Reporter lines based on the gexp02 promoter enable early quantification of sexual conversion rates in the malaria parasite Plasmodium falciparum

Abstract

Transmission of malaria parasites from humans to mosquito vectors requires that some asexual parasites differentiate into sexual forms termed gametocytes. The balance between proliferation in the same host and conversion into transmission forms can be altered by the conditions of the environment. The ability to accurately measure the rate of sexual conversion under different conditions is essential for research addressing the mechanisms underlying sexual conversion, and to assess the impact of environmental factors. Here we describe new Plasmodium falciparum transgenic lines with genome-integrated constructs in which a fluorescent reporter is expressed under the control of the promoter of the gexp02 gene. Using these parasite lines, we developed a sexual conversion assay that shortens considerably the time needed for an accurate determination of sexual conversion rates, and dispenses the need to add chemicals to inhibit parasite replication. Furthermore, we demonstrate that gexp02 is expressed specifically in sexual parasites, with expression starting as early as the sexual ring stage, which makes it a candidate marker for circulating sexual rings in epidemiological studies.

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

“…Compound 2 has been used previously to aid synchronization of P. knowlesi for transfection [44]. Furthermore, ML10 has recently been used to block the proliferation of asexual stages in a P. falciparum culture induced to form gametocytes [60].…”

Exposure to artemisinin at the trophozoite stage increases sexual conversion rates in the malaria parasite Plasmodium falciparum

“…This parasite line expresses the fluorescent reporter tdTomato under the control of the promoter of the sexual stage-specific gene gexp02 (PF3D7_1102500), which allows accurate flow cytometry-based detection of very early gametocytes within a few hours after sexual conversion (Portugaliza et al, 2019). The short drug pulse 170 mimics the short plasma half-life of ARTs (de Vries & Dien, 1996).…”

“…1), using an analogous reporter line generated in the 3D7-E5 genetic background that has lower levels of basal sexual conversion than NF54 (E5-gexp02-Tom line, Supplementary Fig. 2) 195 (Portugaliza et al, 2019), and using a transgenic line with the fluorescent reporter under the control of the etramp10.3 (PF3D7_1016900) gametocyte-specific promoter (NF54-10.3-Tom line) (Portugaliza et al, 2019). Using this latter parasite line, we measured sexual conversion rates by flow cytometry, by immunofluorescence assay (IFA) detecting the Pfs16 (PF3D7_0406200) early gametocyte marker and by light 200 microscopy analysis of Giemsa-stained blood smears.…”

“…Depletion of LysoPC or choline, a downstream metabolite in the same metabolic pathway, have now been used by several groups to stimulate sexual conversion 90 under culture conditions (Brancucci et al, 2017;Filarsky et al, 2018;Portugaliza et al, 2019).…”

“…Given the widespread use of ACTs for malaria treatment and in mass drug administration campaigns aimed at malaria elimination, understanding the impact of ARTs on sexual conversion is an urgent research priority. Here, we developed a robust assay based on recently described gametocyte-reporter parasite lines 155 (Portugaliza et al, 2019) to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. Using this assay, we tested the effect of exposing parasites to dihydroartemisinin (DHA, the active metabolite of all ARTs) and CQ at different stages and under different metabolic conditions, to provide an accurate and comprehensive description of the direct effect 160 of these drugs on sexual conversion rates.…”