Bioluminescence Method for In Vitro Screening of Plasmodium Transmission-Blocking Compounds

Abstract

The sporogonic stage of the life cycle of Plasmodium spp., the causative agents of malaria, occurs inside the parasite's mosquito vector, where a process of fertilization, meiosis, and mitotic divisions culminates in the generation of large numbers of mammalian-infective sporozoites. Efforts to cultivate Plasmodium mosquito stages in vitro have proved challenging and yielded only moderate success. Here, we describe a methodology that simplifies the in vitro screening of much-needed transmission-blocking (TB) compounds employing a bioluminescence-based method to monitor the in vitro development of sporogonic stages of the rodent malaria parasite Plasmodium berghei Our proof-of-principle assessment of the in vitro TB activity of several commonly used antimalarial compounds identified cycloheximide, thiostrepton, and atovaquone as the most active compounds against the parasite's sporogonic stages. The TB activity of these compounds was further confirmed by in vivo studies that validated our newly developed in vitro approach to TB compound screening.

Keywords: Plasmodium berghei; in vitro cultures; malaria; mosquito stages; sporogony.

FIG 1

A new system for culturing and detecting P. berghei mosquito stages. (A) Bioluminescence measured in relative luminescence units (RLU) at different time points throughout the formation and maturation of ookinetes, represented as a percentage of the RLU measured at the start of culture, 0 h. Neg corresponds to the uninfected blood sample used as a negative control. Results are expressed as the mean ± the standard deviation. (B) Western blot analysis of PbCSP protein expression throughout the transformation of gametes into ookinetes in vitro. Salivary gland sporozoites were used as a positive control. (C) Immunofluorescence microscopy analysis of PbCSPGFP-Luc gametes and ookinetes (green, Pbs21 protein; red, PbCSP; blue, nuclei). Scale bars, 5 μm. (D) Bioluminescence measured in RLU throughout 21 days of oocyst culture, represented as a percentage of the RLU at time zero (d0) of culture. Results are expressed as the mean ± the standard deviation. (E) Representative images of immunofluorescence staining of P. berghei tooks, oocysts, sporulating oocysts, and free sporozoites (red, PbCSP; blue, nuclei). Scale bars, 5 μm. (F) Quantification of oocyst numbers and development by live fluorescence microscopy. Results are expressed as the mean ± the standard deviation. d3, day 3. (G) Immunofluorescence staining of PbCSPGFP-Luc ookinetes (time zero [d0]) and live imaging of PbCSPGFP-Luc oocysts (day 3 [d3] to day 21 [d21]) cultured in vitro. Scale bars, 10 μm.

FIG 2

Effects of selected compounds against Plasmodium mosquito stages in vitro. (A) Schematics of the progress of the parasite culturing process highlighting the different schedules of compound treatment employed. d, day. (B) Assessment of compound effects on ookinete formation and development, expressed as percentages of inhibition of P. berghei ookinete formation. (C) In vitro activities of selected compounds against early stages of oocyst development. (D) In vitro activities of selected compounds on young oocysts and subsequent developmental stages. Ten compounds, Az, Ch, DA, Py, Lu, Ha, Po, At, Th, and Cy, were screened at a concentration of 10 μM. Bars correspond to RLU measurements represented as percentages of the RLU of the DMSO control. Results are expressed as the mean ± the standard error of the mean.

FIG 3

Compound activity on P. berghei sporogonic development in infected mosquitoes and sporozoite infectivity of a mammalian host. (A) Schematics of assessment of in vivo compound activity on oocysts and sporozoites and liver parasite loads of mice infected by mosquitoes treated with selected compounds. d, day. (B) In vivo activities of selected compounds on the number of oocysts developing in mosquito midguts. Similar population sizes were analyzed, and results are expressed as the mean ± the standard error of the mean. (C) In vivo activities of selected compounds on oocyst development in mosquito midguts. Oocyst areas in square micrometers are presented for random samples of parasites observed in midguts of treated mosquitoes. Results are expressed as the mean ± the standard error of the mean. (D) In vivo activities of selected compounds on sporozoite formation. Results are expressed as the mean ± the standard deviation. (E) Quantification of in vivo hepatic infection of mice infected by treated mosquitoes. Results are expressed as the mean ± the standard error of the mean. Compounds were screened at a concentration of 50 μM. **, P < 0.0001; ***, P < 0.001; ****, P < 0.005; ns, not significant.