Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction

The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal Fe─O (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of β-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerol-water interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper.     

Malarial pigment-dependent error in the estimation of hemoglobin content in Plasmodium falciparum-infected red cells: implications for metabolic and biochemical studies of the erythrocytic phases of malaria

Measurements of mean corpuscular hemoglobin (MCH) in Plasmodium falciparum-infected red cells cultured in vitro revealed that malarial pigment (hemozoin) interferes with a true estimate of the actual hemoglobin content in Drabkin's reagent. When the hemozoin pigment was removed by passage of the lysate over a Biorex 70 column, a lower MCH value was obtained which allowed one to estimate that, under these conditions, the parasite consumes about 25% of the red cell's initial hemoglobin. Because spectrophotometric examinations of infected red cell lysates in Drabkin's reagent detect the unchanging heme content of infected red cells (hemoglobin + hemozoin), it can be used for expressing enzymatic activity or metabolite content. Results agree with simultaneous measurements on a per cell basis. However, it is suggested that instead of per gram hemoglobin, the activity should be stated as per mmole (or mumole) heme pigment. The ability to estimate accurately the consumption of intracellular hemoglobin will be useful in metabolic and pharmacologic studies of the parasite/red cell interaction.     

Hemozoin Enhances Maturation of Murine Bone Marrow Derived Macrophages and Myeloid Dendritic Cells

Background: Falciparum malaria is a severe health burden worldwide. Antigen presenting cells are reported to be affected by erythrocytic stage of the parasite. Malarial hemozoin (HZ), a metabolite of malaria parasite, has adjuvant properties and may play a role in the induction of immune response against the parasite. Objective: To determine the immunological impact of hemozoin on the capacity of innate immune cells maturation. Methods: Plasmodium falciparum (F32 strain) was cultured in O+ blood group up to 18% parasitemia. Natural hemozoin was extracted from infected red blood cells. Murine bone marrow derived macrophages and myeloid dendritic cells were stimulated with 4 ߤg/mL or 40 ߤg/mL of synthetic hemozoin (β-hematin) or natural hemozoin. We assessed the immunomodulatory role of synthetic or natural hemozoin in vitro by flowcytometric analysis. Results: The maturation markers MHCII, CD80 and CD86 were significantly upregulated (p<0.05) on the surface of murine bone marrow derived macrophages or myeloid dendritic cells. Data confirmed the potential of macrophages or myeloid dendritic cells, through hemozoin activation, to establish an innate immune response against malaria parasites. Conclusion: Both synthetic and natural hemozoin are potent inducers of cellular immunity against malaria infection. However, natural hemozoin is a stronger inducer as compared to synthetic hemozoin.     

Cysteine proteinase inhibitors block early steps in hemoglobin degradation by cultured malaria parasites

Erythrocytic malaria parasites degrade hemoglobin as a source of amino acids for parasite protein synthesis. Cysteine proteinase inhibitors have been shown to block the hydrolysis of globin by cultured parasites, indicating that a malarial cysteine proteinase is required for this process. In the present study, we have evaluated the role of parasite proteinases in earlier steps of hemoglobin degradation, namely the disassociation of the hemoglobin tetramer and the separation of heme from globin. Hemoglobin did not spontaneously denature or release heme under the pH and reducing conditions of the malarial food vacuole, suggesting that parasite enzymatic activity is necessary for early steps in hemoglobin degradation. The incubation of cultured parasites with cysteine proteinase inhibitors inhibited the denaturation of hemoglobin and the release of heme from globin. These results suggest that, in addition to its role in globin hydrolysis, a malarial cysteine proteinase participates in the dissociation of the hemoglobin tetramer and the release of heme from globin. Thus, the malarial cysteine proteinase is a promising target for antimalarial chemotherapy.     

