Dual fluorescent labelling of the human malaria parasite Plasmodium falciparum for the analysis of the ABC type transporter pfmdr2

Abstract

BackgroundThe study of the Plasmodium falciparum heavy metal transporter gene pfmdr2 employed radioactive labelled heavy metal. As the use of radioactive isotopes shrank considerably during the last few years, resulting in the cessation of the production of some isotopes, amongst them Cadmium109 which was used for that purpose, a different approach had to be developed. Herein, a dual fluorescent labelling of heavy metals accumulation in the P. falciparum parasite is proposed as an alternative to the use of radioactive labelled heavy metals.MethodsPlasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl2 concentrations and for different time periods followed by staining of the infected red blood cells with Fluo-3/AM for Cd detection and Hoechst 33342 for parasite DNA labelling. The fluorescent analysis was done by flow cytometry and confocal microscopy.ResultsThe results show that the sensitive strain has a higher Fluo-3/AM fluorescence in a Cd concentration and time dependent manner, whereas in the resistant strain Fluo-3/AM fluorescence levels were negligible and increased only at high concentrations of Cd and at long incubation periods, but to a much lesser extent than the sensitive strain. No Cd uptake is observed in uninfected red blood cells populations originating from cultures infected with either sensitive or resistant strain. In addition, confocal microscopy overlay of Fluo-3/AM and Hoechst staining shows that the Cd metal accumulates in the parasite itself.ConclusionsThe dual fluorescent labelling is a valid method for detecting heavy metal accumulation in P. falciparum. Furthermore, in contrast to the use of radioactive labelled heavy metal, the fluorescent labelling enables us to differentiate between the different populations existing in a P. falciparum infected red blood cells cultures and thus actually study a phenomenon at the level of a single cell.

Characterization of native PfABCG protein in Plasmodium falciparum

“…Moreover, PfMDR1 shown to localize to the vacuolar membrane of the parasite digestive vacuole (DV) confers resistance to mefloquine and other related quinolines [13]. PfMDR2 is localized to the parasite plasma membrane and shown recently to confer resistance to heavy metals (e.g., cadmium) presumably through an efflux mechanism [14,15]. Two additional ABC transporters, PfMRP1 and PfMRP2 (or PfABCC1 and PfABCC2), found at the parasite plasma membrane have been implicated in conferring resistance to quinoline drugs and the transport of glutathione [16,17].…”

Coral cell separation and isolation by fluorescence-activated cell sorting (FACS)

“…This technique, which has been used primarily in biomedical and immunological research, utilizes lasers to analyze and sort different cell types in real time based on specific properties of the cell. Applications of FACS include clinical analysis, cell purification, functional assays, and pathogen detection [ 41 – 47 ]. Although these techniques have not been widely applied to many non-medical systems, they are a powerful methods for cell type discovery and cell activity in comparative and evolutionary research.…”