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The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin ? Subunit is Clathrin Independent and Essential for Schizont Maturation.

ABSTRACT: The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the ? subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2? in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2? is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2? and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2?-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the ? subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.

SUBMITTER: Henrici RC

PROVIDER: S-EPMC7042695 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

Henrici Ryan C RC Edwards Rachel L RL Zoltner Martin M van Schalkwyk Donelly A DA Hart Melissa N MN Mohring Franziska F Moon Robert W RW Nofal Stephanie D SD Patel Avnish A Flueck Christian C Baker David A DA Odom John Audrey R AR Field Mark C MC Sutherland Colin J CJ

mBio 20200225 1

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to misloc ...[more]

PMID: 32098816

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Project description:BACKGROUND: The Plasmodium falciparum genome (3D7 strain) published in 2002, revealed ~5,400 genes, mostly based on in silico predictions. Experimental data is therefore required for structural and functional assessments of P. falciparum genes and expression, and polymorphic data are further necessary to exploit genomic information to further qualify therapeutic target candidates. Here, we undertook a large scale analysis of a P. falciparum FcB1-schizont-EST library previously constructed by suppression subtractive hybridization (SSH) to study genes expressed during merozoite morphogenesis, with the aim of: 1) obtaining an exhaustive collection of schizont specific ESTs, 2) experimentally validating or correcting P. falciparum gene models and 3) pinpointing genes displaying protein polymorphism between the FcB1 and 3D7 strains. RESULTS: A total of 22,125 clones randomly picked from the SSH library were sequenced, yielding 21,805 usable ESTs that were then clustered on the P. falciparum genome. This allowed identification of 243 protein coding genes, including 121 previously annotated as hypothetical. Statistical analysis of GO terms, when available, indicated significant enrichment in genes involved in "entry into host-cells" and "actin cytoskeleton". Although most ESTs do not span full-length gene reading frames, detailed sequence comparison of FcB1-ESTs versus 3D7 genomic sequences allowed the confirmation of exon/intron boundaries in 29 genes, the detection of new boundaries in 14 genes and identification of protein polymorphism for 21 genes. In addition, a large number of non-protein coding ESTs were identified, mainly matching with the two A-type rRNA units (on chromosomes 5 and 7) and to a lower extent, two atypical rRNA loci (on chromosomes 1 and 8), TARE subtelomeric regions (several chromosomes) and the recently described telomerase RNA gene (chromosome 9). CONCLUSION: This FcB1-schizont-EST analysis confirmed the actual expression of 243 protein coding genes, allowing the correction of structural annotations for a quarter of these sequences. In addition, this analysis demonstrated the actual transcription of several remarkable non-protein coding loci: 2 atypical rRNA, TARE region and telomerase RNA gene. Together with other collections of P. falciparum ESTs, usually generated from mixed parasite stages, this collection of FcB1-schizont-ESTs provides valuable data to gain further insight into the P. falciparum gene structure, polymorphism and expression.

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Dataset Information

The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin ? Subunit is Clathrin Independent and Essential for Schizont Maturation.

Publications

The <i>Plasmodium falciparum</i> Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

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