Hemozoin formation in Echinostoma trivolvis rediae

Rediae of the trematode Echinostoma trivolvis, from naturally infected Helisoma trivolvis snails, form a black pigment while inside the snail host. Here we examine the black pigment to show that the insolubility characteristics in detergent and weak base solution are identical to Plasmodium falciparum hemozoin. Laser desorption mass spectrometry of the purified pigment demonstrates the presence of heme. Examination of purified pigment under polarized light microscopy illuminates ordered birefringent crystals. Field emission in lens scanning electron microscopy reveals irregular ovoid crystals of 200-300 nm in diameter. The purified pigment crystals seeded extension of monomeric heme onto the crystal which by Fourier Transform Infrared analysis is beta-hematin. Rediae of a second echinostome parasite, Echinostoma caproni, from experimentally infected Biomphalaria glabrata, do not produce measurable or recoverable heme crystals. These observations are consistent with heme crystal formation by a hematophagous parasite within a non-vertebrate intermediate host.     

Recent advances in understanding the mechanism of hemozoin (malaria pigment) formation

The recent literature on hemozoin/β-hematin formation is reviewed, with an emphasis on the mechanism of its formation. Recent findings from unrelated organisms that produce hemozoin, namely the malaria parasite Plasmodium falciparum, the worm Schistosoma mansoni and the kissing bug Rhodnius prolixus all of which consume human hemoglobin show that the formation of this crystalline substance occurs within or at the surface of lipids. Biomimetic experimental models of the lipid–water interface as well as computational studies indicate that these lipid environments are probably extraordinarily efficient at producing hemozoin. A rethink is now needed, with a new emphasis on Fe(III)PPIX in non-aqueous environments that mimic lipids and indeed within the lipid environment itself. These findings are explored and discussed in the context of earlier studies on β-hematin formation.     

Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update

Digestion of hemoglobin in the food vacuole of the malaria parasite produces very high quantities of redox active toxic free heme. Hemozoin (beta-hematin) formation is a unique process adopted by Plasmodium sp. to detoxify free heme. Hemozoin formation is a validated target for most of the well-known existing antimalarial drugs and considered to be a suitable target to develop new antimalarials. Here we discuss the possible mechanisms of free heme detoxification in the malaria parasite and the mechanistic details of compounds, which offer antimalarial activity by inhibiting hemozoin formation. The chemical nature of new antimalarial compounds showing antimalarial activity through the inhibition of hemozoin formation has also been incorporated, which may help to design future antimalarials with therapeutic potential against multi-drug resistant malaria.     

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.     

On the kinetics of formation of hemozoin, the malaria pigment

We report on the kinetics of formation of hemozoin as a function of hemin concentration and reaction medium. Evidence is presented for the critical role played by interfacial regions in the efficient conversion of hemin to the malaria pigment.     

In search of next generation antimalarials

Compounds 7–10 displayed potency without any apparent toxicity, in animal models of both relapsing and non-relapsing forms of malaria offering hope of a single molecule that can cure both relapsing and non relapsing forms of malaria.     

Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b₅, localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b₅ reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.     

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic β-hematin (βH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to βH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.     

Plasmodium lophurae: composition and properties of hemozoin, the malarial pigment.

Plasmodium lophurae hemozoin (malarial pigment) is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces, and hydrophobic interactions but not by covalent bonding. Hemozoin is not composed of partially degraded globin peptides coordinated to heme, since fragments of molecular size less than that of globin monomers were not observed by SDS-PAGE. Two major polypeptides constituted the macromolecular portion of hemozoin; these had molecular weights of 21,000 and 15,000. The 21,000-molecular-weight protein is probably of parasite origin. The 15,000-molecular-weight polypeptide is believed to consist of globin monomers, and indicates the presence of irreversibly denatured hemoglobin (hemiglobin), as a constituent of hemozoin. The formation of hemozoin is hypothesized to play the following roles: protection of the parasite against molecular oxygen and compartmentation of the iron porphyrin which is a product of hemoglobin digestion by the plasmodium.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.     

