LS-7-P-2902 Three-dimensional analysis of the biogenesis of hemozoin crystals during intra-erythrocytic cycle of Plasmodium chabaudi

Malaria is a disease caused by protozoan parasites from the genus Plasmodium with the highest impact on public health in endemic areas. Morbidity and mortality of malaria results from the asexual replication of Plasmodium in the erythrocyte of the mammalian host. In the course of infection, different developmental stages of the parasite are formed as it progresses from the ring stage to the trophozoite and then to the replicating schizont stage. During its intra-erythrocytic development, malaria parasites internalize massive amounts of hemoglobin from the red blood cell in order to obtain free amino acids and to regulate osmotic pressure. Hemoglobin is digested in a compartment with acidic pH termed the food vacuole, producing aminoacids and others byproducts, namely heme. It is known that free heme can generate free-radicals, causing molecular and cellular damage. In order to avoid these effects, free heme is immobilized and stored in a crystal form known as hemozoin or malaria pigment. Although hemoglobin uptake and heme crystallization are physiological steps used as target for many antimalarial drugs, the fine mechanisms underlying hemoglobin crystallization are still under discussion. In this work we studied the mechanism of hemozoin nucleation in the different stages of the intra-erythrocytic cycle of the rodent parasite Plasmodium chabaudi by transmission electron tomography of cryofixed and freeze substituted cells. Cryofixation of samples generally provided a better preservation of the cells and their hemozoin crystals (figure 1). Electron tomography showed the three-dimensional dispersion of hemozoin crystals within the food vacuole and the cytoplasm of the parasite (figure 2). Results showed that large amounts of hemoglobin are internalized during the early stages after invasion (ring stage and early throphozoite), whereas in late stages (throphozoite), assembly of several vesicles containing hemoglobin spread through the parasite cytoplasm were observed. Small food vacuoles concentrated near the membrane of the parasite were also frequently seen. In the late (schizont) stage, the hemoglobin containing vesicles were drastically reduced and larger food vacuoles were seen, containing large amounts of hemozoin (figure 2). Taking together, these results provide new insights on the mechanisms of hemoglobin uptake and degradation in rodent malaria parasites.