Micromolar Dihydroartemisinin Concentrations Elicit Lipoperoxidation in Plasmodium falciparum-Infected Erythrocytes.

Malaria is still the most important parasitic infectious disease. Numerous substances are known to have antimalarial activity; among them, artemisinin is the most widely used one, and artemisinin-based combination therapy (ACT) is recommended for the treatment of Plasmodium falciparum (P.f.) malaria. Antitumor, immunomodulatory, and other therapeutic applications of artemisinin are under extensive study. Several different mechanisms of action were proposed for dihydroartemisinin (DHA), the active metabolite of artemisinin, such as eliciting oxidative stress in target cells. The goal of this study is to monitor the generation of reactive oxygen species (ROS) and lipid peroxidation product 4-hydroxynonenal (4-HNE) by DHA in P.f.-infected human erythrocytes. Checking ROS and 4-HNE-protein adducts kinetics along the maturation of the parasite, we detected the highest level of 4-HNE in ring forms of P.f. due to DHA treatment. Low micromolar concentrations of DHA quickly induced levels of 4-HNE-adducts which are supposed to be damaging. Mass spectrometry identified the P.f. protein cysteine proteinase falcipain-1 as being heavily modified by 4-HNE, and plausibly, 4-HNE conjugation with vital P.f. proteins might contribute to DHA-elicited parasite death. In conclusion, significant 4-HNE accumulation was detectable after DHA treatment, though, at concentrations well above pharmacologically effective ranges in malaria treatment, but at concentrations described for antitumor activity. Thus, lipid peroxidation with consequent 4-HNE conjugation of functionally relevant proteins might be considered as a uniform mechanism for how DHA potentiates antimalarials' action in ACT and controls the progression of tumors.

Posttranslational Modification of Human Cytochrome CYP4F11 by 4-Hydroxynonenal Impairs ω-Hydroxylation in Malaria Pigment Hemozoin-Fed Monocytes: The Role in Malaria Immunosuppression.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

An Optimized Dihydrodibenzothiazepine Lead Compound (SBI-0797750) as a Potent and Selective Inhibitor of Plasmodium falciparum and P. vivax Glucose 6-Phosphate Dehydrogenase 6-Phosphogluconolactonase.

In Plasmodium, the first two and rate-limiting enzymes of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconolactonase, are bifunctionally fused to a unique enzyme named GluPho, differing structurally and mechanistically from the respective human orthologs. Consistent with the enzyme's essentiality for malaria parasite proliferation and propagation, human G6PD deficiency has immense impact on protection against severe malaria, making PfGluPho an attractive antimalarial drug target. Herein we report on the optimized lead compound N-(((2R,4S)-1-cyclobutyl-4-hydroxypyrrolidin-2-yl)methyl)-6-fluoro-4-methyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]thiazepine-8-carboxamide (SBI-0797750), a potent and fully selective PfGluPho inhibitor with robust nanomolar activity against recombinant PfGluPho, PvG6PD, and P. falciparum blood-stage parasites. Mode-of-action studies have confirmed that SBI-0797750 disturbs the cytosolic glutathione-dependent redox potential, as well as the cytosolic and mitochondrial H2O2 homeostasis of P. falciparum blood stages, at low nanomolar concentrations. Moreover, SBI-0797750 does not harm red blood cell (RBC) integrity and phagocytosis and thus does not promote anemia. SBI-0797750 is therefore a very promising antimalarial lead compound.

Malaria Pigment Hemozoin Impairs GM-CSF Receptor Expression and Function by 4-Hydroxynonenal.