Spectrophotometric determination of de novo hemozoin/beta-hematin formation in an in vitro assay

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.     

Raman imaging of hemozoin within the food vacuole of Plasmodium falciparum trophozoites

Micro-Raman spectra of hemozoin encapsulated within the food vacuole of a Plasmodium falciparum-infected erythrocyte are presented. The spectrum of hemozoin is identical to the spectrum of beta-hematin at all applied excitation wavelengths. The unexpected observation of dramatic band enhancement of A(1g) modes including nu(4) (1374 cm(-1)) observed when applying 780 nm excitation enabled Raman imaging of hemozoin in the food vacuole. This unusual enhancement, resulting from excitonic coupling between linked porphyrin moieties in the extended porphyrin array, enables the investigation of hemozoin within its natural environment for the first time.     

Identification of a Plasmodium falciparum Phospholipid Transfer Protein

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.     

Antimalarial drug synergism and antagonism: mechanistic and clinical significance

Interactions between antimicrobial agents provide clues as to their mechanisms of action and influence the combinations chosen for therapy of infectious diseases. In the treatment of malaria, combinations of drugs, in many cases acting synergistically, are increasingly important in view of the frequency of resistance to single agents. The study of antimalarial drug interactions is therefore of great significance to both treatment and research. It is therefore worrying that the analysis of drug-interaction data is often inadequate, leading in some cases to dubious conclusions about synergism or antagonism. Furthermore, making mechanistic deductions from drug-interaction data is not straightforward and of the many reported instances of antimalarial synergism or antagonism, few have been fully explained biochemically. This review discusses recent findings on antimalarial drug interactions and some pitfalls in their analysis and interpretation. The conclusions are likely to have relevance to other antimicrobial agents.     

The basis of the immunomodulatory activity of malaria pigment (hemozoin)

The most common and deadly form of the malaria parasite, Plasmodium falciparum, is responsible for 1.5–2.7 million deaths and 300–500 million acute illnesses annually [Bremen in J. Trop. Med. Hyg. 64:1–11 (2001); World Health Organization (2002)]. Hemozoin, the biomineral formed to detoxify the free heme produced during parasitic hemoglobin catabolism, has long been suspected of contributing to the pathological immunodeficiencies that occur during malarial infection. While there is a growing consensus in the literature that native hemozoin maintains immunosuppressive activity, there is considerable controversy over the reactivity of the synthetic form, β-hematin (BH). Given the emerging importance of hemozoin in modulating a host immune response to malarial infection, a careful examination of the effects of the constitutive components of the malaria pigment on macrophage response has been made in order to clarify the understanding of this process. Herein, we present evidence that BH alone is unable to inhibit stimulation of NADPH oxidase and inducible nitric oxide synthase, the key enzymes involved in oxidative burst, and is sensitive to the microbicidal agents of these enzymes both in vitro and in vivo. Further, by systematically examining each of the malaria pigment’s components, we were able to dissect their impact on the immune reactivity of a macrophage model cell line. Reactions between BH and red blood cell (RBC) ghosts effectively reconstituted the observed immunomodulatory reactivity of native hemozoin. Together, these results suggest that the interaction between hemozoin and the RBC lipids results in the generation of toxic products and that these products are responsible for disrupting macrophage function in vivo.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Plasmodium falciparum Merozoite Surface Protein 3: OLIGOMERIZATION,SELF-ASSEMBLY,AND HEME COMPLEX FORMATION*

Merozoite surface protein 3 of Plasmodium falciparum, a 40-kDa protein that also binds heme, has been biophysically characterized for its tendency to form highly elongated oligomers. This study aims to systematically analyze the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through size exclusion chromatography, dynamic light scattering, and transmission electron microscopy, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid-like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C terminus of MSP3 may not be the only structural element responsible for oligomerization and that other peptide segments like MSP3(192–196) (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5–15-μm-long fibrillar structures stained by anti-MSP3 antibodies were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV spectrophotometry, HPLC, and electrophoresis. This study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point to the other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.     

