Search

The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3′ untranslated region of a gene encoding a HECT E3 ubiquitin ligase, termed Plasmodium falciparum ubiquitin transferase (PfUT), increased steady state RNA and protein levels 2.5-fold in the human malaria parasite P. falciparum. Overexpression of pfut resulted in an S/M phase-associated lengthening of the parasite's intraerythrocytic developmental cycle and a reduced merozoite invasion efficiency. The addition of glucosamine partially restored the wild type phenotype. Our study suggests a role of PfUT in controlling cell cycle progression and merozoite invasion. Our study further raises awareness regarding unexpected effects on gene expression when inserting the glms ribozyme sequence into a gene locus. [ABSTRACT FROM AUTHOR]

Histones are abundant nuclear core proteins that are present in all eukararyotes and are responsible for linking chromosomes and packaging them into tight chromatin aggregates. The histone H2A, H2B, and H3 genes and a partial sequence of the histone H4 gene from Plasmodium falciparum have been previously identified and share a high level of nucleotide sequence identity. In this study, we compare the histone H4 sequence of the human malaria P. falciparum with the sequences of two mouse malarias, Plasmodium berghei and Plasmodium yoelii, revealing at least 91% identity at the nucleotide level and 100% conservation at the amino acid level. Furthermore, we show the P. falciparum histone H4 is developmentally transcribed in late stage asexual parasites, completing the transcription profile for the genes comprising the histone octamer of P. falciparum and adding support to suggestions that a novel histone mRNA control mechanism exists in this parasite. [ABSTRACT FROM AUTHOR]

During intraerythrocytic development, the human malaria parasite Plasmodium falciparum alters the mechanical deformability of its host cell. The underpinning biological processes involve gain in parasite mass, changes in the membrane protein compositions, reorganization of the cytoskeletons and its coupling to the plasma membrane, and formation of membrane protrusions, termed knobs. The hemoglobinopathies S and C are known to partially protect carriers from severe malaria, possibly through additional changes in the erythrocyte biomechanics, but a detailed quantification of cell mechanics is still missing. Here, we combined flicker spectroscopy and a mathematical model and demonstrated that knob formation strongly suppresses membrane fluctuations by increasing membrane-cytoskeleton coupling. We found that the confinement increased with hemoglobin S but decreases with hemoglobin C in spite of comparable knob densities and diameters. We further found that the membrane bending modulus strongly depends on the hemoglobinopathetic variant, suggesting increased amounts of irreversibly oxidized hemichromes bound to membranes. Fröhlich et al. show that membrane protrusions of malaria parasite-infected blood cells reduce their membrane fluctuation due to the enforced coupling of membrane and cytoskeleton. Differential cellular mechanics of blood cells possessing variant hemoglobins that protect people from malaria suggests that oxidative stress may explain the selective advantage of certain hemoglobin variants. [ABSTRACT FROM AUTHOR]

PfEMP1 (erythrocyte membrane protein 1) adhesins play a pivotal role in the pathophysiology of falciparum malaria, by mediating sequestration of Plasmodium falciparum-infected erythrocytes in the microvasculature. PfEMP1 variants are expressed by var genes and are presented on membrane elevations, termed knobs. However, the organization of PfEMP1 on knobs is largely unclear. Here, we use super-resolution microscopy and genetically altered parasites expressing a modified var2csa gene in which the coding sequence of the photoactivatable mEOS2 was inserted to determine the number and distribution of PfEMP1 on single knobs. The data were verified by quantitative fluorescence-activated cell sorting analysis and immuno-electron microscopy together with stereology methods. We show that knobs contain 3.3 ± 1.7 and 4.3 ± 2.5 PfEMP1 molecules, predominantly placed on the knob tip, in parasitized erythrocytes containing wild type and sickle haemoglobin, respectively. The ramifications of our findings for cytoadhesion and immune evasion are discussed. Cecilia Sanchez et al. investigate the organization of immune-variant adhesins on Plasmodium falciparum-infected erythrocytes by super-resolution microscopy. Using a fluorescently tagged protein, they obtain quantitative and spatial information about the distribution of adhesin molecules on knobs, which are structures used by the malaria parasite to anchor to the host cells. [ABSTRACT FROM AUTHOR]

