Imaging-based high-throughput screening assay to identify new molecules with transmission-blocking potential against Plasmodium falciparum female gamete formation.

In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z' factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 µM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential.

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

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.

Epigenetic control of the variable expression of a Plasmodium falciparum receptor protein for erythrocyte invasion.

Plasmodium falciparum can invade erythrocytes by redundant receptors, some of which have variable expression. A P. falciparum clone Dd2 requiring erythrocyte sialic acid for invasion can be switched to a sialic acid-independent progeny clone Dd2NM by growing the Dd2 clone with neuraminidase-treated erythrocytes. The RH4 gene is transcriptionally up-regulated in Dd2NM compared to Dd2, despite the absence of DNA changes in and around the gene. We determined the epigenetic modifications around the transcription start site (TSS) at the time of expression of RH4 in Dd2NM (44 h) and at an earlier time when RH4 is not expressed (24 h). At 44 h, the occupancy of the +1 nucleosome site downstream of the TSS of the active RH4 gene in Dd2NM was markedly reduced compared to Dd2; no difference was observed at 24 h. At 44 h, histone modifications associated with up-regulation were positively correlated to the active RH4 gene of Dd2NM compared to Dd2; no differences were observed at 24 h. Histone H3K9 trimethylation (a marker for silencing) was higher in Dd2 than Dd2NM along the 5'-UTRs of the RH4 gene at both 44 and 24 h. Our data indicate that the failure of Dd2 to express the sialic acid-independent invasion receptor gene RH4 is associated with the epigenetic silencing mark H3K9 trimethylation present throughout the cycle.

Effect of PCR extension temperature on high-throughput sequencing.

The DNA amplification process can be a source of bias and artifacts, especially when amplifying genomic areas with extreme AT or GC content. The human malaria parasite Plasmodium falciparum has an AT-rich genome, and some of its highly AT-rich regions have been shown to be refractory to polymerase chain reaction (PCR) amplification. Biased amplification may lead to erroneous conclusions for studies investigating genome-wide gene expression, nucleosome position, and copy number variation. Here we compare genome-wide nucleosome coverage in libraries amplified at three different extension temperatures and show that reduction in PCR extension temperature from 70°C to 60°C can greatly increase the fraction of coverage at AT-rich regions of the P. falciparum genome. Our method will improve the efficiency and coverage in sequencing an AT-rich genome.