Gene expression patterns have been demonstrated to be highly variable between similar cell types, for example lab strains and wild strains of Saccharomyces cerevisiae cultured under identical growth conditions exhibit a wide range of expression differences. We have used a genome-wide approach to characterize transcriptional differences between strains of Plasmodium falciparum by characterizing the transcriptome of the 48 h intraerythrocytic developmental cycle (IDC) for two strains, 3D7 and Dd2 and compared these results to our prior work using the HB3 strain. These three strains originate from geographically diverse locations and possess distinct drug sensitivity phenotypes. Our goal was to identify transcriptional differences related to phenotypic properties of these strains including immune evasion and drug sensitivity. We find that the highly streamlined transcriptome is remarkably well conserved among all three strains, and differences in gene expression occur mainly in genes coding for surface antigens involved in parasite-host interactions. Our analysis also detects several transcripts that are unique to individual strains as well as identifying large chromosomal deletions and highly polymorphic regions across strains. The majority of these genes are uncharacterized and have no homology to other species. These tractable transcriptional differences provide important phenotypes for these otherwise highly related strains of Plasmodium.

Background

The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development.

Results

We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages.

Conclusions

DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.

Plasmodium falciparum is the causative agent of the most burdensome form of human malaria, affecting 200-300 million individuals per year worldwide. The recently sequenced genome of P. falciparum revealed over 5,400 genes, of which 60% encode proteins of unknown function. Insights into the biochemical function and regulation of these genes will provide the foundation for future drug and vaccine development efforts toward eradication of this disease. By analyzing the complete asexual intraerythrocytic developmental cycle (IDC) transcriptome of the HB3 strain of P. falciparum, we demonstrate that at least 60% of the genome is transcriptionally active during this stage. Our data demonstrate that this parasite has evolved an extremely specialized mode of transcriptional regulation that produces a continuous cascade of gene expression, beginning with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium-specific functionalities, such as genes involved in erythrocyte invasion. The data reveal that genes contiguous along the chromosomes are rarely coregulated, while transcription from the plastid genome is highly coregulated and likely polycistronic. Comparative genomic hybridization between HB3 and the reference genome strain (3D7) was used to distinguish between genes not expressed during the IDC and genes not detected because of possible sequence variations. Genomic differences between these strains were found almost exclusively in the highly antigenic subtelomeric regions of chromosomes. The simple cascade of gene regulation that directs the asexual development of P. falciparum is unprecedented in eukaryotic biology. The transcriptome of the IDC resembles a "just-in-time" manufacturing process whereby induction of any given gene occurs once per cycle and only at a time when it is required. These data provide to our knowledge the first comprehensive view of the timing of transcription throughout the intraerythrocytic development of P. falciparum and provide a resource for the identification of new chemotherapeutic and vaccine candidates.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 μM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 μM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 μM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.