Abstract
ABSTRACTMalaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. These proteins lack the multiple transmembrane domains typically present in channel-forming proteins, raising doubts about their precise roles. Using the virulent human Plasmodium falciparum parasite, we report that CLAG3 undergoes self-association and that this protein’s expression determines channel phenotype quantitatively. We overcame epigenetic silencing of clag3 paralogs and engineered parasites that express two CLAG3 isoforms simultaneously. Stoichiometric expression of these isoforms yielded intermediate channel phenotypes, in agreement with observed trafficking of both proteins to the host membrane. Coimmunoprecipitation and surface labeling revealed formation of CLAG3 oligomers. In vitro selections applied to these transfectant lines yielded distinct mutants with correlated changes in channel activity. These findings support involvement of the identified oligomers in PSAC formation and parasite nutrient acquisition.
Highlights
Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins
The KC5 parasite line carries a single clag3h gene and was chosen for our transfections to simplify interpretation; in KC5, the crossover occurred between conserved polymorphisms located 607 and 844 bp from the start of clag3h, with the 5= end derived from clag3.2 and the majority of the gene derived from an ancestral clag3.1
CLAG3 was identified as a critical determinant of infected-cell nutrient uptake through classical genetic mapping with ISPA-28, an isolate-specific PSAC inhibitor found through high-throughput screening [4]
Summary
Malaria parasites increase host erythrocyte permeability to ions and nutrients via a broad-selectivity channel known as the plasmodial surface anion channel (PSAC), linked to parasite-encoded CLAG3 and two associated proteins. Forward and reverse genetic screens along with pharmacological studies have recently determined that the increased permeability of most solutes results from a single ion channel termed the plasmodial surface anion channel (PSAC); these studies linked PSAC to the clag multigene family conserved in all examined malaria parasites While these molecular studies have convincingly implicated these parasite genes in nutrient uptake, fundamental questions remain about how the encoded proteins determine PSAC activity and the importance of increased permeability to intracellular parasite development. Molecular studies to define the role of CLAG3 have been complicated by epigenetic silencing, which allows infected cells to express only one clag paralog for several generations before switching to the other paralog [15, 16] While these two genes encode proteins that are 90 to 95% identical, a short polymorphic motif is exposed at the erythrocyte surface and may affect function [8]. Another difficulty is clag3’s large size, which complicates full gene replacements through technologies such as CRISPR-Cas
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