Cellular and Molecular Physiology of Bloodstream Malaria Parasites

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

In 2023, the Apicomplexan Molecular Physiology Section Malaria examined Na+ and H+ homeostasis within the intracellular human malaria parasite, Plasmodium falciparum. This parasite maintains this homeostasis through the action of PfATP4, a cation pump localized to the intracellular parasite plasma membrane. PfATP4 is the target of advanced antimalarial leads, which produce many poorly understood metabolic disturbances within infected erythrocytes. To better understand these disturbances, we have now expressed the mammalian ligand-gated TRPV1 ion channel at the parasite plasma membrane to study ion regulation and examine the effects of cation leak. TRPV1 expression was well-tolerated, consistent with negligible ion flux through the nonactivated channel. TRPV1 ligands produced rapid parasite death in the transfectant line at their activating concentrations, but were harmless to the wild-type parent. Activation triggered cholesterol redistribution at the parasite plasma membrane, reproducing effects of PfATP4 inhibitors and directly implicating cation dysregulation in this process. In contrast to predictions, TRPV1 activation in low Na+ media accentuated parasite killing but a PfATP4 inhibitor had unchanged efficacy. Selection of a ligand-resistant mutant revealed a previously uncharacterized G683V mutation in TRPV1 that occludes the lower channel gate, implicating reduced permeability as a mechanism for parasite resistance to antimalarials targeting ion homeostasis. Our findings provide key insights into malaria parasite ion regulation and will guide mechanism-of-action studies for advanced antimalarial leads that act at the host-pathogen interface. PLoS One 18:e0283776 (2023). PMID: 37014920 In another study, we examined Ca++ uptake and efflux at the erythrocyte membrane. Ca++ is required for numerous cellular developmental activities and is required by intracellular malaria parasites. Despite its requirement, Ca++ is maintained at very low concentrations in human erythrocytes by an efficient PMCA Ca++ extrusion pump. Although much of our knowledge about this Ca++ extrusion pump derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity. Eur. Biophys. J. 52:101-110 (2023). PMID: 36512028