Malarial pigment hemozoin (HZ) generates the lipoperoxidation product 4-hydroxynonenal (4-HNE), which is known to cause dysregulation of the immune response in malaria. The inhibition of granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent differentiation of dendritic cells (DC) by HZ and 4-HNE was previously described in vitro, and the GM-CSF receptor (GM-CSF R) was hypothesised to be a primary target of 4-HNE in monocytes. In this study, we show the functional impact of HZ on GM-CSF R in monocytes and monocyte-derived DC by (i) impairing GM-CSF binding by 50 ± 9% and 65 ± 14%, respectively (n = 3 for both cell types); (ii) decreasing the expression of GM-CSF R functional subunit (CD116) on monocyte's surface by 36 ± 11% (n = 6) and in cell lysate by 58 ± 16% (n = 3); and (iii) binding of 4-HNE to distinct amino acid residues on CD116. The data suggest that defective DC differentiation in malaria is caused by GM-CSF R dysregulation and GM-CSF R modification by lipoperoxidation product 4-HNE via direct interaction with its CD116 subunit.

Relaxometric studies of erythrocyte suspensions infected by Plasmodium falciparum: a tool for staging infection and testing anti-malarial drugs.

PURPOSE: Malaria is a global health problem with the most malignant form caused by Plasmodium falciparum (P. falciparum). Parasite maturation in red blood cells (RBCs) is accompanied by changes including the formation of paramagnetic hemozoin (HZ) nanocrystals, and increased metabolism and variation in membrane lipid composition. Herein, MR relaxometry (MRR) was applied to investigate water exchange across RBCs' membrane and HZ formation in parasitized RBCs. METHODS: Transverse water protons relaxation rate constants (R2 = 1/T2 ) were measured for assessing HZ formation in P. falciparum-parasitized human RBCs. Moreover, water exchange lifetimes across the RBC membrane (τi ) were assessed by measuring longitudinal relaxation rate constants (R1 = 1/T1 ) at 21.5 MHz in the presence of a gadolinium complex dissolved in the suspension medium. RESULTS: τi increased after invasion of parasites (ring stage, mean τi / τi0 = 1.234 ± 0.022) and decreased during maturation to late trophozoite (mean τi / τi0 = 0.960 ± 0.075) and schizont stages (mean τi / τi0 = 1.019 ± 0.065). The HZ accumulation in advanced stages was revealed by T2 -shortening. The curves reporting R2 (1/T2 ) vs. magnetic field showed different slopes for non-parasitized RBCs (npRBCs) and parasitized RBCs (pRBCs), namely 0.003 ± 0.001 for npRBCs, 0.009 ± 0.002, 0.028 ± 0.004 and 0.055 ± 0.002 for pRBCs at ring-, early trophozoite-, and late trophozoite stage, respectively. Antimalarial molecules dihydroartemisinin and chloroquine elicited measurable changes in parasitized RBCs, namely dihydroartemisinin modified τi , whereas the interference of chloroquine with HZ formation was detectable by a significant T2 increase. CONCLUSIONS: MRR can be considered a useful tool for reporting on P. falciparum blood stages and for screening potential antimalarial molecules.

Plasmodium Impairs Antibacterial Innate Immunity to Systemic Infections in Part Through Hemozoin-Bound Bioactive Molecules.

One complication of malaria is increased susceptibility to invasive bacterial infections. Plasmodium infections impair host immunity to non-Typhoid Salmonella (NTS) through heme-oxygenase I (HO-I)-induced release of immature granulocytes and myeloid cell-derived IL-10. Yet, it is not known if these mechanisms are specific to NTS. We show here, that Plasmodium yoelii 17XNL (Py) infected mice had impaired clearance of systemic Listeria monocytogenes (Lm) during both acute parasitemia and up to 2 months after clearance of Py infected red blood cells that was independent of HO-I and IL-10. Py-infected mice were also susceptible to Streptococcus pneumoniae (Sp) bacteremia, a common malaria-bacteria co-infection, with higher blood and spleen bacterial burdens and decreased survival compared to naïve mice. Mechanistically, impaired immunity to Sp was independent of HO-I, but was dependent on Py-induced IL-10. Splenic phagocytes from Py infected mice exhibit an impaired ability to restrict growth of intracellular Lm, and neutrophils from Py-infected mice produce less reactive oxygen species (ROS) in response to Lm or Sp. Analysis also identified a defect in a serum component in Py-infected mice that contributes to reduced production of ROS in response to Sp. Finally, treating naïve mice with Plasmodium-derived hemozoin containing naturally bound bioactive molecules, excluding DNA, impaired clearance of Lm. Collectively, we have demonstrated that Plasmodium infection impairs host immunity to diverse bacteria, including S. pneumoniae, through multiple effects on innate immunity, and that a parasite-specific factor (Hz+bound bioactive molecules) directly contributes to Plasmodium-induced suppression of antibacterial innate immunity.

