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Malaria pathogenicity results from the parasite’s ability to invade, multiply within and then egress from the host red blood cell (RBC). Infected RBCs are remodeled, expressing antigenic variant proteins (such as PfEMP1, coded by thevargene family) for immune evasion and survival. These processes require the concerted actions of many proteins, but the molecular regulation is poorly understood. We have characterized an essentialPlasmodiumspecific Apicomplexan AP2 (ApiAP2) transcription factor inPlasmodium falciparum(PfAP2-MRP; Master Regulator of Pathogenesis) during the intraerythrocytic developmental cycle (IDC). An inducible gene knockout approach showed that PfAP2-MRP is essential for development during the trophozoite stage, and critical forvargene regulation, merozoite development and parasite egress. ChIP-seq experiments performed at 16 hour post invasion (h.p.i.) and 40 h.p.i. matching the two peaks of PfAP2-MRP expression, demonstrate binding of PfAP2-MRP to the promoters of genes controlling trophozoite development and host cell remodeling at 16 h.p.i. and antigenic variation and pathogenicity at 40 h.p.i. Using single-cell RNA-seq and fluorescence-activated cell sorting, we show de-repression of mostvargenes inΔpfap2-mrpparasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, theΔpfap2-mrpparasites overexpress several early gametocyte marker genes at both 16 and 40 h.p.i., indicating a regulatory role in the sexual stage conversion. Using the Chromosomes Conformation Capture experiment (Hi-C), we demonstrate that deletion of PfAP2-MRP results in significant reduction of both intra-chromosomal and inter-chromosomal interactions in heterochromatin clusters. We conclude that PfAP2-MRP is a vital upstream transcriptional regulator controlling essential processes in two distinct developmental stages during the IDC that include parasite growth, chromatin structure andvargene expression.

The complex life cycle ofPlasmodium falciparumrequires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified and shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs inP. falciparumwe explored the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identified a total of 1,768 lncRNAs, of which 58% were identified as novel lncRNAs inP. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs were validated using RNA fluorescence in situ hybridization (RNA-FISH). Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs was explored using Chromatin Isolation by RNA Purification (ChIRP). ChIRP-seq of candidate lncRNAs revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. We further validated the function of one specific lncRNA (lncRNA-ch14) using the CRISPR-Cas9 genome editing tool. Genomic and phenotypic analysis of the △lncRNA-ch14 line demonstrated the importance of this lncRNA in sexual differentiation and sexual reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation. These findings also open new avenues for targeted approaches towards therapeutic strategies against the deadly malaria parasite.

Babesia species are tick-transmitted apicomplexan pathogens and the causative agents of babesiosis, a malaria-like disease of major medical and veterinary importance. Of the different species of Babesia reported so far, Babesia duncani causes severe to lethal infection in patients. Despite the highly virulent nature of this parasite and the risk it may pose as an emerging pathogen, little is known about its biology, metabolic requirements, and pathogenesis. B. duncani is unique among apicomplexan parasites that infect red blood cells in that it can be continuously cultured in vitro in human erythrocytes but can also infect mice leading to fulminant babesiosis infection and death. Here we have taken advantage of the recent advances in the propagation of this parasite in vitro and in vivo to conduct detailed molecular, genomic and transcriptomic analyses and to gain insights into its biology. We report the assembly, 3D structure, and annotation of the nuclear genome of this parasite as well as its transcriptomic profile and an atlas of its metabolism during its intraerythrocytic life cycle. Detailed examination of the B. duncani genome and comparative genomic analyses identified new classes of candidate virulence factors, suitable antigens for diagnosis of active infection, and several attractive drug targets. Translational analyses and efficacy studies identified highly potent inhibitors of B. duncani thus enriching the pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Over the last decades, novel methods have been developed to study the role of chromosome positioning within the nucleus may play in gene regulation. Established proximity ligation-based chromosome conformation capture (3C) techniques such as Hi-C have revealed the existence of chromosome territories, functional nuclear landmarks, and topologically associating domains (TAPs) in most eukaryotic organisms. Adaptation of these methods in apicomplexan parasites has recently uncovered new aspects of 3D genome biology in virulence factors. Here, we describe the Hi-C protocol in the human malaria parasite, Plasmodium falciparum. This method can determine the genome organization in malaria parasites and its role in gene regulation and virulence.

Over the last decades, novel methods have been developed to study the role of chromosome positioning within the nucleus may play in gene regulation. Established proximity ligation-based chromosome conformation capture (3C) techniques such as Hi-C have revealed the existence of chromosome territories, functional nuclear landmarks, and topologically associating domains (TAPs) in most eukaryotic organisms. Adaptation of these methods in apicomplexan parasites has recently uncovered new aspects of 3D genome biology in virulence factors. Here, we describe the Hi-C protocol in the human malaria parasite, Plasmodium falciparum. This method can determine the genome organization in malaria parasites and its role in gene regulation and virulence.

Multiple hosts and various life cycle stages prompt the human malaria parasite, Plasmodium falciparum, to acquire sophisticated molecular mechanisms to ensure its survival, spread, and transmission to its next host. To face these environmental challenges, increasing evidence suggests that the parasite has developed complex and complementary layers of regulatory mechanisms controlling gene expression. Here, we discuss the recent developments in the discovery of molecular components that contribute to cell replication and differentiation and highlight the major contributions of epigenetics, transcription factors, and nuclear architecture in controlling gene regulation and life cycle progression in Plasmodium spp.

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii. In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.