Oriented nucleation of hemozoin at the digestive vacuole membrane in Plasmodium falciparum

Heme detoxification is a critical step in the life cycle of malaria-causing parasites, achieved by crystallization into physiologically insoluble hemozoin. The mode of nucleation has profound implications for understanding the mechanism of action of antimalarial drugs that inhibit hemozoin growth. Several lines of evidence point to involvement of acylglycerol lipids in the nucleation process. Hemozoin crystals have been reported to form within lipid nanospheres; alternatively, it has been found in vitro that they are nucleated at an acylglycerol lipid-water interface. We have applied cryogenic soft X-ray tomography and three-dimensional electron microscopy to address the location and orientation of hemozoin crystals within the digestive vacuole (DV), as a signature of their nucleation and growth processes. Cryogenic soft X-ray tomography in the "water window" is particularly advantageous because contrast generation is based inherently on atomic absorption. We find that hemozoin nucleation occurs at the DV inner membrane, with crystallization occurring in the aqueous rather than lipid phase. The crystal morphology indicates a common {100} orientation facing the membrane as expected of templated nucleation. This is consistent with conclusions reached by X-ray fluorescence and diffraction in a companion work. Uniform dark spheres observed in the parasite were identified as hemoglobin transport vesicles. Their analysis supports a model of hemozoin nucleation primarily in the DV. Modeling of the contrast at the DV membrane indicates a 4-nm thickness with patches about three times thicker, possibly implicated in the nucleation.     

On the molecular mechanism of chloroquine''s antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.     

Inhibition of heme aggregation by chloroquine reduces Schistosoma mansoni infection

Adult Schistosoma mansoni digest large amounts of host hemoglobin and release potentially toxic heme inside their guts. We have previously demonstrated that free heme in S. mansoni is detoxified through aggregation, forming hemozoin (Hz). Possible mechanisms of heme aggregation and the effects of chloroquine (CLQ) on formation of Hz and on the viability of this parasite have now been investigated. Different fractions isolated from S. mansoni, such as crude whole-worm homogenates, total lipid extracts, and Hz itself promoted heme aggregation in vitro in a CLQ-sensitive manner. Treatment of S. mansoni-infected mice with CLQ led to remarkable decreases in total protein, Hz content, and viability of the worms, as well as in parasitemia and deposition of eggs in mouse livers. These results indicate that inhibition of formation of Hz in S. mansoni, by CLQ, led to an important decrease in the overall severity of experimental murine schistosomiasis. Taken together, the results presented here suggest that formation of Hz is a major mechanism of heme detoxification and a potential target for chemotherapy in S. mansoni.     

Nuclear Magnetic Resonance: A Tool for Malaria Diagnosis?

Malaria control can be improved by rapid, sensitive, low-cost detection of infection. Several such strategies are being pursued. Rapid diagnostic tests can detect infections at parasite densities above 200 μL(-1). Polymerase chain reaction methods can detect low parasite densities, but are slow and prone to contamination under field conditions. Methods that detect hemozoin presence in blood have been proposed as alternatives for rapid detection of infection. In this study, we used a benchtop nuclear magnetic resonance (NMR) device to detect hemozoin. This device could be deployed in malaria-endemic settings. We measured synthetic hemozoin in phosphate-buffered saline and malaria parasites in human blood. The NMR detected hemozoin in suspensions of 4 ng μL(-1) and parasites at densities of 8,000-10,000 μL(-1) (0.2% parasitemia). Thus, our preliminary NMR approach, although providing very rapid measurements, is unlikely to achieve the required sensitivity and specificity for malaria diagnosis, unless a preliminary concentration step is performed.     

Biochemical characterization of Plasmodium falciparum hemozoin

Hemozoin, the pigment granule which develops within the blood stage food vacuole of the malaria parasite Plasmodium falciparum, was biochemically characterized. Hemozoin was found to be composed of 65% protein, 16% ferriprotoporphyrin-IX (hematin), 6% carbohydrate, and trace amounts of lipid and nucleic acids. The overwhelming majority of the protein component is a mixture of native and denatured human globin non-covalently associated with the metalloporphyrin. Immunoelectron microscopy, employing anti-human hemoglobin as a probe, identified in situ association of hemoglobin with hemozoin. Hemozoin produced within diabetic blood had a higher proportion of carbohydrate, suggesting that the carbohydrate component comes from non-enzymatic glycosylation of hemoglobin.     

Routes to S-nitroso-hemoglobin formation with heme redox and preferential reactivity in the beta subunits

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the alpha subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the beta subunit (cysbeta-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of S-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the beta subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of beta subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme-iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.     

