A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

Lyko, Brian; Hammershaimb, E. Adrianne; Nguitragool, Wang; Wellems, Thomas E.; Desai, Sanjay A.

doi:10.1186/1475-2875-11-124

Cited by 22 publications

(24 citation statements)

References 9 publications

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“…By identifying gluconate as a nontoxic substitute anion, this study also found that parasites tolerate significant reductions in external Cl − ( EC 50 of 31 mM). Combined with another study showing that the parasite tolerates a broad range of extracellular H + concentrations [79], these studies indicate that the parasite has a significantly greater tolerance of changes in monovalent ion concentrations than cultured mammalian cells. This tolerance parallels that seen with dinoflagellates, to which apicomplexan parasites are closely related [80].…”

Section: Proposed Roles For Increased Permeabilitymentioning

confidence: 83%

Why do malaria parasites increase host erythrocyte permeability?

Desai

2014

Trends in Parasitology

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Malaria parasites increase erythrocyte permeability to diverse solutes including anions, some cations, and organic solutes, as characterized with several independent methods. Over the last decade, patch-clamp studies have determined that the permeability results from one or more ion channels on the infected erythrocyte host membrane. However, the biological role(s) served by these channels, if any, remain controversial. Recent studies implicate the plasmodial surface anion channel (PSAC) and a role in parasite nutrient acquisition. A debated alternative role in remodeling host ion composition for the benefit of the parasite appears to be nonessential. Because both channel activity and the associated clag3 genes are strictly conserved in malaria parasites, channel-mediated permeability is an attractive target for development of new therapies.

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Section: Proposed Roles For Increased Permeabilitymentioning

confidence: 83%

Why do malaria parasites increase host erythrocyte permeability?

Desai

2014

Trends in Parasitology

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“…The transfected culture was selected with 2.5 nM WR99210 and screened for homologous recombination by PCR. Integration into the clag3.1 gene was detected after 6 months; the HB3-c3A1210T clone was generated by limiting dilution (25).…”

Section: Methodsmentioning

confidence: 99%

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

et al. 2015

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Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted ␣-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.T he human malaria parasite Plasmodium falciparum remodels its host erythrocyte by exporting many proteins, generating membranous structures in the host cytosol, and increasing erythrocyte permeability to many solutes. Studies by multiple groups have determined that anions, sugars, purines, organic cations, and some vitamins have increased permeability after infection (1-4). The increase in permeability is primarily mediated by a parasitederived ion and nutrient channel known as the plasmodial surface anion channel (PSAC) (5). Importantly, both PSAC single-channel properties and the relative increases in solute permeabilities are conserved in divergent malaria parasites (6). Because Babesia parasites do not induce PSAC-like activity in erythrocytes that they invade (7), this channel is thought to be restricted to the genus Plasmodium.The clag multigene family, also conserved in and restricted to malaria parasites (8), has recently been linked to PSAC activity (9-11). Two paralogs on parasite chromosome 3, known as clag3.1 and clag3.2, appear to play a critical role, as identified by genetic mapping experiments with ISPA-28, an isolate-specific PSAC antagonist that blocks channels on the Dd2 laboratory clone but is ineffective against channels from other lines. These genes undergo epigenetic switching to encode a single protein that is initially packaged in specialized organelles known as rh...

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“…Growth inhibition studies with the HB3 3rec parasite were performed after transport-based selection with ISPA-28, to achieve expression of the chimeric clag3 gene generated through allelic exchange transfection. Limiting-dilution cultures to obtain parasite clones were performed in 96-well microplates; positive wells were detected by using the 5(and 6)-carboxy-seminaphthorhodafluor-1 method (Lyko et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Solute Restriction Reveals an Essential Role forclag3-Associated Channels in Malaria Parasite Nutrient Acquisition

et al. 2012

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The plasmodial surface anion channel (PSAC) increases erythrocyte permeability to many solutes in malaria but has uncertain physiological significance. We used a PSAC inhibitor with different efficacies against channels from two Plasmodium falciparum parasite lines and found concordant effects on transport and in vitro parasite growth when external nutrient concentrations were reduced. Linkage analysis using this growth inhibition phenotype in the Dd2 ϫ HB3 genetic cross mapped the clag3 genomic locus, consistent with a role for two clag3 genes in PSAC-mediated transport. Altered inhibitor efficacy, achieved through allelic exchange or expression switching between the clag3 genes, indicated that the inhibitor kills parasites through direct action on PSAC. In a parasite unable to undergo expression switching, the inhibitor selected for ectopic homologous recombination between the clag3 genes to increase the diversity of available channel isoforms. Broad-spectrum inhibitors, which presumably interact with conserved sites on the channel, also exhibited improved efficacy with nutrient restriction. These findings indicate that PSAC functions in nutrient acquisition for intracellular parasites. Although key questions regarding the channel and its biological role remain, antimalarial drug development targeting PSAC should be pursued.

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A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

Cited by 22 publications

References 9 publications

Why do malaria parasites increase host erythrocyte permeability?

Why do malaria parasites increase host erythrocyte permeability?

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

Solute Restriction Reveals an Essential Role forclag3-Associated Channels in Malaria Parasite Nutrient Acquisition

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