Oxidative Stress, Lipid Peroxidation, and Loss of Hyaluronic Acid in the Human Vitreous Affected by Synchysis Scintillans.

The aim of this study was to assess the oxidative stress status in eyes affected by synchysis scintillans and to compare it to vitreoretinal disorders without synchysis scintillans. Human aqueous and vitreous humors were obtained during vitrectomy from thirty-seven otherwise healthy patients that were randomly chosen among patients that had to undergo a 25-gauge pars plana vitrectomy from the central vitreous cavity, for either synchysis scintillans (n = 16) or vitreoretinal disorders without synchysis scintillans (n = 21), such as idiopathic epimacular membrane (n = 12), macular hole (n = 5), or rhegmatogenous retinal detachment (n = 4). The redox parameters thiobarbituric acid reactive substances (TBARS), a measurement of lipid peroxidation, nitrite concentration, an estimate of nitric oxide (NO) production, 4-hydroxynonenal (4-HNE)-protein conjugates, a structural protein modification by lipid peroxidation product 4-HNE, and the antioxidative activities of Cu,Zn-superoxide dismutase (SOD), and catalase were measured in aqueous and vitreous humors and compared between synchysis scintillans affected and not-affected patients. TBARS and nitrite levels of the vitreous humor were significantly higher in patients with synchysis scintillans as compared to patients affected by vitreoretinal disorders without synchysis scintillans. Synchysis scintillans patients had significantly lower activities of SOD and catalase both in aqueous and vitreous humors than patients with vitreoretinal disorders without synchysis. The consequently higher lipoperoxide-dependent 4-HNE production in synchysis scintillans was detectable in aqueous and vitreous humors as a significant increased accumulation of 4-HNE-protein conjugates vs nonsynchysis vitreoretinal disorders. Additionally, hyaluronic acid (HA) was significantly decreased in the vitreous body of synchysis scintillans patients. The data consistently show that synchisis scintillans is accompanied by a redox imbalance with increased oxidative modifications of 4-HNE proteins and loss of HA, both of likely importance for remote damages of the retina. It remains to be proven whether a therapeutic strategy which targets oxidative stress may be effective in the treatment of synchysis patients.

Neutrophil extracellular traps drive inflammatory pathogenesis in malaria.

Neutrophils are essential innate immune cells that extrude chromatin in the form of neutrophil extracellular traps (NETs) when they die. This form of cell death has potent immunostimulatory activity. We show that heme-induced NETs are essential for malaria pathogenesis. Using patient samples and a mouse model, we define two mechanisms of NET-mediated inflammation of the vasculature: activation of emergency granulopoiesis via granulocyte colony-stimulating factor production and induction of the endothelial cytoadhesion receptor intercellular adhesion molecule-1. Soluble NET components facilitate parasite sequestration and mediate tissue destruction. We demonstrate that neutrophils have a key role in malaria immunopathology and propose inhibition of NETs as a treatment strategy in vascular infections.

Clinical impact of the two ART resistance markers, K13 gene mutations and DPC3 in Vietnam.

