Role of Rabenosyn-5 and Rab5b in host cell cytosol uptake reveals conservation of endosomal transport in malaria parasites.

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of Plasmodium falciparum blood-stage parasites. Endocytosis of host cell cytosol (HCC) provides nutrients and room for parasite growth and is critical for the action of antimalarial drugs and parasite drug resistance. Previous work showed that PfVPS45 functions in endosomal transport of HCC to the parasite's food vacuole, raising the possibility that malaria parasites possess a canonical endolysosomal system. However, the seeming absence of VPS45-typical functional interactors such as rabenosyn 5 (Rbsn5) and the repurposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here, we identified a parasite Rbsn5-like protein and show that it functions with VPS45 in the endosomal transport of HCC. We also show that PfRab5b but not PfRab5a is involved in the same process. Inactivation of PfRbsn5L resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall, this indicates that despite the low sequence conservation of PfRbsn5L and the unusual N-terminal modification of PfRab5b, principles of endosomal transport in malaria parasite are similar to that of model organisms. Using a conditional double protein inactivation system, we further provide evidence that the PfKelch13 compartment, an unusual apicomplexa-specific endocytosis structure at the parasite plasma membrane, is connected upstream of the Rbsn5L/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC uptake consists of a highly parasite-specific part that feeds endocytosed material into an endosomal system containing more canonical elements, leading to the delivery of HCC to the food vacuole.

A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites.

Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite's Kelch13 protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance.

Identification of novel parasitophorous vacuole proteins in P. falciparum parasites using BioID.

Malaria blood stage parasites develop within red blood cells where they are contained in a vacuolar compartment known as the parasitophorous vacuole (PV). This compartment holds a key role in the interaction of the parasite with its host cell. However, the proteome of this compartment has so far not been comprehensively analysed. Here we used BioID in asexual blood stages of the most virulent human malaria parasite Plasmodium falciparum to identify new proteins of the PV. The resulting proteome contained many of the already known PV proteins and validation by GFP-knock-in of 10 previously in P. falciparum uncharacterised hits revealed 5 new PV proteins and two with a partial PV localisation. This included proteins peripherally attached to the inner face of the PV membrane as well as proteins anchored in the parasite plasma membrane that protrude into the PV. Using selectable targeted gene disruption we generated mutants for 2 of the 10 candidates. In contrast we could not select parasites with disruptions for another 3 candidates, strongly suggesting that they are important for parasite growth. Interestingly, one of these included the orthologue of UIS2, a protein previously proposed to regulate protein translation in the parasite cytoplasm but here shown to be an essential PV protein. This work extends the number of known PV proteins and provides a starting point for further functional analyses of this compartment.