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.
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Acknowledgements
We thank D. Ravel for critical reading of the manuscript. This work has been supported through a career development grant from the Burroughs Wellcome Fund and by US National Institutes of Health (NIH) grants R21 AI105328 and RC1AI086222 (M.M.). N.M.B.B. is supported by a postdoctoral fellowship from the Swiss National Science Foundation and K.B. was supported through a Feodor Lynen postdoctoral fellowship from the Alexander von Humboldt Foundation.
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I.G., N.M.B.B. and M.M. wrote the manuscript, with input from all coauthors. K.B. and M.M. conceived and developed the originally published protocol. I.G. conceived and performed preliminary experiments and N.M.B.B. conceived and performed later experiments leading to improvements in the protocol. N.M.B.B. and K.W. generated the data shown in the figures. All authors revised the manuscript and contributed to interpretation of the results.
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Integrated supplementary information
Supplementary Figure 1 Cytostatic drugs cause a faux activation of the tdTomato reporter.
a. Bar graphs showing the proportion of red fluorescent parasites (Pf2004/164-tdTom) after treatment with different concentrations of dihydroartemisinin (upper panel) and methylene blue (lower panel). Except for the CM-induced control parasites, cells were cultured in absence of conditioned medium, i.e. under conditions that do not induce gametocyte production. High concentrations of methylene blue induce a short-lived activation of the tdTom reporter (highlighted in red). Note that this faux induction precedes proper reporter expression in early gametocytes of the control cells (highlighted in green). Drugs were added for 24 hours (from 28±4hpi of the preceding IDC to 4±4hpi). Bars show the mean of technical triplicates b. Effect of methylene blue on asexual parasite multiplication (mean ± SEM, n=3). Concentrations triggering a faux induction of the tdTom reporter are highlighted.
Supplementary Figure 2 Conditions during CM production hamper parasite development.
Diff-Quick stained blood smears show the morphology of cells used to produce CM. Smears were made at the time point of CM collection (44±4hpi). Parasitemia is indicated. Note that the development of CM-producing parasites is delayed compared to control cells kept at a low parasitemia. In the assay, highest rates of sexual conversion were achieved by using CM produced from cells at a parasitemia of 5.6% (data not shown).
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Brancucci, N., Goldowitz, I., Buchholz, K. et al. An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development. Nat Protoc 10, 1131–1142 (2015). https://doi.org/10.1038/nprot.2015.072
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DOI: https://doi.org/10.1038/nprot.2015.072
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