BACKGROUND: In Vietnam, a rapid decline of P. falciparum malaria cases has been documented in the past years, the number of Plasmodium falciparum malaria cases has rapidly decreased passing from 19.638 in 2012 to 4.073 cases in 2016. Concomitantly, the spread of artemisinin resistance markers is raising concern on the future efficacy of the ACTs. An evaluation of the clinical impact of the artemisinin resistance markers is therefore of interest. METHODS: The clinical effectiveness of dihydroartemisinin-piperaquine therapy (DHA-PPQ) has been evaluated in three districts characterized by different rates of ART resistance markers: K13(C580Y) mutation and delayed parasite clearance on day 3 (DPC3). Patients were stratified in 3 groups a) no markers, b) one marker (suspected resistance), c) co-presence of both markers (confirmed resistance). In the studied areas, the clinical effectiveness of DHA-PPQ has been estimated as malaria recrudescence within 60 days. RESULTS: The rate of K13(C580Y) ranged from 75.8% in Krong Pa to 1.2% in Huong Hoa district. DPC3 prevalence was higher in Krong Pa than in Huong Hoa (86.2% vs 39.3%). In the two districts, the prevalence of confirmed resistance was found in 69.0% and 1.2% of patients, respectively. In Thuan Bac district, we found intermediate prevalence of confirmed resistance. Treatment failure was not evidenced in any district. PPQ resistance was not evidenced. Confirmed resistance was associated to the persistence of parasites on day 28 and to 3.4-fold higher parasite density at diagnosis. The effectiveness of malaria control strategies was very high in the studied districts. CONCLUSION: No treatment failure has been observed in presence of high prevalence of ART resistance and in absence of PPQ resistance. K13(C580Y) was strongly associated to higher parasitemia at admission, on days 3 and 28. Slower parasite clearance was also observed in younger patients.

Induction of oxidative stress in human aqueous and vitreous humors by Nd:YAG laser posterior capsulotomy.

AIM: To evaluate whether the Q-switched Nd:YAG laser treatment applied in routine capsulotomy elicits oxidative stress in aqueous and vitreous humors. METHODS: Thirty-six patients who had to undergo a 25 gauge pars plana vitrectomy due to vitreoretinal disorders were enrolled, 15 of them underwent a Q-switched Nd:YAG laser capsulotomy 7d before vitrectomy due to posterior capsule opacification (PCO) (Nd:YAG laser group) while the remaining 21 patients were not laser treated before vitrectomy (no Nd:YAG laser group). Samples of the aqueous and vitreous humors were collected during vitrectomy from all patients for the assessment of oxidative parameters which were compared between the Nd:YAG laser group and no Nd:YAG laser group. Thiobarbituric acid reactive substances (TBARS), a product of membrane lipid peroxidation, nitrite levels, the antioxidative activities of SOD and catalase, the 4-HNE-protein conjugate formation, indicating structural modifications in proteins due to lipoperoxidation, were assessed in aqueous and vitreous samples. RESULTS: In the human vitreous humor TBARS levels are significantly higher in the Nd:YAG laser group compared to the no Nd:YAG laser group and importantly, there is a significant correlation between the TBARS levels and the total energy of Nd:YAG laser used during capsulotomy. Moreover the anti-oxidative activities of SOD and catalase were significantly decreased by Nd:YAG laser treatment, both in aqueous and vitreous humors. In accordance with the TBARS data and anti-oxidative enzyme activities, significantly higher levels of proteins were conjugated with the lipoperoxidation product 4-HNE in the aqueous and vitreous humors in the Nd:YAG laser-treated group in comparison to no Nd:YAG laser group. CONCLUSION: These data, clearly suggest that any change that Q-switched Nd:YAG photo disruption may cause in the aqueous and vitreous compartments, resulting in a higher level of oxidative damage might be of considerable clinical significance particularly by accelerating the aging of the anterior and posterior segments of the eye and by worsening the intraocular pressure, the uveal, the retinal (especially macular) pathologies.

Publisher Correction: Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity.

