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.

Current systems to study essential genes in the human malaria parasite Plasmodium falciparum are often inefficient and time intensive, and they depend on the genetic modification of the target locus, a process hindered by the low frequency of integration of episomal DNA into the genome. Here, we introduce a method, termed selection-linked integration (SLI), to rapidly select for genomic integration. SLI allowed us to functionally analyze targets at the gene and protein levels, thus permitting mislocalization of native proteins, a strategy known as knock sideways, floxing to induce diCre-based excision of genes and knocking in altered gene copies. We demonstrated the power and robustness of this approach by validating it for more than 12 targets, including eight essential ones. We also localized and inducibly inactivated Kelch13, the protein associated with artemisinin resistance. We expect this system to be widely applicable for P. falciparum and other organisms with limited genetic tractability.

Highlights d Inactivation of VPS45 abolishes the growth of malaria blood stage parasites d VPS45 is located near the parasite's food vacuole and Golgi d VPS45 is needed for the transport of host cell cytosol to the parasite's food vacuole d Host cell cytosol-filled transport vesicles display the endosomal marker PI(3)P

No abstract

BackgroundThe family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation.MethodsIn this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo.ResultsBoth investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony.ConclusionsOur results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0982-3) contains supplementary material, which is available to authorized users.

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility ofP. falciparumparasites to Artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in ART resistance. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins are among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by identifying four novel proteins at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these, and previously identified proteins, showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance and endocytosis, strengthening the link to endocytosis. Despite this, most showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation. A second group of proteins did not influence endocytosis or ART resistance and was characterised by a lack of vesicle trafficking domains. We here identified the first essential protein of the second group and showed that it is needed in late-stage parasites. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of the endocytosis in malaria parasites.

Homologous recombination-based integration of plasmids into the genome of Plasmodium falciparum parasites is inefficient. The traditionally used drug cycling to obtain parasites with such integrations ('integrants') is time consuming and not always successful. Here we provide a protocol for the rapid selection of integrants and describe how to use it for endogenous gene tagging or to select parasites with specific gene disruptions. Using the appropriate tags, the gene product can then be functionally analysed using knock sideways. A protocol for a flow cytometry (FC) assay to assess the impact of inactivating the gene product on parasite development is also provided. These protocols accompany

Vesicular trafficking, including secretion and endocytosis, plays fundamental roles in the unique biology of P. 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 re-purposing of Rab5 isoforms and other endolysosomal proteins for secretion in apicomplexans question this idea. Here we identified the likely parasite Rbsn5 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 PfRbsn5 resulted in PI3P and PfRab5b decorated HCC-filled vesicles, typical for endosomal compartments. Overall this indicates that despite the low sequence conservation of PfRbsn5 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 Rbsn5/VPS45/Rab5b-dependent endosomal route. Altogether, this work indicates that HCC-uptake consists of a highly parasite-specific part that feeds endocytosed material into a more canonical endosomal system, leading to the delivery of HCC to the food vacuole.