Cellular and Molecular Physiology of Bloodstream Malaria Parasites

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

In 2025, the Apicomplexan Molecular Physiology Section (AMPS) reported the first successful patch-clamp of an intracellular organelle in bloodstream malaria parasites. We isolated the P. falciparum digestive vacuole (DV), a specialized organelle that is the target of many antimalarial drugs and the site of hemoglobin digestion. Single DV patch-clamp was then used to identify a novel large conductance anion cannel as the primary conductive pathway for ion flux across the DV membrane. This channel, termed the Big Vacuolar Anion Channel (BVAC), is primarily open at the DV resting membrane potential and has broad permeability to anions including Cl- and glutamate-. The channel is conserved on geographically divergent strains and is not blocked by antimalarials targeting essential DV activities, implicating an unexploited antimalarial target. A conditional knockdown strategy excluded links to PfCRT and PfMDR1, two drug-resistance transporters with poorly defined transport activities. We propose that BVAC functions to maintain electroneutrality during H+ uptake, allowing DV acidification and efficient hemoglobin digestion. The channel also facilitates amino acid salvage, providing essential building blocks for parasite growth. Direct transport measurements at the DV membrane provide foundational insights into vacuolar physiology, should help clarify antimalarial action and drug resistance, and will guide therapy development against the parasite's metabolic powerhouse. PLoS Biology (2025). 23:e3003202. PMID: 40446075. In another project, AMPS examined export and host membrane insertion of CLAG3, a key determinant of the Plasmodial Surface Anion Channel (PSAC), an essential nutrient acquisition channel identified and characterized by our group. We used gene-editing to insert the HiBit NanoLuc fragment into a surface-exposed loop on CLAG3 and detected host membrane insertion through a luminescence signal resulting from complementation. We then miniaturized this assay into 384-well microplate format and screened ~52,000 small molecules and uncovered 65 chemically diverse inhibitors. Hits that inhibit the NanoLuc reporter without blocking protein export were filtered out by a secondary screen whose signal does not depend on protein export. Because chemicals that interfere with parasite maturation were found to compromise CLAG3 export indirectly, a third screen using a NanoLuc reporter-tagged intracellular protein was used to evaluate nonspecific toxicity. Although our relatively small chemical screen did not identify bona fide inhibitors of CLAG3 host membrane insertion, these studies establish a framework for larger screens to identify novel export inhibitors. Such novel inhibitors will provide important insights into how Plasmodia remodel their host cells and may seed the development of therapies that block the export and membrane insertion of proteins needed for intracellular parasite survival. Pathogens (2025). 14:520. PMID: 40559528 We also collaborated with Swee Lay Thein and Kang Le of NHLBI, NIH to examine mechanism of action of AG-348 (mitapivat), a pyruvate kinase inhibitor that is an advanced drug lead for sickle cell disease therapies. We investigated possible mechanisms for improved red blood cell (RBC) survival after AG-348 cessation. We evaluated frozen whole blood samples from patients in the phase 1 study and analyzed Band 3 tyrosine phosphorylation (Tyr-p-bd3), ankyrin-1, and intact (active) protein tyrosine phosphatase 1B (PTP1B) levels. We observed a significant dose-dependent decrease in mean Tyr-p-bd3 from baseline in the patients, accompanied by an increase in the levels of membrane-associated ankyrin-1 and intact PTP1B. Because PTP1B is cleaved and inactivated by intracellular Ca2+-dependent calpain, we next measured the effect of AG-348 on ATP production and calpain activity and the plasma membrane Ca2+ ATPase pump-mediated efflux kinetics in HbAA and HbSS erythrocytes. AG-348 treatment increased ATP levels, decreased calpain activity, and increased Ca2+ efflux. These findings capitalized on Ca2+ ATPase pump assays developed in the AMPS. Our data indicate that ATP increase is a key mechanism underlying the increase in hemoglobin levels upon pyruvate kinase activation in sickle cell disease. This trial was registered at www.clinicaltrials.gov as #NCT04000165. Blood Advances (2024). 8:5653-5662. PMID: 39265169. In another study, we collaborated with the Chakrabarti lab of U. Central Florida to characterize mechanism of action of compound 12, a type II kinase inhibitor with potent antimalarial activity, fast action, and in vivo action in the P. berghei rodent malaria model. Our studies implicated two primary mechanisms of action: inhibition of β-hematin and hemozoin formation in the digestive vacuole and action on the P. falciparum protein kinase 6 (PfPK6). Our studies repurpose a safe human kinase inhibitor as a potent, fast-acting antimalarial with established in vivo efficacy. Cell Chemical Biology (2025). 32:926-941. PMID: 40628257.