Sickle cell trait, a common hereditary blood disorder, protects carriers from severe disease in infections with the human malaria parasite Plasmodium falciparum. Protection is associated with a reduced capacity of parasitized erythrocytes to cytoadhere to the microvascular endothelium and cause vaso-occlusive events. However, the underpinning cellular and biomechanical processes are only partly understood and the impact on endothelial cell activation is unclear. Here, we show, by combining quantitative flow chamber experiments with multiscale computer simulations of deformable cells in hydrodynamic flow, that parasitized erythrocytes containing the sickle cell haemoglobin displayed altered adhesion dynamics, resulting in restricted contact footprints on the endothelium. Main determinants were cell shape, knob density and membrane bending. As a consequence, the extent of endothelial cell activation was decreased. Our findings provide a quantitative understanding of how the sickle cell trait affects the dynamic cytoadhesion behavior of parasitized erythrocytes and, in turn, endothelial cell activation. Christine Lansche, Anil Dasanna et al. investigate the dynamic cytoadhesion behavior of erythrocytes infected with malaria parasite using quantitative flow chamber experiments and computer simulations. They find that parasitized erythrocytes have altered adhesion dynamics, mainly due to differences in cell shape, knob density and membrane bending. [ABSTRACT FROM AUTHOR]

Background: The increased resistance of the human malaria parasite Plasmodium falciparum to currently employed drugs creates an urgent call for novel anti-malarial drugs. Particularly, efforts should be devoted to developing fast-acting anti-malarial compounds in case clinical resistance increases to the first-line artemisinin-based combination therapy. SC83288, an amicarbalide derivative, is a clinical development candidate for the treatment of severe malaria. SC83288 is fast-acting and able to clear P. falciparum parasites at low nanomolar concentrations in vitro, as well as in a humanized SCID mouse model system in vivo. In this study, the antiplasmodial activity of SC83288 against artemisinins was profiled in order to assess its potential to replace, or be combined with, artemisinin derivatives. Results: Based on growth inhibition and ring survival assays, no cross-resistance was observed between artemisinins and SC83288, using parasite lines that were resistant to either one of these drugs. In addition, no synergistic or antagonistic interaction was observed between the two drugs. This study further confirmed that SC83288 is a fast acting drug in several independent assays. Combinations of SC83288 and artesunate maintained the rapid parasite killing activities of both components. Conclusion: The results obtained in this study are consistent with artemisinins and SC83288 having distinct modes of action and different mechanisms of resistance. This study further supports efforts to continue the clinical development of SC83288 against severe malaria as an alternative to artemisinins in areas critically affected by artemisinin-resistance. Considering its fast antiplasmodial activity, SC83288 could be combined with a slow-acting anti-malarial drug. [ABSTRACT FROM AUTHOR]

The article reports on the malarial secretome. Malaria, caused by the Plasmodium protozoan parasite, kills more than 1 million children in Africa alone each year. New interventions are needed to relieve the burden that malaria places on public health and economic development, particularly in Africa, which bears the brunt of the disease. Malarial parasites not only have a complex life cycle alternating between a vertebrate and invertebrate host, but also are capable of developing within highly specialized red blood cells — a challenge met by only a few intracellular pathogens.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs. [ABSTRACT FROM AUTHOR]

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ. Author summary: Chloroquine (CQ) used to be the drug of choice against malaria until the parasite responsible for the disease became resistant. In the 1970s, piperaquine (PPQ) was introduced in areas where resistance to CQ was wide spread. In the following decade, an estimated 140 million doses were distributed, which substantially reduced the malaria burden, particularly in China, but created an environment in which PPQ resistant strains of the human malaria parasite Plasmodium falciparum emerged and spread. Interestingly, the PPQ resistant parasites displayed an increased CQ sensitivity. The main genetic determinant of both CQ and PPQ resistance in P. falciparum is a drug transporter, termed PfCRT. In this study, we used biochemical and bioinformatics approaches to understand how mutational changes in PfCRT influence the interaction of the carrier with CQ and PPQ. We found that PfCRT from CQ resistant parasites is better at transporting CQ than are PfCRT variants from PPQ resistant parasites, while the opposite is true for PPQ. We also found that PfCRT can be engineered such that it transports both antimalarials equally well. Our study offers insights into how PfCRT has evolved in response to changing drug pressure, and raises concerns regarding how it may evolve in the future. [ABSTRACT FROM AUTHOR]

Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood–brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood–brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = − 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months' follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics. [ABSTRACT FROM AUTHOR]

Red blood cells can withstand the harsh mechanical conditions in the vasculature only because the bending rigidity of their plasma membrane is complemented by the shear elasticity of the underlying spectrin-actin network. During an infection by the malaria parasite Plasmodium falciparum, the parasite mines host actin from the junctional complexes and establishes a system of adhesive knobs, whose main structural component is the knob-associated histidine rich protein (KAHRP) secreted by the parasite. Here we aim at a mechanistic understanding of this dramatic transformation process. We have developed a particle-based computational model for the cytoskeleton of red blood cells and simulated it with Brownian dynamics to predict the mechanical changes resulting from actin mining and KAHRP-clustering. Our simulations include the three-dimensional conformations of the semi-flexible spectrin chains, the capping of the actin protofilaments and several established binding sites for KAHRP. For the healthy red blood cell, we find that incorporation of actin protofilaments leads to two regimes in the shear response. Actin mining decreases the shear modulus, but knob formation increases it. We show that dynamical changes in KAHRP binding affinities can explain the experimentally observed relocalization of KAHRP from ankyrin to actin complexes and demonstrate good qualitative agreement with experiments by measuring pair cross-correlations both in the computer simulations and in super-resolution imaging experiments. Author summary: Malaria is one of the deadliest infectious diseases and its symptoms are related to the blood stage, when the parasite multiplies within red blood cells. In order to avoid clearance by the spleen, the parasite produces specific factors like the adhesion receptor PfEMP1 and the multifunctional protein KAHRP that lead to the formation of adhesive knobs on the surface of the red blood cells and thus increase residence time in the vasculature. We have developed a computational model for the parasite-induced remodelling of the actin-spectrin network to quantitatively predict the dynamical changes in the mechanical properties of the infected red blood cells and the spatial distribution of the different protein components of the membrane skeleton. Our simulations show that KAHRP can relocate to actin junctions due to dynamical changes in binding affinities, in good qualitative agreement with super-resolution imaging experiments. In the future, our simulation framework can be used to gain further mechanistic insight into the way malaria parasites attack the red blood cell cytoskeleton. [ABSTRACT FROM AUTHOR]

Abstract: Resistance to antimalarial drugs has often threatened malaria elimination efforts and historically has led to the short-term resurgence of malaria incidences and deaths. With concentrated malaria eradication efforts currently underway, monitoring drug resistance in clinical settings complemented by in vitro drug susceptibility assays and analysis of resistance markers, becomes critical to the implementation of an effective antimalarial drug policy. Understanding of the factors, which lead to the development and spread of drug resistance, is necessary to design optimal prevention and treatment strategies. This review attempts to summarize the unique factors presented by malarial parasites that lead to the emergence and spread of drug resistance, and gives an overview of known resistance mechanisms to currently used antimalarial drugs. [Copyright &y& Elsevier]

Abstract: The ability to control expression of a specific gene is a prerequisite to understand the function of essential genes. Many gene regulation systems operate on the transcriptional level by employing heterologous cis- and trans-acting elements. Recently, novel approaches employing autocatalytic RNA have been reported for different organisms. Here we show specific downregulation of gene expression in the apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum, employing self-cleaving ribozymes integrated into the transcriptional unit of different genes. Moreover, we demonstrate the potential to specifically upregulate reporter gene expression by employment of the recently identified ribozyme inhibitor toyocamycin. At the RNA-level, we were able to significantly stabilise the mRNA in T. gondii. Furthermore we show that the adenosine analogue toyocamycin needs to be phosphorylated by adenosine kinase in order to act as an inhibitor for hammerhead ribozymes, since neither upregulation of reporter gene expression nor a toxic effect of toyocamycin can be detected in parasites that do not express the enzyme adenosine kinase. [Copyright &y& Elsevier]