The original version of this Article contained errors in Fig. 3. In panel a, bars from a chart depicting the percentage of antibody-positive individuals in non-infectious and infectious groups were inadvertently included in place of bars depicting the percentage of infectious individuals, as described in the Article and figure legend. However, the p values reported in the Figure and the resulting conclusions were based on the correct dataset. The corrected Fig. 3a now shows the percentage of infectious individuals in antibody-negative and -positive groups, in both the PDF and HTML versions of the Article. The incorrect and correct versions of Figure 3a are also presented for comparison in the accompanying Publisher Correction as Figure 1.The HTML version of the Article also omitted a link to Supplementary Data 6. The error has now been fixed and Supplementary Data 6 is available to download.

Unravelling the immune signature of Plasmodium falciparum transmission-reducing immunity.

Infection with Plasmodium can elicit antibodies that inhibit parasite survival in the mosquito, when they are ingested in an infectious blood meal. Here, we determine the transmission-reducing activity (TRA) of naturally acquired antibodies from 648 malaria-exposed individuals using lab-based mosquito-feeding assays. Transmission inhibition is significantly associated with antibody responses to Pfs48/45, Pfs230, and to 43 novel gametocyte proteins assessed by protein microarray. In field-based mosquito-feeding assays the likelihood and rate of mosquito infection are significantly lower for individuals reactive to Pfs48/45, Pfs230 or to combinations of the novel TRA-associated proteins. We also show that naturally acquired purified antibodies against key transmission-blocking epitopes of Pfs48/45 and Pfs230 are mechanistically involved in TRA, whereas sera depleted of these antibodies retain high-level, complement-independent TRA. Our analysis demonstrates that host antibody responses to gametocyte proteins are associated with reduced malaria transmission efficiency from humans to mosquitoes.

Syk inhibitors interfere with erythrocyte membrane modification during P falciparum growth and suppress parasite egress.

Band 3 (also known as the anion exchanger, SLCA1, AE1) constitutes the major attachment site of the spectrin-based cytoskeleton to the erythrocyte's lipid bilayer and thereby contributes critically to the stability of the red cell membrane. During the intraerythrocytic stage of Plasmodium falciparum's lifecycle, band 3 becomes tyrosine phosphorylated in response to oxidative stress, leading to a decrease in its affinity for the spectrin/actin cytoskeleton and causing global membrane destabilization. Because this membrane weakening is hypothesized to facilitate parasite egress and the consequent dissemination of released merozoites throughout the bloodstream, we decided to explore which tyrosine kinase inhibitors might block the kinase-induced membrane destabilization. We demonstrate here that multiple Syk kinase inhibitors both prevent parasite-induced band 3 tyrosine phosphorylation and inhibit parasite-promoted membrane destabilization. We also show that the same Syk kinase inhibitors suppress merozoite egress near the end of the parasite's intraerythrocytic lifecycle. Because the entrapped merozoites die when prevented from escaping their host erythrocytes and because some Syk inhibitors have displayed long-term safety in human clinical trials, we suggest Syk kinase inhibitors constitute a promising class of antimalarial drugs that can suppress parasitemia by inhibiting a host target that cannot be mutated by the parasite to evolve drug resistance.

Malaria-derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells.

Plasmodium falciparum (P. falciparum)-induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long-lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen-presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte-derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, ß-hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP-1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and ß-hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not ß-hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to ß-hematin.

Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells.

The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione.

Oxidative stress-mediated antimalarial activity of plakortin, a natural endoperoxide from the tropical sponge Plakortis simplex.

