BackgroundIt has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown.ResultsWe have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized.ConclusionsIt is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

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 (IC 50 s), 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 (ϳ3 to 5 females:1 male), current gametocyte assays without a sexspecific 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. Malaria is a disease of devastating economic and health burdens, with 216 million cases and 655,000 fatalities per year, among which most are either pregnant women or children of less than 5 years of age (1). The recent appreciation that local elimination and global eradication of malaria will require interventions that prevent parasite transmission from the human host to the vector (2) has revitalized the search for transmission-blocking drugs (3-7). One target of such drugs is the gametocyte, which is the parasite stage uniquely responsible for Plasmodium transmission to the mosquito.Plasmodium asexual parasites form gametocytes at a low frequency (0.2 to 1%) (8), with sexually committed merozoites from one precommitted schizont all forming gametocytes of the same sex (9). In Plasmodium falciparum, gametocytes develop over a period of 12 days, during which they are initially susceptible to schizonticidal antimalarials (stages I to III), but for the final part of their maturation process (stages IV and V), they become broadly insensitive to most antimalarial drugs, except for primaquine and methylene blue (6, 10-13). Mature stage V gametocytes, when considered as a single population, are developmentally arrested, and curr...

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.epigenetic mark | histone modification | nucleosome T he causative agent for the most severe human malaria, Plasmodium falciparum, invades erythrocytes by multiple redundant pathways (1, 2). Two P. falciparum receptor protein families, the Duffy Binding-Like (DBL) family and the Reticulocyte Homology (RH) family, play key roles in parasite invasion (1). A unique parasite clone Dd2 that initially invaded erythrocytes requiring sialic acid was able to be selected to a sialic acid-independent clone (Dd2NM) after maintaining the parental clone Dd2 in culture with neuraminidase-treated erythrocytes (3). Comparing the gene expression of Dd2 and Dd2NM, only RH4 and the DBL pseudogene PEBL, are transcriptionally up-regulated in Dd2NM (4, 5). The two genes are located contiguously on P. falciparum chromosome 4 and transcribed in opposite directions. The sequences of the exons and introns of the two genes, the 1.8 kb between the two ORFs, and 1.1 kb of genomic DNA 3′ to each gene's coding region were identical in Dd2 and Dd2NM (4), suggesting that transcription of the two genes is epigenetically regulated.Because a transcription-associated histone acetyltransferase from Tetrahymena was identified in 1996 (6), diverse histone modifications on different amino acid residues in the core histones have been discovered to play important roles in transcriptional regulation in various eukaryotic organisms (7). This led to a hypothesis of a program of epigenetic marks consisting of covalent histone tail modifications, chromatin remodeling, noncoding RNA, and DNA methylation (8). Thus far, no evidence for DNA methylation has been observed in P. falciparum. There are eight histone genes in P. falciparum that allow formation of ...

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 M 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. D espite the efforts made over decades of scientific research, malaria still remains a major health problem in tropical and subtropical areas, with more than 220 million cases and 600,000 deaths being registered per year (1). This parasitic disease is caused by Plasmodium infection through the bite of infected Anopheles female mosquitoes, with Plasmodium falciparum being responsible for the highest mortality rates (2).Traditionally, pharmacological antimalarial treatments have targeted parasite asexual reproduction inside erythrocytes, which leads to the clinical symptoms of malaria. However, a small proportion of these asexual blood stages (0.2 to 1%) are committed to develop into sexual stages: male and female gametocytes (3, 4). Their differentiation process inside erythrocytes takes 8 to 12 days for P. falciparum, and inside erythrocytes they undergo a series of morphological and metabolic changes classically categorized into five stages of maturation (5, 6). While most schizonticides, such as chloroquine, affect young gametocytes (stages I, II, and III), gametocytes at late stages of maturation are not sensitive to them (7). These insensitive stage V gametocytes, which are responsible for ...

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 70ºC to 60ºC 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.

Keywords 22Plasmodium falciparum; Plasmodium berghei; malaria; ookinete; transmission. 24not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/216721 doi: bioRxiv preprint first posted online Nov. 10, 2017; Note: Supplementary data associated with this article