Although quinine, the active ingredient of chinchona bark, has been used in the treatment of malaria for several centuries, there is little information regarding the interactions of this drug with the human malaria parasite Plasmodium falciparum. To better understand quinine's mode of action and the mechanism underpinning reduced responsiveness, we have investigated the factors that contribute to quinine accumulation by parasites that differ in their susceptibility to quinine. Interestingly, passive distribution, in accordance with the intracellular pH gradients, and intracellular binding could account for only a small fraction of the high amount of quinine accumulated by the parasites investigated. The results of trans-stimulation kinetics suggest that high accumulation of quinine is brought about by a carrier-mediated import system. This import system seems to be weakened in parasites with reduced quinine susceptibility. Other data show that polymorphisms within PfCRT are causatively linked with an increased verapamil-sensitive quinine efflux that, depending on the genetic background, resulted in reduced quinine accumulation. The polymorphisms within PfMDR1 investigated did not affect quinine accumulation. Our data are consistent with the model that several factors, including acidotropic trapping, binding to intracellular sites and carrier-mediated import and export transport systems, contribute to steady-state intracellular quinine accumulation. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ), an antimalarial drug with a long history, now frequently fails in the field owing to the rapid spread of resistant Plasmodium falciparum strains. CQ resistance is linked to a K76T mutation in PfCRT, a membrane-located food vacuolar protein and member of the drug-metabolite transporter superfamily, but there is as yet no agreed mechanism of how mutated PfCRT brings about CQ resistance. Current models suggest that mutated PfCRT acts either as a channel or a transporter of CQ, enabling CQ to leave the digestive food vacuole of the parasite, in which the CQ accumulates. Here, we review the pros and cons of the carrier and transporter models in light of recent developments in the field. [Copyright &y& Elsevier]

The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite- encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold. [ABSTRACT FROM AUTHOR]

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently considered models to explain the resistance phenotype include acquisition of a chloroquine efflux pump, changes in intracellular chloroquine partitioning, diminished binding affinity of chloroquine to its intracellular target, heme, and changes in heme crystallization. To challenge these different models, we have investigated chloroquine accumulation under trans-stimulation conditions and in the presence and absence of glucose. We show that, in chloroquinesensitive strains, labeled chloroquine accumulation is steadily reduced as the pre-equilibrated chloroquine concentration is raised. In the resistant cells, the extent of accumulation is, strikingly, raised at the lower levels of preloading, in comparison with resistant controls in the absence of chloroquine. The transstimulation effect observed in chloroquine-resistant cells is strictly energy-dependent. The data are interpreted in terms of a model in which chloroquine is bound to intracellular binding sites, not different as between sensitive and resistant cells, but where, in resistant cells, there exists an energy-dependent carrier that moves chloroquine out of this intracellular compartment. A mathematical model describing the kinetics of these processes is presented. [ABSTRACT FROM AUTHOR]

Examines the subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum. Derivation of the shikimate pathway of an apicomplexan parasite; Immunological cross-reactivity of chorismate synthase; Presence of apicoplast in apicomplexans.

The article presents the structure of the merozoite surface protein 1 from Plasmodium falciparum. Topics include the merozoite surface protein 1 (MSP-1) is the most abundant protein on the surface of the erythrocyte-invading Plasmodium merozoite, the MSP-1 is essential for merozoite formation, and MSP-1 adopts an unusual fold with a large central cavity.

Abstract: Phosphoinositides comprise a group of essential phospholipids that control a variety of cellular functions. In the case of the human malaria parasite Plasmodium falciparum, phosphoinositides have been shown to trigger exflagellation and to affect haemoglobin endocytosis and maturation of the parasite''s digestive vacuole. A central enzyme in the formation of phosphoinositides is the phosphatidylinositol 4-kinase that catalyzes the production of phosphatidylinositol 4-phosphate from phosphatidylinositol. Here we have identified and characterized a phosphatidylinositol 4-kinase from P. falciparum. Our data show that the corresponding P. falciparum gene, termed PFE0485w, can functionally complement a yeast temperature-sensitive pik1 mutation. Our data add to the concept that P. falciparum maintains its own phospholipids biosynthesis pathway. [Copyright &y& Elsevier]