Artemisinin Enhances Heme-Catalysed Oxidation of Lipid Membranes

Artemisinin, a sesquiterpene endoperoxide derived from a traditional Chinese herbal remedy for fevers, is a promising new antimalarial drug, particularly useful against multidrug resistant strains of P. falciparum. Despite widespread clinical use, its mode of action remains uncertain. We investigated whether its antimalarial properties could be explained by an ability to enhance the redox activity of heme, formed in the parasite food vacuole from digested hemoglobin. Artemisinin caused a sustained threefold increase, followed by a gradual decline, in the peroxidase activity of heme. It also enhanced the ability of heme to oxidize membrane lipids about sixfold. An unexpected finding was the potentiation of heme-catalysed membrane lipid oxidation by Vitamin E. The changes in redox-catalytic activity induced by artemisinin were paralleled by major changes in the absorption spectrum of heme, culminating in loss of the Soret band. We propose a model in which artemisinin binds irreversibly to heme in the parasite food vacuole, preventing its polymerization to chemically inert hemozoin, and promoting heme-catalysed oxidation of the vacuolar membrane by molecular oxygen, which leads, ultimately, to vacuole rupture and parasite autodigestion. © 1997 Elsevier Science Inc.     

Antimalarial drugs and heme in detergent micelles: An NMR study

Proton nuclear magnetic resonance relaxation times were measured for the protons of micelles formed by the detergents sodium dodecyl sulfate, dodecyltrimethyl ammonium bromide, and polyethylene glycol sorbitan monolaureate in the presence of ferriprotoporphyrin IX and the antimalarial drugs chloroquine, 7-chloro-4-quinolyl 4-N,N-diethylaminobutyl sulfide, and primaquine. Diffusion coefficients were extracted from pulsed gradient NMR experiments to evaluate the degree of association of these drugs with the detergent micelles. Results indicate that at low or neutral pH when the quinolyl N is protonated, chloroquine does not associate with neutral or cationic detergent micelles. For this reason, chloroquine’s interaction with heme perturbs the partitioning of heme between the aqueous medium and detergent micelles.     

Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs

Two subclasses of quinoline antimalarial drugs are used clinically. Both act on the endolysosomal system of malaria parasites, but in different ways. Treatment with 4-aminoquinoline drugs, such as chloroquine, causes morphologic changes and hemoglobin accumulation in endocytic vesicles. Treatment with quinoline-4-methanol drugs, such as quinine and mefloquine, also causes morphologic changes, but does not cause hemoglobin accumulation. In addition, chloroquine causes undimerized ferriprotoporphyrin IX (ferric heme) to accumulate whereas quinine and mefloquine do not. On the contrary, treatment with quinine or mefloquine prevents and reverses chloroquine-induced accumulation of hemoglobin and undimerized ferriprotoporphyrin IX. This difference is of particular interest since there is convincing evidence that undimerized ferriprotoporphyrin IX in malaria parasites would interact with and serve as a target for chloroquine. According to the ferriprotoporphyrin IX interaction hypothesis, chloroquine would bind to undimerized ferriprotoporphyrin IX, delay its detoxification, cause it to accumulate, and allow it to exert its intrinsic biological toxicities. The ferriprotoporphyrin IX interaction hypothesis appears to explain the antimalarial action of chloroquine, but a drug target in addition to ferriprotoporphyrin IX is suggested by the antimalarial actions of quinine and mefloquine. This article summarizes current knowledge of the role of ferriprotoporphyrin IX in the antimalarial actions of quinoline drugs and evaluates the currently available evidence in support of phospholipids as a second target for quinine, mefloquine and, possibly, the chloroquine-ferriprotoporphyrin IX complex.