensitive strains with various copy numbers for PfMRD1 mutations: Dd2, K1. 3D7 will serve as a control
(4) to perform immuno-gold labelling experiments to assess the transporter expression density on the food vacuolar membrane
(5) to implement the kinetics and steady state data in multi-compartment models to predict solute fluxes in live parasites, i.e. passive diffusion-driven vs. active pump rates and extract the pump rates for drug resistance per molecule, for the first time
(6) to test for new drug efficiencies in isolated food vacuoles using previously identified new artemisinin-derived 2nd generation trioxane-ferrocene hybrids
Within the project period, confocal experiments are performed in the Canadian labs while all multiphoton imaging experiments will be carried out in the new PC2 research facilities of the German partner. Fluo-4 Ca2+ recordings will be performed by tracing fluorescence uptake kinetics in the prepared multi-compartment systems. Infected RBCs represent the full compartmentalised system with intact parasites and digestive vacuoles (DV). Bioseparation procedures established by us allow enrichment of isolated DVs and intact parasites in bioreactors. After harvesting parasitic material from resistant (K1, Dd2) and sensitive strains (3D7), Fluo-4 will be added and time-lapse imaging for compartmental fluorescence performed. From that, we will be able to extract dye uptake curves and calculate overall pump rates for drug resistant strains and correlate those to the PfMDR1 copy number. The DV isolation procedure has been adopted from our Canadian partner. In extension of our previous work, obtaining the overall pump rates for PfMDR1 to extrude solutes and drugs from the DV, is still biased by the unknown expression density of the transporter. This missing link will be provided by immuno-gold-electron microscopy which has been established in the lab of the Canadian partner to be adopted during the project in the German labs. Using plate reader assays, imaging information is translated into high-throughput technology, in particular for the isolated DVs, where spatial information is no longer compromised by outer cellular compartments. For novel drug efficiency testing, the isolated DV plate reader assay will be used to test for 2nd generation trioxane-ferrocene compounds.