Virulence and drug resistance are traits that pathogens can acquire independently, albeit these traits can influence each other. A recent publication has reported on the co-evolution of virulence and pyrimethamine resistance in malaria parasites. Here, we discuss this finding in the context of the folate biosynthesis pathway and explain how mutational changes in this pathway can affect both parasite replication rates and the development of drug resistance. [Copyright &y& Elsevier]

The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance-conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ~50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT. [ABSTRACT FROM AUTHOR]

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes. [ABSTRACT FROM AUTHOR]

With the aim of increasing the structural diversity on the early antimalarial drug plasmodione, an efficient and versatile procedure to prepare a series of biaryl- and N-arylalkylamines as plasmodione analogues is described. Using the naturally occurring and commercially available menadione as starting material, a 2-step sequence using a Kochi-Anderson reaction and subsequent Pd-catalyzed Suzuki-Miyaura coupling was developed to prepare three representative biphenyl derivatives in good yields for antimalarial evaluation. In addition, synthetic methodologies to afford 3-benzylmenadione derivatives bearing a terminal -N(Me)2 or -N(Et)2 in different positions (ortho, meta and para) on the aryl ring of the benzylic chain of plasmodione were investigated through reductive amination was used as the optimal route to prepare these protonable N-arylalkylamine privileged scaffolds. The antimalarial activities were evaluated and discussed in light of their physicochemical properties. Among the newly synthesized compounds, the para-position of the substituent remains the most favourable position on the benzyl chain and the carbamate -NHBoc was found active both in vitro (42 nM versus 29 nM for plasmodione) and in vivo in Plasmodium berghei-infected mice. The measured acido-basic features of these new molecules support the cytosol-food vacuole shuttling properties of non-protonable plasmodione derivatives essential for redox-cycling. These findings may be useful in antimalarial drug optimization. [ABSTRACT FROM AUTHOR]

Life under aerobic conditions has shaped peroxiredoxins (Prx) as ubiquitous thiol-dependent hydroperoxidases and redox sensors. Structural features that balance the catalytically active or inactive redox states of Prx, and, therefore, their hydroperoxidase or sensor function, have so far been analyzed predominantly for Prx1-type enzymes. Here we identify and characterize two modulatory residues of the Prx5-type model enzyme Pf AOP from the malaria parasite Plasmodium falciparum . Gain- and loss-of-function mutants reveal a correlation between the enzyme parameters and the inactivation susceptibility of Pf AOP with the size of residue 109 and the presence or absence of a catalytically relevant but nonessential cysteine residue. Based on our kinetic data and the crystal structure of Pf AOP L109M , we suggest a novel mechanism for balancing the hydroperoxidase activity and inactivation susceptibility of Prx5-type enzymes. Our study provides unexpected insights into Prx structure–function relationships and contributes to our understanding of what makes Prx good enzymes or redox sensors. [ABSTRACT FROM AUTHOR]

Infections with the human malaria parasite Plasmodium falciparum during pregnancy can lead to severe complications for both mother and child, resulting from the cytoadhesion of parasitized erythrocytes in the intervillous space of the placenta. Cytoadherence is conferred by the specific interaction of the parasite-encoded adhesin VAR2CSA with chondroitin-4-sulfate (CSA) present on placental proteoglycans. CSA presented elsewhere in the microvasculature does not afford VAR2CSA-mediated cytoadhesion of parasitized erythrocytes. To address the placenta-specific binding tropism, we investigated the effect of the receptor/ligand arrangement on cytoadhesion, using artificial membranes with different CSA spacing intervals. We found that cytoadhesion is strongly dependent on the CSA distance, with half-maximal adhesion occurring at a CSA distance of 9 ± 1 nm at all hydrodynamic conditions. Moreover, binding to CSA was cooperative and shear stress induced. These findings suggest that the CSA density, together with allosteric effects in VAR2CSA, aid in discriminating between different CSA milieus. [ABSTRACT FROM AUTHOR]

