Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue

Abstract

Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose- and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine and piperaquine showed activity only against immature gametocytes. Our data also identify methylene blue as a potent inhibitor of gametocyte development across all stages. This thiazine dye almost fully abolishes P. falciparum transmission to mosquitoes at concentrations readily achievable in humans, highlighting the potential of this chemical class to reduce the spread of malaria.

Fig. 1.

Integration of an unmarked attB site for site-specific recombination in P. falciparum NF54 parasites. (A) Integration of the plasmid pCC-cg6-attB into the cg6 gene and subsequent intralocus recombination leading to removal of the selectable markers from the recombinant locus. This plasmid expresses human dihydrofolate reductase (hdhfr) and Saccharomyces cerevisiae cytosine deaminase–uracyl phosphotransferase (cdup) for positive and negative selection, respectively (58). cg6 5′ and 3′ coding sequences were placed on either side of an attB site to permit single cross-over homologous recombination between the pCC-cg6-attB plasmid and the endogenous cg6 gene. Single cross-overs were identified between the cg6 3′ homologous regions, leading to plasmid integration into the genome. Selection with 5-fluorocytosine, which is activated into a toxic compound by CDUP, was used to obtain parasites that have lost the hdhfr and cdup selectable markers. This selection was achieved through intralocus recombination between the duplicated cg6 5′ sequences, leading to the formation of a single unmarked attB locus. The black rectangle illustrates the 0.4-kb cg6 probe used for Southern blot analysis, and restriction digest fragment sizes are indicated. (B) Southern blot analysis confirming pCC-cg6-attB plasmid integration into NF54 parasites. DNAs were digested with SalI/NruI (S/N) and hybridized with the cg6 probe. Bulk cultures contained a mixture of integrants that yielded band sizes of 6.6 and 4.9 kb (characteristic of plasmid integration in the 3′ region of cg6) as well as episomal transfectants that showed the 4.9-kb plasmid band and the 8.2-kb nonrecombinant cg6 locus. (C) Southern blot analysis confirming unmarking of attB⁺ parasites. DNAs were digested with SalI/EcoRV (S/E) or S/N and hybridized with the cg6 probe. This process produced bands of 6.8 and 8.2 kb, respectively, in NF54 wild-type parasites. In comparison, band sizes of 6.3 and 2.7 or 6.6 and 4.9 kb were detected in marked attB⁺ recombinant parasites digested with S/E or S/N, respectively. Unmarked NF54^attB parasites yielded a single band of 3.3-kb on digestion with either restriction enzyme pair, consistent with loop-out recombination and excision of the selectable markers.

Fig. 2.

Engineering and characterization of gametocytogenesis-specific GFP-luciferase reporter lines. (A) Schematic of the Bxb1 integrase (INT) -mediated genomic insertion of attP-containing plasmids that used gametocyte-specific promoters (pfs16, pfs48/45, or mal8p1.16) to express the GFP-luciferase (GFP-LUC) fusion. Cloned unmarked NF54^attB parasites were cotransformed with the integrase-expressing plasmid pINT (14) and GFP-LUC-attP plasmids. After integration, the attB site was destroyed, leaving left (attL) and right (attR) flanking regions. (B) Assessment of pfs16, pfs48/45, and mal8p1.16 promoter-driven luciferase expression throughout gametocytogenesis. Luciferase signals were measured daily from triplicate wells harvested from the parasite lines NF54^pfs16, NF54^pfs48/45, and NF54^mal8p1.16. Values were normalized to gametocyte numbers and plotted as a proportion of peak promoter activity (normalized to 1.0; mean ± SEM peak luciferase values were 152,398 ± 1,722, 52,052 ± 897, and 2,081 ± 506 for NF54^pfs16, NF54^pfs48/45, and NF54^mal8p1.16). Days of gametocytogenesis are represented on the x axis. (C) Assessment of gametocyte promoter-driven GFP expression by fluorescence microscopy. NF54^pfs16, NF54^pfs48/45, and NF54^mal8p1.16 parasites were imaged as gametocyte stages V, III, and V, respectively.

