An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites

Abstract

Plasmodium falciparum genotyping has recently undergone a revolution, and genome-wide genotype datasets are now being collected for large numbers of parasite isolates. By contrast, phenotyping technologies have lagged behind, with few high throughput phenotyping platforms available. Invasion of human erythrocytes by Plasmodium falciparum is a phenotype of particular interest because of its central role in parasite development. Invasion is a variable phenotype influenced by natural genetic variation in both the parasite and host and is governed by multiple overlapping and in some instances redundant parasite-erythrocyte interactions. To facilitate the scale-up of erythrocyte invasion phenotyping, we have developed a novel platform based on two-color flow cytometry that distinguishes parasite invasion from parasite growth. Target cells that had one or more receptors removed using enzymatic treatment were prelabeled with intracellular dyes CFDA-SE or DDAO-SE, incubated with P. falciparum parasites, and parasites that had invaded either labeled or unlabeled cells were detected with fluorescent DNA-intercalating dyes Hoechst 33342 or SYBR Green I. Neither cell label interfered with erythrocyte invasion, and the combination of cell and parasite dyes recapitulated known invasion phenotypes for three standard laboratory strains. Three different dye combinations with minimal overlap have been validated, meaning the same assay can be adapted to instruments harboring several different combinations of laser lines. The assay is sensitive, operates in a 96-well format, and can be used to quantitate the impact of natural or experimental genetic variation on erythrocyte invasion efficiency.

Figure 1

Staining of pRBC with fluorescent DNA-binding dyes. pRBC were detected by flow cytometry after staining with either 10 μg/mL EB, 2 μM Hoechst 33342, or 1:5,000 SYBR Green I. a: Uninfected RBC and pRBC were directly stained with the DNA dyes. b: Uninfected RBC and pRBC were fixed, permeabilized, and treated with RNase before staining with the DNA dyes.

Figure 2

Accuracy of flow cytometry in determining parasitemia. Correlation between parasitemia determined by flow cytometry, using either Hoechst 33342 (direct staining) or SYBR Green I (staining post RNase treatment), and by light microscopy, using Giemsa staining, for a serial dilution of a Dd2 parasite culture. Standard error bars are represented on the horizontal axis for microscopy counts and vertical axis for flow cytometry counts.

Figure 3

Labeling of target RBC with fluorescent dyes. RBC were labeled with either 20 μM CFDA-SE or 10 μM DDAO-SE and coincubated with an approximately equal quantity of unlabeled RBC for 48 h at +37°C under standard P. falciparum culture conditions. RBC were then harvested and fluorescence detected either by confocal microscopy (top panels) or flow cytometry (bottom panels).

Figure 4

P. falciparum invasion in labeled cells. Target RBC labeled with fluorescent dyes CFDA-SE or DDAO-SE were coincubated with unlabeled P. falciparum Dd2 strain cultures, containing a mix of uninfected and pRBC, for 48 h under standard P. falciparum culture conditions. Cultures were then harvested and stained with Hoechst 33342 or SYBR Green I, respectively. a: Cultures were observed by confocal microscopy (first 4 panels) and flow cytometry (last panel on the right). In the microscopy pictures, yellow (Hoechst 33342) and blue (SYBR Green I) arrows point to parasites detected inside unlabeled RBC, whereas white arrows point to parasites detected inside fluorescently labeled RBC. In the dotplot representation of the data generated by flow cytometry, four populations can be readily distinguished: unlabeled, uninfected RBC (lower left); labeled, uninfected RBC (lower right); unlabeled, infected RBC (upper left); and labeled, infected RBCs (upper right). b: Effect of the starting parasitemia on the final parasitemia in labeled RBC. Parasitemia of DDAO-SE-labeled RBC was determined by SYBR Green I staining using flow cytometry, for different starting parasitemia using donor unlabeled population with increasing parasitemia. c: Effect of the unlabeled to labeled RBC ratio on the final parasitemia in labeled RBC. Parasitemia of DDAO-SE-labeled RBC was determined by SYBR Green I staining using flow cytometry, in a mixed unlabeled donor pRBC/label target RBC culture with a volume of 100 μL and a starting parasitemia of 1%.

Figure 5

Invasion phenotypes of three laboratory strains of P. falciparum. Unlabeled pRBC were incubated with CFDA-SE- or DDAO-SE-labeled RBC, at an unlabeled-to-labeled ratio of 1:2 and a starting parasitemia of 1%. After 48 h, parasites were stained with Hoechst 33342 or SYBR Green I, respectively, and final parasitemia in the target population was determined by gating on the fluorescently labeled RBC population using flow cytometry. Invasion efficiencies were determined as a percentage of the final parasitemia of a mock-treated-labeled positive control RBC group.