Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography

Abstract

A key drug target for malaria has been the detoxification pathway of the iron-containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element-specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X-ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X-ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non-crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug development.

Antimalarial Quinoline Drugs Inhibit β-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases

“…Plasmodium uses a system to detoxify heme (Fe 3+ ) called biocrystallization based on the formation of hemozoin pigment which appears as a dark black crystalline spot (a dark brown pigment) in red blood cells of infected patients 14–18 . Hemozoin is chemically and structurally identical to β-hematin, a heme dimer that crystallizes under the acidic conditions of digestive vacuole of Plasmodium (pH values of 4.8–5.0) 18–20 . It contains two heme (Fe 3+ ) monomers reciprocally linked through coordination complexes between the carboxyl group of a propionate side chain of one monomer and the iron (Fe 3+ ) atom in the porphyrin ring of another monomer 19,21 .…”

Mode of action of quinoline antimalarial drugs in red blood cells infected by Plasmodium falciparum revealed in vivo

“…The heme is rendered inert by crystallization into hemozoin. We have recently shown that the parasite stores large quantities of hemoglobin in its digestive vacuole (4). For the parasite to survive, the rate of heme liberation via hemoglobin digestion must not exceed the rate of hemozoin crystallization.…”

Imaging cell morphology and physiology using X-rays

“…Such access to the ultrastructural organization in whole cells placed X-ray microscopy as a valuable tool to study cell-parasite interactions. Structural development of malaria parasite, for example, was extensively studied by several groups by 2D soft X-ray microscopy [119–121], soft X-ray coherent diffraction imaging }[122] and X-ray fluorescence diffraction [123, 124]. Spatial localization of vaccinia and herpes viral factories was likewise observed by X-ray microscopy [125–127].…”