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The human malaria parasite Plasmodium falciparum invades red blood cells (RBCs) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, protecting iRBCs from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to in vivo studies. The P. berghei iRBC also sequesters by CD36-binding via an unknown parasite ligand and few parasite proteins, including EMAP1 and EMAP2, have been localised to the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it likely interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not a major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen in vivo for putative inhibitors of P. falciparum cytoadherence.

Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite’s biology at scale.

Malaria remains a major global health concern that threatens 40% of the world’s population, particularly in low-income tropical and subtropical regions. High malaria transmission countries experience a 1.3% reduction in economic growth due to the consequences of the disease. Additionally, young children and pregnant women are at the highest risk of developing severe disease. Despite advancements in the development of antimalarial drugs and vector control strategies, the disease is yet to be eradicated. The emergence of drug resistance, the lack of a highly effective vaccine, and incomplete transmission control emphasize the relevance of continued research towards malaria eradication. This body of work addresses critical gaps in understanding Plasmodium biology and host-parasite interactions that underpin malaria pathogenesis.

A better understanding of the parasite’s underlying biology is crucial to developing interventions for malaria. Nearly half of the Plasmodium genome is predicted to be essential for parasite survival, yet much of it remains uncharacterized—highlighting a pool of potential targets for novel treatments and vaccines. Uncovering the function of essential genes in Plasmodium has been difficult due to the low genetic tractability of the parasite. In manuscript 1, we present a scalable CRISPR-based genome editing system for the ubiquitously used rodent malaria model parasite Plasmodium berghei called PbHiT. This platform enables efficient and high-throughput functional genetic screening of parasite genes, using knockout or epitope tagging vectors that efficiently integrate into the target locus, providing a novel tool for malaria research that can be tailored to study essential genes. We provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents the first tool for high-throughput CRISPR screens in Plasmodium for studying the parasite’s biology at scale.

Malaria pathogenesis and the development of symptoms are closely tied to the asexual blood stage of the parasite. The parasite invades and remodels host red blood cells (RBCs), exporting approximately 10% of its proteome to alter host cell structure and function to support its survival. These modifications enhance nutrient uptake, increase cell rigidity, and enable cytoadherence of infected RBCs (iRBCs) to vascular endothelium. Cytoadherence promotes parasite growth through organ sequestration and immune evasion by prevention of clearance by the spleen and contributes to severe disease. While proteins such as PfEMP1 and host receptor CD36 are known to mediate cytoadherence in the human malaria parasite Plasmodium falciparum, the analogous parasite factors in the rodent model P. berghei remain largely undefined. In manuscript 2, we identify a novel P. berghei protein, EMAP3, which localizes to the iRBC membrane and interacts with the exported protein EMAP1. Deletion of EMAP3 does not affect sequestration, however, its external localization makes it a potentially valuable scaffold for displaying P. falciparum proteins, enabling in vivo screening of cytoadherence inhibitors. Continuing the focus on parasite proteins that mediate interactions with the host, manuscript 3 investigates two previously uncharacterized rhoptry-associated proteins, MAP1 and RhoSH, shown to be implicated in parasite virulence. These proteins are localized to parasite secretory organelles called the rhoptries and are likely injected into the host cell upon infection where they function to form or maintain the protective vacuole the parasite creates upon infection.  Notably, MAP1 contributes to adipose tissue sequestration and MAP1 knockout parasites fail to induce host leptin production, which is associated with severe disease. Together, these findings advance our understanding of parasite-host interactions during the blood stage and help identify new molecular targets for therapeutic intervention and malaria eradication strategies.

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.

Juvenile open-angle glaucoma (JOAG) is a severe type of glaucoma with onset before age 40 and dominant inheritance. Using exome sequencing we identified 3 independent families from the Philippines with novel EFEMP1 variants (c.238A>T, p.Asn80Tyr; c.1480T>C, p.Ter494Glnext*29; and c.1429C>T, p.Arg477Cys) co-segregating with disease. Affected variant carriers (N = 34) exhibited severe disease with average age of onset of 16 years and with 76% developing blindness. To investigate functional effects, we transfected COS7 cells with vectors expressing the three novel EFEMP1 variants and showed that all three variants found in JOAG patients caused significant intracellular protein aggregation and retention compared to wild type and also compared to EFEMP1 variants associated with other ocular phenotypes including an early-onset form of macular degeneration, Malattia Leventinese/Doyne's Honeycomb retinal dystrophy. These results suggest that rare EFEMP1 coding variants can cause JOAG through a mechanism involving protein aggregation and retention, and that the extent of intracellular retention correlates with disease phenotype. This is the first report of EFEMP1 variants causing JOAG, expanding the EFEMP1 disease spectrum. Our results suggest that EFEMP1 mutations appear to be a relatively common cause of JOAG in Filipino families, an ethnically diverse population.

Malaria, caused by Plasmodium parasites, claims over 600,000 deaths annually. Parasite invasion of red blood cells (RBCs) involves protein secretion from specialised organelles— micronemes, rhoptries, and dense granules—to facilitate host cell entry and establish a protective parasitophorous vacuole (PV). Despite the critical role of rhoptry proteins in infection, many remain poorly characterised due to the absence of recognisable trafficking motifs and dispensability in vitro. Here, we leverage spatial proteomics from Plasmodium falciparum to identify two novel Plasmodium berghei ortholog proteins associated with the PV (MAP1, PBANKA_1425900 and RhoSH, PBANKA_1001500) both containing hydrolase domains. Ultra-expansion microscopy reveals their localisation to the rhoptries in late schizogony, while co-immunoprecipitation shows their interaction. In vivo studies demonstrate that these proteins help the parasite evade spleen-mediated clearance and contribute to disease progression. One protein, MAP1, mediates sequestration to adipose tissue, and conditional knockdown of its P. falciparum ortholog results in reduced CD36-mediated cytoadhesion, suggesting a mechanism for immune evasion and sustained infection. Our findings identify MAP1 and RhoSH as key mediators of Plasmodium virulence. Takingadvantage of an in vivo approach, this work provides valuable insights toward global malaria eradication efforts as it lays the groundwork for novel therapeutic strategies, positioning mainly MAP1 but also RhoSH as promising targets, including their use as antigens in recombinant vaccines, attenuated live vaccine candidates, or enzyme-inhibiting drugs.

The human malaria parasite Plasmodium falciparum invades red blood cells (RBC) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate uptake of nutrients and cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, thus protecting the iRBC from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to in vivo studies. P. berghei iRBC also sequesters in small blood vessels, mediated by binding to CD36. However, the parasite proteins binding to CD36 are unknown and only very few parasite proteins, including EMAP1 and EMAP2, have been identified that are present at the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not the major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen in vivo putative inhibitors of P. falciparum cytoadherence.