Fig. 3.

Activity of the active artemisinin metabolite DHA and ACT partner drugs on gametocyte maturation in vitro. (A) Experimental scheme to assess impact of antimalarials at different stages of gametocyte maturation. After gametocyte induction by limiting nutrients, cultures were treated for 6 d with NAG to eliminate asexual parasites, and gametocytes were magnet-purified on day 2 postinduction. Drug treatments of 3-d duration were initiated on days 2, 5, 8, or 11 (corresponding to stages I and II, III, IV, and V). Results are shown for (B) DHA, (C) lumefantrine (LMF), (D) mdAQ, and (E) piperaquine (PPQ). Drugs were tested at 0.5×, 1×, and 5× the IC₅₀ concentration that produced a 50% inhibition of growth of asexual blood stage parasites (Table 1). Assays were performed in triplicate on two to five independent occasions. The gametocytocidal effect was measured relative to the luciferase signal emitted by untreated gametocyte controls cultured in parallel. Drug-specific effects (represented as means ± SEM) were calculated for up to 12 d after the beginning of treatment at day 0.

Fig. 4.

Identification of potent gametocytocidal activity of MB against P. falciparum gametocytes. Compounds were tested against gametocytes at different developmental stages, and results are illustrated as described in Fig. 3. Compounds were (A) pyronaridine (PND), (B) atovaquone (ATQ), (C) primaquine (PMQ), (D) tafenoquine (TFQ), and (E) MB. Drugs were tested at 5×, 1×, and 0.5× the IC₅₀ concentration that produced a 50% inhibition of growth of asexual blood stage parasites (Table 1).

Fig. 5.

Transmission-blocking activity of selected antimalarial drugs. P. falciparum NF54 stage V gametocytes were cultured in the presence of drug for 3 d followed by culture in the absence of drug for 3–4 d before feeding to female A. stephensi mosquitoes. Midguts of blood-fed mosquitoes were dissected 6–7 d postfeeding, and oocyst numbers were scored. (A) Percent reduction in oocyst formation in mosquitoes fed on drug-treated gametocytes compared with solvent-treated controls (negative reduction equates to enhanced oocyst numbers). (B) Percent block in transmission in mosquitoes fed drug-treated gametocytes. This value denotes the percentage of mosquitoes in which no oocysts were observed after ingestion of drug-treated parasites; all mosquitoes fed on nontreated or mock-treated parasites had oocysts in their midguts. Transmission assays were performed in duplicate, with compounds evaluated at the concentrations indicated. Values were calculated from an average of 20 mosquitoes (range = 14–25) per drug treatment or untreated or DMSO mock-treated control (details provided in Tables S1–S3).

Fig. P1.

An experimental system to measure inhibition of P. falciparum gametocyte development and subsequent transmission to mosquitoes. (A) Luciferase-expressing parasite reporter lines were used to define the rate of reduction of gametocyte viability at each developmental stage for a panel of antimalarials. Log₁₀ reductions were derived from the luciferase data measured daily from in vitro drug-treated gametocytes. Stage-specific effects were mathematically analyzed and combined to form one inhibitory curve normalized to untreated gametocytes. MB exerted the greatest reduction in gametocyte viability over the full course of development. (B) Effects of antimalarials on parasite development within mosquitoes fed on drug-treated gametocytes. These in vivo studies revealed potent transmission-blocking properties of MB, which showed near-complete suppression of oocyst formation. ATQ, atovaquone; DHA, dihydroartemisinin; LMF, lumefantrine; MB, methylene blue; mdAQ, monodesethyl-amodiaquine; PMQ, primaquine; PND, pyronaridine; PPQ, piperaquine; TFQ, tafenoquine.