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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.