Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs

Abstract

It is the mature gametocytes of Plasmodium that are solely responsible for parasite transmission from the mammalian host to the mosquito. They are therefore a logical target for transmission-blocking antimalarial interventions, which aim to break the cycle of reinfection and reduce the prevalence of malaria cases. Gametocytes, however, are not a homogeneous cell population. They are sexually dimorphic, and both males and females are required for parasite transmission. Using two bioassays, we explored the effects of 20 antimalarials on the functional viability of both male and female mature gametocytes of Plasmodium falciparum. We show that mature male gametocytes (as reported by their ability to produce male gametes, i.e., to exflagellate) are sensitive to antifolates, some endoperoxides, methylene blue, and thiostrepton, with submicromolar 50% inhibitory concentrations (IC50s), whereas female gametocytes (as reported by their ability to activate and form gametes expressing the marker Pfs25) are much less sensitive to antimalarial intervention, with only methylene blue and thiostrepton showing any significant activity. These findings show firstly that the antimalarial responses of male and female gametocytes differ and secondly that the mature male gametocyte should be considered a more vulnerable target than the female gametocyte for transmission-blocking drugs. Given the female-biased sex ratio of Plasmodium falciparum (∼3 to 5 females:1 male), current gametocyte assays without a sex-specific readout are unlikely to identify male-targeted compounds and prioritize them for further development. Both assays reported here are being scaled up to at least medium throughput and will permit identification of key transmission-blocking molecules that have been overlooked by other screening campaigns.

Fig 1

Exflagellation can be detected and quantified by computer-aided identification of moving exflagellation centers. Exflagellation of day 14 gametocyte cultures was triggered by incubation with ookinete medium containing 100 μM xanthurenic acid and a temperature decrease to room temperature for 20 min in a disposable hemocytometer. (A) Under these conditions, exflagellation centers can be identified manually and clearly as clusters of vigorous movement (highlighted by red circles) by using bright-field illumination and a 10× objective. Bar = 400 μm. (B) By recording a 4-s, 20-frame time-lapse image, subtracting each frame from the previous, and then combining the subtracted images, it is possible to identify exflagellation centers as intense white dots (regions of movement) on a black background (regions that are still). (C) Applying standard image processing to images permits exflagellation centers to be identified and counted. (D) By imaging the same field of view over time, the progression of exflagellation was recorded for three independent cultures (data shown in red, green, and blue), with each culture being sampled five times. Exflagellation progressed with a reproducible pattern, first being observed at 16 to 18 min posttriggering and reaching a maximum at 24 to 25 min. It then immediately diminished, with a half-life of ∼12 min, until 40 min posttriggering, after which exflagellation reduced by an inconsistent rate.

Fig 2

Female gamete production can be detected by surface Pfs25 expression. Activation of day 16 gametocyte cultures was triggered by addition of ookinete medium containing 100 μM xanthurenic acid to a final 1:20 dilution and a temperature decrease to room temperature. (A) The activated culture was sampled at 2, 6, and 24 h posttriggering and stained 1:200 with anti-Pfs25–Cy3 antibody (red) for 30 min. Cell nuclei were then counterstained with Hoechst dye (blue) and then visualized with a 100× objective. With increasing activation time, Pfs25 staining became progressively brighter. Bar = 10 μm. (B) At 24 h posttriggering, Pfs25 staining was intense enough to visualize activated female gametes in an untreated sample with a 10× objective, while no staining was observed in a sample treated with 10 μM methylene blue. Bar = 200 μm.

Fig 3

Response of a panel of antimalarial drugs in both male and female assays. (A) Twenty antimalarial drugs were evaluated at 1 μM in the male and female assays, in triplicate experiments (see compounds evaluated and corresponding percentages of inhibition in Table 1). Drug activities in both assays were compared by plotting male activity along the x axis and female activity along the y axis. At 1 μM, no drug showed appreciable activity in the female assay, while thiostrepton, methylene blue, pyrimethamine, cycloguanil, artesunate, artemisinin, and DHA all showed activity in the male assay. (B) The 20 antimalarial drugs were retested at 10 μM in the female assay and compared again to the 1 μM male assay data. At 10 μM, only pyronaridine, thiostrepton, and methylene blue showed activity.