Hemozoin is a key factor in the induction of malaria-associated immunosuppression

Infection-associated immunoincompetence during malaria might result from macrophage dysfunction. In the present study, we investigated the role of macrophages as target for immunosuppression during infection, using the murine Plasmodium c. chabaudi model. Special attention has been paid to the analysis of processing/presentation of protein antigens and presentation of peptides, using cocultures of peritoneal exudate cells (PECs) from infected mice and antigen-specific T-cell hybridomas. The results obtained indicate a defective processing of protein antigens that becomes maximal at acute parasitemias. In addition, macrophages from acutely infected mice suppress the interleukin-2 production by the antigen-activated T-cell hybridomas. This effect was independent of prostaglandin and nitric oxide production by the macrophage. The possible role of parasite components in the impaired accessory cell function of PECs was investigated and hemozoin, the end-product of the hemoglobin catabolism by intraerythrocytic malaria parasites, was found to induce similar infection-associated deficiencies in vitro. Moreover, hemozoin, was shown to mimic the immunosuppressive effects induced in PECs during in-vivo infections with P. chabaudi. In conclusion, we propose that hemozoin is a key factor in the malaria-associated immunosuppression, affecting both the antigen processing and immunomodulatory functions of macrophages.     

Functional Nonequivalence of α and β Hemes in Human Adult Hemoglobin

Nuclear magnetic resonance studies of the contact-shifted spectra of heme protons in deoxyhemoglobin A from human adults show conclusively that oxygen binds to the alpha hemes in preference to the beta hemes. The preferential binding is produced in 10% hemoglobin solution at neutral pH by either a 15-fold molar excess of 2,3-diphosphoglycerate or a 5-fold molar excess of inositol hexaphosphate. Preferential binding is not observable in the absence of the organic phosphates. The results indicate that the oxygenation of hemoglobin may be described by a sequential model, or by a concerted model that allows the alpha hemes to bind ligand first.     

Linearity of the hemoglobin oxidation bohr effect.

The hemoglobin oxidation Bohr effect below pH 7 is essentially proportional to the fraction of hemes oxidized, just as the ligation Bohr effect is proportional to fractional heme ligation. The reported nonlinear proton release during oxidation [(1965) J. Biol. Chem. 240, 3317-3324] is shown to be an artifact resulting from the use of ferricyanide as oxidant. Published forms of the two-state allosteric transition model for hemoglobin function have used several proton linkage schemes, and none are compatible with a linear proton release upon oxidation.     

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.     

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ?-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ?-hematin synthesis (I?HS) and hemoglobin proteolysis (IHbP) in vitro. The ?-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5%, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 μM and 14.4 ± 1.4 μM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0% and 55.3 ± 3.6%, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia (%P) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0% compared to 26.6 ± 3.7%, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.     

Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27 degrees C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H(2)O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H(2)O) as a function of oxygenation, the amount of oxygen bound to the alpha and beta chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential O(2) binding to the alpha or beta chains; (ii) in the presence of organic phosphates, the alpha hemes have a higher affinity for O(2) as compared to the beta hemes; (iii) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (iv) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.     

Malaria-parasitized erythrocytes and hemozoin nonenzymatically generate large amounts of hydroxy fatty acids that inhibit monocyte functions

