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Vacuole Membrane in Plasmodium Falciparum Oriented nucleation of hemozoin at the digestive vacuole membrane in Plasmodium falciparum Sergey Kapishnikova,1, Allon Weinera,1, Eyal Shimonib, Peter Guttmannc, Gerd Schneiderc, Noa Dahan-Pasternakd, Ron Dzikowskid, Leslie Leiserowitza,2, and Michael Elbauma,2 aDepartment of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel; bChemical Research Support Department, Weizmann Institute of Science, Rehovot 76100, Israel; cHelmholtz Zentrum Berlin für Materialien und Energie GmbH, Wilhelm-Conrad-Röntgen Campus, 12489 Berlin, Germany; and dDepartment of Microbiology and Molecular Genetics, The Kuvin Center for the Study of Infectious and Tropical Diseases, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel Edited by* Ada Yonath, Weizmann Institue, Rehovot, Israel, and approved May 10, 2012 (received for review November 3, 2011) Heme detoxification is a critical step in the life cycle of malaria- the mature DV (9), or in a single large engulfment of RBC causing parasites, achieved by crystallization into physiologically cytoplasm (10). insoluble hemozoin. The mode of nucleation has profound implica- Our hypothesis has been that hemozoin nucleates at an acyl- tions for understanding the mechanism of action of antimalarial glycerol lipid–aqueous interface (3–5, 11), based on analogy drugs that inhibit hemozoin growth. Several lines of evidence to the wide literature on templated nucleation (12). A specific point to involvement of acylglycerol lipids in the nucleation pro- expectation is that the relative orientation of mature crystals cess. Hemozoin crystals have been reported to form within lipid should reflect their initial nucleation. In the companion report nanospheres; alternatively, it has been found in vitro that they (13), hemozoin alignment was characterized using nanoprobe are nucleated at an acylglycerol lipid–water interface. We have ap- beam X-ray Fe-fluorescence and crystal diffraction. Crystals were plied cryogenic soft X-ray tomography and three-dimensional elec- found in clusters with the c axis of individual needles aligned in tron microscopy to address the location and orientation of hemo- parallel. The diffraction pattern indicated alignment along a zoin crystals within the digestive vacuole (DV), as a signature curved surface, with {100} faces adjoint to that surface. of their nucleation and growth processes. Cryogenic soft X-ray To establish the crystal orientation with respect to such lipids, BIOPHYSICS AND tomography in the “water window” is particularly advantageous we have here applied real-space electron and X-ray microscopy COMPUTATIONAL BIOLOGY because contrast generation is based inherently on atomic absorp- tools in order to locate the lipid structures possibly implicated in tion. We find that hemozoin nucleation occurs at the DV inner hemozoin nucleation. Cryogenic soft X-ray tomography (cryo- membrane, with crystallization occurring in the aqueous rather XT) (14) is uniquely suited to examine the above questions be- than lipid phase. The crystal morphology indicates a common {100} cause contrast can be interpreted quantitatively. We have applied orientation facing the membrane as expected of templated nuclea- such analysis in detail to the content of HTVs and to the thickness tion. This is consistent with conclusions reached by X-ray fluores- of the DV membrane. Cryo-XT has been applied recently by cence and diffraction in a companion work. Uniform dark spheres others to study stage progression and hemoglobin consumption Plasmodium observed in the parasite were identified as hemoglobin transport in (15, 16). Working with X-ray illumination energies vesicles. Their analysis supports a model of hemozoin nucleation between atomic absorption transitions of carbon and oxygen, the “ ” primarily in the DV. Modeling of the contrast at the DV membrane so-called water window from 280 to 530 eV, lipid bodies and indicates a 4-nm thickness with patches about three times thicker, dense organic matter appear dark while the aqueous medium possibly implicated in the nucleation. is relatively bright. As implemented at the electron storage ring BESSY II (Berlin, Germany), cryo-XT offers in-plane resolution heterogeneous crystal nucleation ∣ beta-hematin ∣ X-ray microscopy ∣ of approximately 30 nm, fully cryogenic sample handling, and 3D focused ion beam ∣ quinoline reconstruction (14). The basis for material analysis appears in Fig. 1A. The long μ alaria is caused by parasites of the Plasmodium genus that penetration length of the X-rays in water, reaching 10 m, per- Minvade host red blood cells and feed on the hemoglobin. mits direct observation of intact cells in the vitrified state and The released heme byproduct Fe(II)-protoporphyrin