Following invasion of human red blood cells (RBCs) by the malaria parasite, Plasmodium falciparum, a remarkable process of remodeling occurs in the host cell mediated by trafficking of several hundred effector proteins to the RBC compartment. The exported virulence protein, P falciparum erythrocyte membrane protein 1 (PfEMP1), is responsible for cytoadherence of infected cells to host endothelial receptors. Maurer clefts are organelles essential for protein trafficking, sorting, and assembly of protein complexes. Here we demonstrate that disruption of PfEMP1 trafficking protein 1 (PfPTP1) function leads to severe alterations in the architecture of Maurer's clefts. Furthermore, 2 major surface antigen families, PfEMP1 and STEVOR, are no longer displayed on the host cell surface leading to ablation of cytoadherence to host receptors. PfPTP1 functions in a large complex of proteins and is required for linking of Maurer's clefts to the host actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Objective Sulphadoxine-pyrimethamine ( SP) is widely used as intermittent preventive treatment ( IPT) for malaria in pregnant women in Sub- Saharan Africa. There are reports of wide-spread SP resistance in countries where SP had once been used as a first-line treatment. It is unclear whether the development of SP resistance also affects countries where SP is mainly used in the context of IPT, as is the case in Burkina Faso. To assess the efficacy of SP-based IPT, we monitored the prevalence of SP conferring genetic mutations in the genes dhfr and dhps in Plasmodium falciparum populations in a rural area of Burkina Faso over a period of 13 years. Methods Molecular epidemiological study consisted of six consecutive cross-sectional surveys of rainy and dry seasons (2009-2012). Data from the rainy season in 2000 served as a baseline. Mutations in dhfr and dhps associated with SP resistance were analysed by pyrosequencing in 861 parasite-positive samples. Results The prevalence of the SP resistance conferring triple dhfr mutation 51I, 59R, 108N increased from 1.3% in the rainy season of 2000 to 35.3% in 2009, and 54.3% in 2011 ( P ≤ 0.001). Comparing rainy and dry seasons, we observed an increasing step-like pattern with higher prevalence of the dhfr triple mutant in the respective dry season compared with the preceding rainy season. The proportion of the dhps 437Gly mutation in the rainy season of 2000 was 53.2% and subsequently increased to 77.6% in 2009 ( P ≤ 0.001). Conclusion The increase in molecular markers linked with SP resistance jeopardises the efficacy of IPTp and the planned IPTi interventions in Burkina Faso, calling for careful monitoring of genotypic resistance markers and in vivo validation of IPT efficacy. [ABSTRACT FROM AUTHOR]

The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors. [ABSTRACT FROM AUTHOR]

Many intracellular pathogens remodel the actin of their host cells, and the human malaria parasite Plasmodium falciparum is no exception to this rule. The surprising finding is that several hemoglobinopathies that protect carriers from severe malaria may do so by interfering with host actin reorganization. Here we discuss our current understanding of actin remodeling in P. falciparum-infected erythrocytes, how hemoglobinopathies interfere with this process, and how impaired host actin remodeling affects the virulence of P. falciparum. [ABSTRACT FROM AUTHOR]

The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the Maurer's clefts with the host cell membrane and to which transport vesicles were attached. The actin cytoskeleton and the Maurer's clefts were aberrant in erythrocytes containing hemoglobin S or C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin polymerization in vitro and may account for the protective role in malaria. [ABSTRACT FROM AUTHOR]

Summary Resistance to quinoline antimalarial drugs has emerged in different parts of the world and involves sets of discrete mutational changes in pfcrt and pfmdr1 in the human malaria parasite Plasmodium falciparum. To better understand how the different polymorphic haplotypes of pfmdr1 and pfcrt contribute to drug resistance, we have conducted a linkage analysis in the F1 progeny of a genetic cross where we assess both the susceptibility and the amount of accumulation of chloroquine, amodiaquine, quinine and quinidine. Our data show that the different pfcrt and pfmdr1 haplotypes confer drug-specific responses which, depending on the drug, may affect drug accumulation or susceptibility or both. These findings suggest that PfCRT and PfMDR1 are carriers of antimalarial drugs, but that the interaction with a drug interferes with the carriers' natural transport function such that they are now themselves targets of these drugs. How well a mutant PfCRT and PfMDR1 type copes with its competing transport functions is determined by its specific sets of amino acid substitutions. [ABSTRACT FROM AUTHOR]