Plasmodium falciparum digests up to 75% of erythrocyte (red blood cell [RBC]) hemoglobin and forms hemozoin. Phagocytosed hemozoin and trophozoites inhibit important monocyte functions. Delipidized trophozoites and hemozoin were remarkably less toxic to monocytes. Parasitized RBCs and hemozoin contained large amounts of mostly esterified monohydroxy derivatives (OH-PUFAs), the stable end products of peroxidation of polyenoic fatty acids. The concentrations of OH-PUFA were 1.8 micromoles per liter RBCs in nonparasitized RBCs, 11.1 micromoles per liter RBCs in rings, 35 micromoles per liter RBCs in trophozoites; and approximately 90 micromoles per liter RBC equivalents in hemozoin. In parasitized RBCs and hemozoin a complex mixture of monohydroxy derivatives of arachidonic (HETEs) and linoleic (HODEs) acid was determined. Respectively, 13- and 9-HODE and 9- and 12-HETE were predominant in hemozoin and parasitized RBCs. The estimated concentrations of all HETE isomers were 33 and 39 micromoles per liter RBCs or RBC equivalents in trophozoites and hemozoin, respectively. No evidence of lipoxygenase activity was found, whereas the large number of positional and optical isomers, the racemic structure, and their generation by incubation of arachidonic acid with hemozoin indicated nonenzymatic origin via heme-catalysis. Sub/low micromolar concentrations of 12- and 15-HETE were toxic to monocytes, whereas HODE isomers were ineffective. Low micromolar concentrations of HETE isomers were estimated to be similarly present in monocytes after phagocytosis of trophozoites or hemozoin. Thus, specific products of heme-catalyzed lipid peroxidation appear to contribute to hemozoin toxicity to phagocytes and may thus play a role in increased cytoadherence, vascular permeability, and chemotaxis, as well as in immunodepression in malaria.     

Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite derives much of its requirement for amino acids from the digestion of the hemoglobin of its host cell. However, one amino acid, isoleucine, is absent from adult human hemoglobin and must therefore be obtained from the extracellular medium. In this study we have characterized the mechanisms involved in the uptake of isoleucine by the intraerythrocytic parasite. Under physiologic conditions the rate of transport of isoleucine into human erythrocytes infected with mature trophozoite-stage Plasmodium falciparum parasites is increased to approximately 5-fold that in uninfected cells, with the increased flux being via the new permeability pathways (NPPs) induced by the parasite in the host cell membrane. Transport via the NPPs ensures that protein synthesis is not rate limited by the flux of isoleucine across the erythrocyte membrane. On entering the infected erythrocyte, isoleucine is taken up into the parasite via a saturable, ATP-, Na+-, and H+-independent system which has the capacity to mediate the influx of isoleucine in exchange for leucine (liberated from hemoglobin). The accumulation of radiolabeled isoleucine within the parasite is mediated by a second (high-affinity, ATP-dependent) mechanism, perhaps involving metabolism and/or the concentration of isoleucine within an intracellular organelle.     

Expression of fully functional tetrameric human hemoglobin in Escherichia coli.

Synthetic genes encoding the human alpha- and beta-globin polypeptides have been expressed from a single operon in Escherichia coli. The alpha- and beta-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to greater than 5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of alpha- and beta-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0. The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains. We have also expressed the alpha- and beta-globin genes separately and found that the expression of the alpha-globin gene alone results in a marked decrease in the accumulation of alpha-globin in the cell. Separate expression of the beta-globin gene results in high levels of insoluble beta-globin. These observations suggest that the presence of alpha- and beta-globin in the same cell stabilizes alpha-globin and aids the correct folding of beta-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.     

Hemoglobin E and Glucose-6-Phosphate Dehydrogenase Deficiency and Plasmodium falciparum Malaria in the Chittagong Hill Districts of Bangladesh

Hemoglobin E is largely confined to south and southeast Asia. The association between hemoglobin E (HbE) and malaria is less clear than that of hemoglobin S and C. As part of a malaria study in the Chittagong Hill Districts of Bangladesh, an initial random sample of 202 individuals showed that 39% and 49% of Marma and Khyang ethnic groups, respectively, were positive for either heterozygous or homozygous hemoglobin E. In this group, 6.4% were also found to be severely deficient and 35% mildly deficient for glucose-6-phosphate dehydrogenase (G6PD). In a separate Plasmodium falciparum malaria case–uninfected control study, the odds of having homozygous hemoglobin E (HbEE) compared with normal hemoglobin (HbAA) were higher among malaria cases detected by passive surveillance than age and location matched uninfected controls (odds ratio [OR] = 5.0, 95% confidence interval [CI] = 1.07–46.93). The odds of heterozygous hemoglobin E (HbAE) compared with HbAA were similar between malaria cases and uninfected controls (OR = 0.71, 95% CI = 0.42–1.19). No association by hemoglobin type was found in the initial parasite density or the proportion parasite negative after 2 days of artemether/lumefantrine treatment. HbEE, but not HbAE status was associated with increased passive case detection of malaria.