An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development

Abstract

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

Figure 1

Flowchart depicting individual steps of the protocol. The timeline represents consecutive parasite developmental cycles (IDCs). IDC-I: parasite cultures are sorbitol-treated twice to synchronize the population to an 8-h age window. IDC-II: the population is split into lower (LP) and higher parasitemia (HP) cultures containing below 1% and 1.5–2% infected red blood cells, respectively. IDC-III: the LP culture is sorbitol-synchronized at 4 ± 4 h.p.i. The HP culture is adjusted to a parasitemia of 5.5–6.5% at 28 ± 4 h.p.i. and incubated for 16 h before collecting the CM by three centrifugation steps. IDC-IV: the LP culture is sorbitol-synchronized at 20 ± 4 h.p.i. and adjusted to a parasitemia of 0.3%. At 28 ± 4 h.p.i., the cells are washed and resuspended in 90% (vol/vol) CM (complemented with complete medium) and seeded into wells of a 96-well plate. These conditions will induce sexual conversion in a high proportion of forming schizonts, and the compound to be screened may be either added at this time point or during the subsequent IDC (indicated by blue stars). Parasites cultured in the absence of the compound and under noninducing conditions, i.e., with complete medium, serve as positive and negative controls, respectively. IDC-V: after the parasites have reinvaded fresh erythrocytes (4 ± 4 h.p.i.), the culture medium is exchanged with complete medium. The medium is henceforth exchanged daily. At 28 ± 4 h.p.i., parasitemia is determined by flow cytometry using an aliquot of SYBR Green–stained cells. At 66 ± 4 h.p.i., young gametocytes have acquired enough tdTom fluorescence for cytometry-based quantification.

Figure 2

Cytometric readout of gametocyte production and survival. (a) Gametocyte induction. Bar graphs quantifying the number of sexual cells produced under gametocyte-inducing and control conditions for cell lines 3D7/164-tdTom and Pf2004/164-tdTom (mean ± s.d., n = 3) in example experiments. The time point of flow cytometry–based quantification is indicated in h.p.i. Live cell images are shown for Pf2004/164-tdTom gametocytes and illustrate tdTom reporter expression (red) at 66 ± 4 h.p.i. and 90 ± 4 h.p.i. (b) Cytometry-based gametocyte quantification. Example of cytometric scatterplots quantifying SYBR Green (x axis) and tdTom (y axis) fluorescence for Pf2004/164-tdTom parasites cultured under control (left plots) and gametocyte-inducing (right plots) conditions. Flow cytometry was carried out at 66 ± 4 and 90 ± 4 h.p.i. Gates indicate tdTom-positive gametocytes within the infected RBC (iRBC) population. DIC, differential interference contrast.

Figure 3

Drug screening: DHA treatment affects asexually replicating parasites and early gametocytes. Determination of half-maximal inhibitory concentration (IC₅₀). Shown is the effect of DHA on asexual parasite multiplication (left) and early gametocyte development (right). Mitotic proliferation and gametocyte survival was monitored 48 h after administering DHA at 28 ± 4 h.p.i. Note that both cell lines reveal similar dose–response curves. Curves were generated using a four-parameter nonlinear regression fit. The presented data exemplifies the outcome of a single screening run (mean ± s.e.m., n = 3), as described. Specific dose-response information gained by using the presented assay is available from Buchholz et al..