Heather J. Painter

Sexual maturation and fertility of the malaria-causing parasite, Plasmodium spp., are critical to transmission between hosts. This developmental process involves an intricate gene expression regulatory network that drives gametocyte commitment, development, and the production of male and female gametes. Here, we characterize the role of the CCCH-type zinc-finger protein Plasmodium falciparum Male Development 3 ( Pf MD3) in the formation of gametes. Genetic disruption of Pf MD3 reveals a male fertility defect that decreases infectivity to mosquitoes. Molecular characterization of Pf MD3 reveals its binding to mRNA transcripts is associated with male gamete development through a specific RNA sequence. Gametocyte proteins encoded by these Pf MD3-bound transcripts decrease significantly in Pf MD3 knockout parasites, suggesting that Pf MD3 impacts translation. In addition to RNA-binding, Pf MD3 also interacts with proteins involved in mRNA processing and nuclear export that influence translational regulation. This study establishes Pf MD3 as an RNA-binding protein that impacts lineage-specific translation and male gamete fertility.

Plasmodium vivax (Pv) forms non-replicating liver stages called hypnozoites, which activate after primary infection, and cause relapses of symptomatic blood-stage malaria. We hypothesize that hypnozoites must actively suppress schizogony to maintain a quiescent state. Differential transcriptome prospecting identifies two hypnozoite-expressed transcripts encoding putative RNA-binding proteins. We assess the functional role of the two encoded proteins in Plasmodium yoelii (Py) , a rodent malaria parasite that naturally does not form hypnozoites. Strikingly, individual expression of each protein in Py liver stages blocks liver stage schizogony, with parasites remaining small and uninucleate. A screen of RNA sequences that interact with the putative RNA-binding domains of these proteins shows enrichment of distinct, highly specific motifs, indicating that they might block schizogony by binding RNAs containing these motifs. Our findings provide the unprecedented functional evidence for one potential molecular mechanism of hypnozoite formation. Based on their function we name these proteins IESI-1 and IESI-2 (Initiation of Exo-erythrocytic Schizogony Inhibited).

Sexual maturation and fertility of the malaria causing parasite, Plasmodium spp., is critical to transmission between hosts. This developmental process involves an intricate gene expression regulatory network that drives gametocyte commitment, development, and the production of male and female gametes. Here, we characterized the role of the CCCH-type zinc-finger protein Plasmodium falciparum Male Development 3 ( Pf MD3) in the formation of gametes. Genetic disruption of Pf MD3 revealed a male fertility defect that decreased infectivity to mosquitoes. Molecular characterization of Pf MD3 revealed its binding to mRNA transcripts associated with male gamete development through a specific RNA sequence. Gametocyte proteins encoded by these Pf MD3-bound transcripts decreased significantly in Pf MD3 knockout parasites, suggesting that Pf MD3 impacts translation. In addition to RNA-binding, Pf MD3 also interacts with proteins involved in mRNA processing and nuclear export that influence translational regulation. This study establishes Pf MD3 as an RNA-binding protein that impacts lineage-specific translation and male gamete fertility.

Plasmodium vivax (Pv) forms non-replicating liver stages called hypnozoites, which activate after primary infection, and cause relapses of symptomatic blood-stage malaria. We hypothesized that hypnozoites must actively suppress schizogony to maintain a quiescent state. Differential transcriptome prospecting idenfied two hypnozoite-expressed transcripts encoding putative RNA-binding proteins. We assessed the functional role of the two encoded proteins in Plasmodium yoelii (Py), a rodent malaria parasite that naturally does not form hypnozoites. Strikingly, individual expression of each protein in Py liver stages blocked liver stage schizogony, with parasites remaining small and uninucleate. A screen of RNA sequences that interact with the putative RNA-binding domains of these proteins showed enrichment of distinct, highly specific motifs, indicating that these protein might block schizogony by binding RNAs containing these motifs. Our findings provide the unprecedented functional evidence for one potential molecular mechanism of hypnozoite formation. Based on their function we named these proteins IESI-1 and IESI-2 (Initiation of Exo-erythrocytic Schizogony inhibited).

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B “pauses” the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc ₁ complex, which is an essential component of the mitochondrial electron transport chain (mtETC) required for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis.

Differentiation from asexual blood stages to mature sexual gametocytes is required for the transmission of malaria parasites. Here, we report that the ApiAP2 transcription factor, PfAP2‐G2 (PF3D7_1408200) plays a critical role in the maturation of Plasmodium falciparum gametocytes. PfAP2‐G2 binds to the promoters of a wide array of genes that are expressed at many stages of the parasite life cycle. Interestingly, we also find binding of PfAP2‐G2 within the gene body of almost 3,000 genes, which strongly correlates with the location of H3K36me3 and several other histone modifications as well as Heterochromatin Protein 1 (HP1), suggesting that occupancy of PfAP2‐G2 in gene bodies may serve as an alternative regulatory mechanism. Disruption of pfap2‐g2 does not impact asexual development, but the majority of sexual parasites are unable to mature beyond stage III gametocytes. The absence of pfap2‐g2 leads to overexpression of 28% of the genes bound by PfAP2‐G2 and none of the PfAP2‐G2 bound genes are downregulated, suggesting that it is a repressor. We also find that PfAP2‐G2 interacts with chromatin remodeling proteins, a microrchidia (MORC) protein, and another ApiAP2 protein (PF3D7_1139300). Overall our data demonstrate that PfAP2‐G2 establishes an essential gametocyte maturation program in association with other chromatin‐related proteins.

The continued emergence of drug-resistant Plasmodium falciparum parasites hinders global attempts to eradicate malaria, emphasizing the need to identify new antimalarial drugs. Attractive targets for chemotherapeutic intervention are the cytochrome (cyt) bc ₁ complex, which is an essential component of the mitochondrial electron transport chain (mtETC) necessary for ubiquinone recycling and mitochondrially localized dihydroorotate dehydrogenase (DHODH) critical for de novo pyrimidine synthesis. Despite the essentiality of this complex, resistance to a novel acridone class of compounds targeting cyt bc ₁ was readily attained, resulting in a parasite strain (SB1-A6) that was pan-resistant to both mtETC and DHODH inhibitors. Here we describe the molecular mechanism behind the resistance of the SB1-A6 parasite line which lacks the common cyt bc ₁ point mutations characteristic of resistance to mtETC inhibitors. Using Illumina whole-genome sequencing, we have identified both a copy number variation (∼2x) and a single-nucleotide polymorphism (C276F) associated with pfdhodh in SB1-A6. We have characterized the role of both genetic lesions by mimicking the copy number variation via episomal expression of pfdhodh and introducing the identified SNP using CRISPR/Cas9 and assessed their contributions to drug resistance. Although both of these genetic polymorphisms have been previously identified as contributing to both DSM-1 (1) and atovaquone resistance (2, 3), SB1-A6 represents a unique genotype in which both alterations are present in a single line, suggesting that the combination contributes to the pan-resistant phenotype. This novel mechanism of resistance to mtETC inhibition has critical implications for the development of future drugs targeting the bc ₁ complex or de novo pyrimidine synthesis that could help guide future anti-malarial combination therapies and reduce the rapid development of drug resistance in the field.

Background: Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome ofPlasmodium falciparumas it commits to and develops through gametocytogenesis.Results: The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis inP. falciparum,which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors.Conclusions: The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage ofPlasmodiumdevelopment, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.

Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis. The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The striking transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum , which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors. The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.