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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.