Complex nutrient channel phenotypes despite Mendelian inheritance in a Plasmodium falciparum genetic cross
Open Access
- 18 February 2020
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLoS Pathogens
- Vol. 16 (2), e1008363
- https://doi.org/10.1371/journal.ppat.1008363
Abstract
Malaria parasites activate a broad-selectivity ion channel on their host erythrocyte membrane to obtain essential nutrients from the bloodstream. This conserved channel, known as the plasmodial surface anion channel (PSAC), has been linked to parasite clag3 genes in P. falciparum, but epigenetic switching between the two copies of this gene hinders clear understanding of how the encoded protein determines PSAC activity. Here, we used linkage analysis in a P. falciparum cross where one parent carries a single clag3 gene to overcome the effects of switching and confirm a primary role of the clag3 product with high confidence. Despite Mendelian inheritance, CLAG3 conditional knockdown revealed remarkably preserved nutrient and solute uptake. Even more surprisingly, transport remained sensitive to a CLAG3 isoform-specific inhibitor despite quantitative knockdown, indicating that low doses of the CLAG3 transgene are sufficient to confer block. We then produced a complete CLAG3 knockout line and found it exhibits an incomplete loss of transport activity, in contrast to rhoph2 and rhoph3, two PSAC-associated genes that cannot be disrupted because nutrient uptake is abolished in their absence. Although the CLAG3 knockout did not incur a fitness cost under standard nutrient-rich culture conditions, this parasite could not be propagated in a modified medium that more closely resembles human plasma. These studies implicate oligomerization of CLAG paralogs encoded by various chromosomes in channel formation. They also reveal that CLAG3 is dispensable under standard in vitro conditions but required for propagation under physiological conditions. Malaria, a globally important infectious disease, is caused by parasites that invade and grow in circulating red blood cells to avoid host immune attack. Infected red blood cells have increased uptake of diverse nutrients, fueling parasite growth; this uptake is mediated by an ion channel that transports essential nutrients across the red blood cell membrane. Three proteins made by the parasite have been linked to this channel, but how they increase uptake is unknown. Here, we used mapping in a genetic cross of two strains of the virulent human malaria parasite to confirm a primary role of one protein known as CLAG3. We then used gene editing to produce a parasite that has reduced CLAG3 levels when a stabilizing chemical is removed; surprisingly, solute transport was minimally changed despite a 90% reduction in CLAG3. Gene editing was also used to make a parasite without any CLAG3. This knockout parasite had reduced nutrient uptake, but it still grew normally in media with high nutrient levels; it was unable to grow when nutrient levels were lowered to levels like those in the human bloodstream. The complex effects of channel inhibitors on these genetically modified parasites suggests that CLAG3 and the two other proteins interact with each other to form large protein clusters in the red blood cell membrane; these clusters may form the nutrient uptake pore. Our studies indicate that CLAG3 is required for parasite survival and growth in the bloodstream and that the channel it produces can be targeted to make new antimalarial drugs.Keywords
Funding Information
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases (ZIA AI000882)
This publication has 74 references indexed in Scilit:
- Plasmodium falciparum: Multidrug resistanceChemical Biology & Drug Design, 2019
- The antimalarial pipelineCurrent Opinion in Pharmacology, 2018
- Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort studyThe Lancet Infectious Diseases, 2018
- Molecular mechanisms of deformability of Plasmodium -infected erythrocytesCurrent Opinion in Microbiology, 2017
- Host immune evasion strategies of malaria blood stage parasiteMolecular BioSystems, 2017
- Variant surface antigens of Plasmodium falciparum and their roles in severe malariaNature Reviews Microbiology, 2017
- Critical Steps in Protein Export of Plasmodium falciparum Blood StagesTrends in Parasitology, 2015
- The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015Nature, 2015
- Maurer's clefts, the enigma of Plasmodium falciparumProceedings of the National Academy of Sciences of the United States of America, 2013
- Moving in and renovating: exporting proteins from Plasmodium into host erythrocytesNature Reviews Microbiology, 2010