Plakortin, a polyketide endoperoxide from the sponge Plakortis simplex has antiparasitic activity against P. falciparum. Similar to artemisinin, its activity depends on the peroxide functionality. Plakortin induced stage-, dose- and time-dependent morphologic anomalies, early maturation delay, ROS generation and lipid peroxidation in the parasite. Ring damage by 1 and 10 µM plakortin led to parasite death before schizogony at 20 and 95%, respectively. Treatment of late schizonts with 1, 2, 5 and 10 µM plakortin resulted in decreased reinfection rates by 30, 50, 61 and 65%, respectively. In both rings and trophozoites, plakortin induced a dose- and time-dependent ROS production as well as a significant lipid peroxidation and up to 4-fold increase of the lipoperoxide breakdown product 4-hydroxynonenal (4-HNE). Antioxidants and the free radical scavengers trolox and N-acetylcysteine significantly attenuated the parasite damage. Plakortin generated 4-HNE conjugates with the P. falciparum proteins: heat shock protein Hsp70-1, endoplasmatic reticulum-standing Hsp70-2 (BiP analogue), V-type proton ATPase catalytic subunit A, enolase, the putative vacuolar protein sorting-associated protein 11, and the dynein heavy chain-like protein, whose specific binding sites were identified by mass spectrometry. These proteins are crucially involved in protein trafficking, transmembrane and vesicular transport and parasite survival. We hypothesize that binding of 4-HNE to functionally relevant parasite proteins may explain the observed plakortin-induced morphologic aberrations and parasite death. The identification of 4-HNE-protein conjugates may generate a novel paradigm to explain the mechanism of action of pro-oxidant, peroxide-based antimalarials such as plakortin, artemisinins and synthetic endoperoxides.

Role of the lipoperoxidation product 4-hydroxynonenal in the pathogenesis of severe malaria anemia and malaria immunodepression.

Oxidative stress plays an important role in the pathogenesis of falciparum malaria, a disease still claiming close to 1 million deaths and 200 million new cases per year. Most frequent complications are severe anemia, cerebral malaria, and immunodepression, the latter being constantly present in all forms of malaria. Complications are associated with oxidative stress and lipoperoxidation. Its final product 4-hydroxynonenal (4-HNE), a stable yet very reactive and diffusible molecule, forms covalent conjugates with proteins, DNA, and phospholipids and modulates important cell functions at very low concentrations. Since oxidative stress plays important roles in the pathogenesis of severe malaria, it appears important to explore the role of 4-HNE in two important malaria complications such as malaria anemia and malaria immunodepression where oxidative stress is considered to be involved. In this review we will summarize data about 4-HNE chemistry, its biologically relevant chemical properties, and its role as regulator of physiologic processes and as pathogenic factor. We will review studies documenting the role of 4-HNE in severe malaria with emphasis on malaria anemia and immunodepression. Data from other diseases qualify 4-HNE both as oxidative stress marker and as pathomechanistically important molecule. Further studies are needed to establish 4-HNE as accepted pathogenic factor in severe malaria.

Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes.

Antimalarial NADPH-Consuming Redox-Cyclers As Superior Glucose-6-Phosphate Dehydrogenase Deficiency Copycats.

AIMS: Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum were shown to protect G6PD-deficient populations from severe malaria. Here, we investigated the mechanism of a novel antimalarial series, namely 3-[substituted-benzyl]-menadiones, to understand whether these NADPH-consuming redox-cyclers, which induce oxidative stress, mimic the natural protection of G6PD deficiency. RESULTS: We demonstrated that the key benzoylmenadione metabolite of the lead compound acts as an efficient redox-cycler in NADPH-dependent methaemoglobin reduction, leading to the continuous formation of reactive oxygen species, ferrylhaemoglobin, and subsequent haemichrome precipitation. Structure-activity relationships evidenced that both drug metabolites and haemoglobin catabolites contribute to potentiate drug effects and inhibit parasite development. Disruption of redox homeostasis by the lead benzylmenadione was specifically induced in Plasmodium falciparum parasitized erythrocytes and not in non-infected cells, and was visualized via changes in the glutathione redox potential of living parasite cytosols. Furthermore, the redox-cycler shows additive and synergistic effects in combination with compounds affecting the NADPH flux in vivo. INNOVATION: The lead benzylmenadione 1c is the first example of a novel redox-active agent that mimics the behavior of a falciparum parasite developing inside a G6PD-deficient red blood cell (RBC) giving rise to malaria protection, and it exerts specific additive effects that are inhibitory to parasite development, without harm for non-infected G6PD-sufficient or -deficient RBCs. CONCLUSION: This strategy offers an innovative perspective for the development of future antimalarial drugs for G6PD-sufficient and -deficient populations.