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

BACKGROUND: The 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 Cadmium(109) 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. METHODS: Plasmodium falciparum Cd resistant and sensitive strains at the trophozoite stage were used in this study. The cells were cultured at different CdCl(2) 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. RESULTS: The 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. CONCLUSIONS: The 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.