Chromatin structure and var2csa - a tango in regulation of var gene expression in the human malaria parasite, Plasmodium falciparum?

Over the last few decades, novel methods have been developed to study how chromosome positioning within the nucleus may play a role in gene regulation. Adaptation of these methods in the human malaria parasite, Plasmodium falciparum, has recently led to the discovery that the three-dimensional structure of chromatin within the nucleus may be critical in controlling expression of virulence genes (var genes). Recent work has implicated an unusual, highly conserved var gene called var2csa in contributing to coordinated transcriptional switching, however how this gene functions in this capacity is unknown. To further understand how var2csa influences var gene switching, targeted DNA double-strand breaks (DSBs) within the sub-telomeric region of chromosome 12 were used to delete the gene and the surrounding chromosomal region. To characterize the changes in chromatin architecture stemming from this deletion and how these changes could affect var gene expression, we used a combination of RNA-seq, Chip-seq and Hi-C to pinpoint epigenetic and chromatin structural modifications in regions of differential gene expression. We observed a net gain of interactions in sub-telomeric regions and internal var gene regions following var2csa knockout, indicating an increase of tightly controlled heterochromatin structures. Our results suggest that disruption of var2csa results not only in changes in var gene transcriptional regulation but also a significant tightening of heterochromatin clusters thereby disrupting coordinated activation of var genes throughout the genome. Altogether our result confirms a strong link between the var2csa locus, chromatin structure and var gene expression.

Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

DNA-binding protein PfAP2-P regulates parasite pathogenesis during malaria parasite blood stages.

Malaria-associated pathogenesis such as parasite invasion, egress, host cell remodelling and antigenic variation requires concerted action by many proteins, but the molecular regulation is poorly understood. Here we have characterized an essential Plasmodium-specific Apicomplexan AP2 transcription factor in Plasmodium falciparum (PfAP2-P; pathogenesis) during the blood-stage development with two peaks of expression. An inducible knockout of gene function showed that PfAP2-P is essential for trophozoite development, and critical for var gene regulation, merozoite development and parasite egress. Chromatin immunoprecipitation sequencing data collected at timepoints matching the two peaks of pfap2-p expression demonstrate PfAP2-P binding to promoters of genes controlling trophozoite development, host cell remodelling, antigenic variation and pathogenicity. Single-cell RNA sequencing and fluorescence-activated cell sorting revealed de-repression of most var genes in Δpfap2-p parasites. Δpfap2-p parasites also overexpress early gametocyte marker genes, indicating a regulatory role in sexual stage conversion. We conclude that PfAP2-P is an essential upstream transcriptional regulator at two distinct stages of the intra-erythrocytic development cycle.

Novel insights into the role of long non-coding RNA in the human malaria parasite, Plasmodium falciparum.

The complex life cycle of Plasmodium falciparum requires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum we explore the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identify a total of 1768 lncRNAs, of which 718 (~41%) are novels in P. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs are validated using RNA-FISH. Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs is explored using ChIRP. The results reveal that lncRNA occupancy sites are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis and sexual differentiation. Genomic and phenotypic analysis of one specific lncRNA demonstrate its importance in sexual differentiation and reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation, opening new avenues for targeted therapeutic strategies against the deadly malaria parasite.

PfAP2-MRP DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages.

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 the var gene 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 essential Plasmodium specific Apicomplexan AP2 (ApiAP2) transcription factor in Plasmodium 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 for var gene 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 most var genes in Δpfap2-mrp parasites that express multiple PfEMP1 proteins on the surface of infected RBCs. In addition, the Δpfap2-mrp parasites 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 and var gene expression.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Chromosomes Conformation Capture Coupled with Next-Generation Sequencing (Hi-C) in Plasmodium falciparum.

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.

Three-dimensional chromatin in infectious disease-A role for gene regulation and pathogenicity?

The recent Coronavirus Disease 2019 pandemic has once again reminded us the importance of understanding infectious diseases. One important but understudied area in infectious disease research is the role of nuclear architecture or the physical arrangement of the genome in the nucleus in controlling gene regulation and pathogenicity. Recent advances in research methods, such as Genome-wide chromosome conformation capture using high-throughput sequencing (Hi-C), have allowed for easier analysis of nuclear architecture and chromosomal reorganization in both the infectious disease agents themselves as well as in their host cells. This review will discuss broadly on what is known about nuclear architecture in infectious disease, with an emphasis on chromosomal reorganization, and briefly discuss what steps are required next in the field.

Dynamic Chromatin Structure and Epigenetics Control the Fate of Malaria Parasites.

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.

Three-Dimensional Genome Organization and Virulence in Apicomplexan Parasites.

Mounting evidence supports the idea that epigenetic, and the overall 3-dimensional (3D) architecture of the genome, plays an important role in gene expression for eukaryotic organisms. We recently used Hi-C methodologies to generate and compare the 3D genome of 7 different apicomplexan parasites, including several pathogenic and less pathogenic malaria parasites as well as related human parasites Babesia microti and Toxoplasma gondii. Our goal was to understand the possible relationship between genome organization, gene expression, and pathogenicity of these infectious agents. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes that are unique in their effect on chromosome folding, indicating a link between genome organization and gene expression in more virulent pathogens.