Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in Plasmodium falciparum parasites.

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in kelch13 (k13) but also in other genes, like ubp1. K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. IMPORTANCE: Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including k13, were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene ubp1. Such high fitness costs may explain why mutations in ubp1 and other genes functioning in the same pathway as k13 are rare. This highlights that k13 mutations are unique in their ability to cause resistance at a comparably low fitness cost.

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.

Gene-by-gene screen of the unknown proteins encoded on Plasmodium falciparum chromosome 3.

Taxon-specific proteins are key determinants defining the biology of all organisms and represent prime drug targets in pathogens. However, lacking comparability with proteins in other lineages makes them particularly difficult to study. In malaria parasites, this is exacerbated by technical limitations. Here, we analyzed the cellular location, essentiality, function, and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nucleus was the most common localization, indicating that it is a hotspot of parasite-specific biology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The mitosis proteins defined a possible orphan complex and a highly diverged complex needed for spindle-kinetochore connection. Structure-function comparisons indicated that the taxon-specific proteins evolved by different mechanisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biology of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

The ZIP Code of Vesicle Trafficking in Apicomplexa: SEC1/Munc18 and SNARE Proteins.

Apicomplexans are obligate intracellular parasites harboring three sets of unique secretory organelles termed micronemes, rhoptries, and dense granules that are dedicated to the establishment of infection in the host cell. Apicomplexans rely on the endolysosomal system to generate the secretory organelles and to ingest and digest host cell proteins. These parasites also possess a metabolically relevant secondary endosymbiotic organelle, the apicoplast, which relies on vesicular trafficking for correct incorporation of nuclear-encoded proteins into the organelle. Here, we demonstrate that the trafficking and destination of vesicles to the unique and specialized parasite compartments depend on SNARE proteins that interact with tethering factors. Specifically, all secreted proteins depend on the function of SLY1 at the Golgi. In addition to a critical role in trafficking of endocytosed host proteins, TgVps45 is implicated in the biogenesis of the inner membrane complex (alveoli) in both Toxoplasma gondii and Plasmodium falciparum, likely acting in a coordinated manner with Stx16 and Stx6. Finally, Stx12 localizes to the endosomal-like compartment and is involved in the trafficking of proteins to the apical secretory organelles rhoptries and micronemes as well as to the apicoplast.IMPORTANCE The phylum of Apicomplexa groups medically relevant parasites such as those responsible for malaria and toxoplasmosis. As members of the Alveolata superphylum, these protozoans possess specialized organelles in addition to those found in all members of the eukaryotic kingdom. Vesicular trafficking is the major route of communication between membranous organelles. Neither the molecular mechanism that allows communication between organelles nor the vesicular fusion events that underlie it are completely understood in Apicomplexa. Here, we assessed the function of SEC1/Munc18 and SNARE proteins to identify factors involved in the trafficking of vesicles between these various organelles. We show that SEC1/Munc18 in interaction with SNARE proteins allows targeting of vesicles to the inner membrane complex, prerhoptries, micronemes, apicoplast, and vacuolar compartment from the endoplasmic reticulum, Golgi apparatus, or endosomal-like compartment. These data provide an exciting look at the "ZIP code" of vesicular trafficking in apicomplexans, essential for precise organelle biogenesis, homeostasis, and inheritance.

Endocytosis in Plasmodium and Toxoplasma Parasites.

Endocytosis is critical for many functions in eukaryotic cells. Uptake of host cell cytosol, an indispensable endocytic process in malaria blood-stage parasites, has been known for a long time. However, it is only recently that the proteins involved in this process have started to emerge. Unexpectedly, some of these proteins revealed a critical role for endocytosis in artemisinin resistance. More recently, endocytosis was discovered in both intracellular and extracellular Toxoplasma gondii parasites. Here, we review these findings, compare the endolysosomal systems of Toxoplasma and Plasmodium parasites, and present current knowledge about endocytic mechanisms in apicomplexans.

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.

PfVPS45 Is Required for Host Cell Cytosol Uptake by Malaria Blood Stage Parasites.

During development in human erythrocytes, the malaria parasite Plasmodium falciparum internalizes a large part of the cellular content of the host cell. The internalized cytosol, consisting largely of hemoglobin, is transported to the parasite's food vacuole where it is degraded, providing nutrients and space for growth. This host cell cytosol uptake (HCCU) is crucial for parasite survival but the parasite proteins mediating this process remain obscure. Here, we identify P. falciparum VPS45 as an essential factor in HCCU. Conditional inactivation of PfVPS45 led to an accumulation of host cell cytosol-filled vesicles within the parasite and inhibited the delivery of hemoglobin to the parasite's digestive vacuole, resulting in arrested parasite growth. A proportion of these HCCU vesicle intermediates was positive for phosphatidylinositol 3-phosphate, suggesting endosomal characteristics. Thus PfVPS45 provides insight into the elusive machinery of the ingestion pathway in a parasite that contains an endolysosomal system heavily repurposed for protein secretion.