Decoding the multifaceted role of erythrocyte PMCA4b in oxidative stress-mediated malaria protection and artemisinin resistance.

The ATP2B4 gene, encoding the PMCA4b Ca²+-ATPase in erythrocytes, has been linked to malaria protection via genome-wide association studies, though the proposed dehydration mechanism remains unclear. This study evaluates ATP2B4 genotypes and PMCA4b expression in malaria susceptibility and artemisinin sensitivity. ATP2B4 genotypes were compared across severe malaria, uncomplicated malaria, and healthy controls in Indian population. PMCA4b expression, intra-erythrocytic calcium, oxidative stress markers, Gardos channel activity, and Plasmodium falciparum growth dynamics were analyzed. Artemisinin sensitivity was assessed using growth inhibition and ring survival assays. ATP2B4 genotypes showed no significant association with malaria protection. Regardless of genotype, low PMCA4b expression correlated with increased intra-erythrocytic calcium, oxidative stress, and reduced in-vitro parasite growth. Gardos channel activity inversely correlated with PMCA4b but did not induce dehydration. Instead, oxidative-stress regulation by PMCA4b emerged as a key factor in malaria protection. Treatment of RBCs with resveratrol, a PMCA inhibitor, further validates the functional role of PMCA in regulation of intracellular calcium and oxidative stress. Notably, low PMCA4b expression also reduced P. falciparum artemisinin sensitivity, providing evidence that host genetic variation can influence drug efficacy. These findings suggest a role for PMCA4b in oxidative stress and drug response as critical to malaria pathophysiology. We further emphasize that many redox modulating RBC polymorphisms in malaria endemic areas could also influence artemisinin efficacy and serve as potential biomarkers for predicting therapeutic response.IMPORTANCEDiscovery of the mechanism by which human host variations affect the sensitivity of artemisinin in parasite provides an interestingly important view for optimizing anti-malarial treatment strategies. The human host and malaria parasite share a closely interconnected relationship; hence, we propose that parasite physiology or drug resistance cannot be studied alone without considering the host's biological factors. This study demonstrates that increase in intracellular calcium of RBCs is associated with a proportionate increase in intracellular oxidative stress, which affects the artemisinin sensitivity. Thus, variations in PMCA4b expression, the primary calcium efflux pump in RBCs, significantly alter the erythrocytic ROS levels, thereby affecting the P. falciparum growth and artemisinin sensitivity. Notably, intracellular redox imbalance is a common phenotype of multiple erythrocytic polymorphisms prevalent in malaria endemic areas, such as sickle cell, thalassemia, G6PD deficiency, etc. This study advocates the need for a widespread population-based investigation that associates the importance of host erythrocyte oxidative microenvironment surveillance in monitoring antimalarial drug resistance.