IX, which avoids the stages of chemical fixation, dehydration, embedding, is toxic to the parasite, is sequestered by formation of a cyclic staining, and sectioning normally required for electron micro- ð Þ─ ð Þ scopy. Hemozoin, by virtue of its Fe and high carbon content dimer (1) linked via reciprocal Fe III O propionate bonds, 1 49 ∕ 3 which assembles into physiologically insoluble crystals of hemo- and density ( . g cm ), is dark. For reference, the theoretical zoin. Common quinoline drugs against malaria interfere with growth morphology of hemozoin (17) and its spatial relationship to the molecular packing of synthetic hemozoin (1) are depicted heme detoxification by inhibiting this process. Extensive evidence B C points to involvement of acylglycerol lipids in the crystallization in Fig. 1 and . of hemozoin. This includes promotion of formation of the syn- thetic analogue of hemozoin, β-hematin, in acylglycerol suspen- Author contributions: S.K., A.W., L.L., and M.E. designed research; S.K., A.W., and M.E. sions (2) and at various lipid–solution interfaces (3–5), mass performed research; E.S., P.G., G.S., N.D.-P., R.D., and L.L. contributed new reagents/ analytic tools; S.K. contributed in cryo-XT; A.W. contributed in EM and preparation for spectroscopic analysis of lipids adsorbed on hemozoin crystals cryo-XT; S.K., A.W., L.L., and M.E. analyzed data; and S.K., A.W., L.L., and M.E. wrote the isolated from infected RBCs (6), and transmission electron mi- paper. croscopy (TEM) of histologically stained thin sections (6, 7). The authors declare no conflict of interest. Plasmodium falciparum Within the parasite , hemozoin crystals *This Direct Submission article had a prearranged editor. are found prominently in the DV.Some authors have claimed that Freely available online through the PNAS open access option. hemozoin nucleation occurs within neutral lipid droplets inside 1S.K. and A.W. contributed equally to this work. the DV (6, 7). Others have suggested that hemoglobin digestion 2To whom correspondence may be addressed. E-mail: [email protected] or and hemozoin formation may occur primarily in hemoglobin [email protected]. transport vesicles (HTVs) that later fuse with the DV (8), or This article contains supporting information online at www.pnas.org/lookup/suppl/ may be initiated in pre-DV compartments that coalesce to form doi:10.1073/pnas.1118120109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118120109 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. X-ray cryotomography of hemozoin within P. falciparum cells. (A–C) For reference: (A) relative transmitted intensity at 510 eV energy as a function of material thickness for vitreous ice, cytoplasmic hemoglobin (Hgb), diacylglycerol lipid layer, and hemozoin, together with display gray value; the X-ray absorp- tion coefficients of these materials are, respectively, 0.109, 0.427, 1.031, and 1.618 μm−1.(B) Theoretical growth habit of hemozoin, displaying the dominant faces. (C) Packing arrangement of synthetic hemozoin. (D) Midtrophozoite-stage parasite with a single nucleus (Nu), and large DV enclosing numerous hemozoin (Hz) crystals. (D, l) A slice through the volume reconstruction; the unreconstructed projection appears in SI Text.(D, c and r) Surface renderings from orthogonal view angles of the DV (maroon), Hz (red), and hemoglobin transport vesicles. Note the flattened shape of the DV. (E) Surface rendering of a DV from a second late-trophozoite, or perhaps early-schizont, with nearly spherical DV; note the large crystals at the center and the smaller ones lying along the inner membrane. (F) Multinuclear schizont-stage parasite with a compacted DV showing Hz crystals primarily at the center. Insets show virtual slices through the crystals in the volume reconstructions at similar orientations to those of the respective surface renderings. All scale bars, 1 μm. Various modes of EM have also been applied. These included Fig. 2A shows an early trophozoite imaged by three-dimen- freeze-fracture cryo-SEM, serial surface view (SSV) microscopy sional SSV microscopy. Three-dimensional rendering reveals a (18) in a dual-head focused ion beam—scanning electron micro- flattened, disk-like DV with numerous hemozoin crystals distrib- scope (19, 20), and scanning transmission electron (STEM) uted around the inner surface. Fig. 2 B shows similar views from a tomography (21). Like the cryo-XT, the latter two offer 3D infor- schizont-stage parasite in which the DV has inflated to a more mation, albeit with embedded samples. A glossary of technical spherical shape. Large crystals fill the interior whereas small crys- terms relating to electron microscopy and Plasmodium, as well tals are found along the boundary. Fig. 2C
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