Redefining the role of the Plasmodium heme detoxification protein: From hemozoin formation to mitochondrial protein synthesis

Abstract

Throughout their intraerythrocytic development, malaria parasites digest up to 80% of the host cells hemoglobin within a specialized degradative compartment known as the digestive vacuole. This process releases heme, which is detoxified by sequestration into bioinert hemozoin crystals. Although heme biomineralization is essential for blood-stage survival and a validated drug target, its underlying mechanisms remain unclear. Initially identified as a potent inducer of {beta}-hematin crystallization in vitro, the parasites Heme Detoxification Protein (HDP) has been proposed to execute a similar role in the formation of hemozoin crystals in cellulo. Here, we investigate the function of HDP in live Plasmodium falciparum parasites, integrating experimental genetic approaches with quantitative microscopy, cellular bioenergetics and whole-proteome profiling. Endogenous tagging revealed that HDP localizes to the mitochondrion rather than the digestive vacuole. Conditional inactivation of HDP resulted in a gradual loss of mitochondrial membrane potential, preceding developmental arrest. Bypassing the essential role of the respiratory chain in pyrimidine biosynthesis - either through exogenous electron acceptors or expression of a ubiquinone-independent dihydroorotate dehydrogenase - rescued HDP-deficient parasites, indicating a role in maintaining respiratory chain activity. Consistent with this, electron flow through complex IV was abolished in rescued HDP-null parasites, rendering them hypersensitive to proguanil, an antimalarial that synergizes with respiratory chain inhibitors. We found that loss of HDP leads to a marked reduction of complexes III and IV, whose integrity depends on mitochondrial protein biosynthesis. Integration of quantitative proteomic data with structure-guided homology modelling supports a role for HDP as part of the large mitoribosomal subunit at the inter-subunit contact site. By contrast, HDP loss did not affect the quantity of hemozoin or other heme species, crystal morphology, or sensitivity to the hemozoin-targeting drug chloroquine. Together, these findings challenge previous models linking HDP to hemozoin formation and instead reveal an essential role for HDP in mitochondrial protein biosynthesis.