Abstract: Previously we described a series of 5-acylaminobenzophenones with considerable antimalarial activity. Unfortunately, most compounds also displayed high cytotoxicity resulting in low selectivity towards malaria parasites. Through the replacement of the 5-acylamino moiety by simple chlorine and further modifications of the 2-acylamino residue we could obtain inhibitors with improved selectivity towards malaria parasites combined with an acceptable reduction of antimalarial activity. [Copyright &y& Elsevier]

Abstract: Drug resistance represents a major obstacle in the radical control of malaria. Drug resistance can arise in many different ways, but recent developments highlight the importance of mutations in transporter molecules as being major contributors to drug resistance in the human malaria parasite Plasmodium falciparum. While approximately 2.5% of the P. falciparum genome encodes membrane transporters, this review concentrates on three transporters, namely the chloroquine resistance transporter PfCRT, the multi-drug resistance transporter 1 PfMDR1, and the multi-drug resistance-associated protein PfMRP, which have been strongly associated with resistance to the major antimalarial drugs. The studies that identified these entities as contributors to resistance, and the possible molecular mechanisms that can bring about this phenotype, are discussed. A deep understanding of the underpinning mechanisms, and of the structural specificities of the players themselves, is a necessary basis for the development of the new drugs that will be needed for the future armamentarium against malaria. [Copyright &y& Elsevier]

Resistance to chloroquine has been linked to polymorphisms within the pfcrt gene of the human malarial parasite Plasmodium falciparum. Here, we have investigated the prevalence of the pfcrt allele associated with chloroquine resistance in the peripheral blood and the placenta of pregnant women diagnosed with a P. falciparum infection. Our molecular epidemiological data show an unequal distribution with a significant under-representation of parasites carrying the mutated pfcrt allele in the placenta, as compared to the peripheral blood. In comparison, no differences were seen with regard to pfmdr1 polymorphisms of these parasites. Our data suggest a selective disadvantage of the polymorphic and a selective advantage of the wild-type pfcrt haplotype in the placenta, supporting the model that the human host provides various microenvironments that favor genetically distinct P. falciparum populations. [ABSTRACT FROM AUTHOR]

The digestive vacuole plays an important role in the pathophysiology of the human malaria parasite Plasmodium falciparum. It is a terminal degradation organelle involved in the proteolysis of the host erythrocyte's haemoglobin; it is the site of action of several antimalarial drugs and its membrane harbours transporters implicated in drug resistance. How the digestive vacuole recruits residential proteins is largely unknown. Here, we have investigated the mechanism underpinning trafficking of the chloroquine resistance transporter, PfCRT, to the digestive vacuolar membrane. Nested deletion analysis and site-directed mutagenesis identified threonine 416 as a functional residue for sorting PfCRT to its site of residence. Mass spectroscopy demonstrated that threonine 416 can be phosphorylated. Further phosphorylation was detected at serine 411. Our data establish PfCRT as a phosphoprotein and suggest that phosphorylation of threonine 416 is a possible deciding signal for the sorting of PfCRT to the digestive vacuolar membrane. [ABSTRACT FROM AUTHOR]

Abstract: Here, we describe a series of readily obtainable benzophenone derivatives with antimalarial and antitrypanosomal activity. The most active compounds display submicromolar activity against Plasmodium falciparum. Micromolar activity is obtained against Trypanosoma brucei. Main problem of the compounds is low selectivity. However, there are indications that separation of antimalarial and cytotoxic activity might by possible. In addition, some compounds inhibit human ABC transporter with nanomolar activity. [Copyright &y& Elsevier]

The human malaria parasite Plasmodium falciparum exports determinants of virulence and pathology to destinations within the host erythrocyte, including the erythrocyte cytoplasm, plasma membrane and membrane profiles of parasite origin termed Maurer’s clefts. Most of the exported proteins contain a conserved pentameric motif termed plasmodial export element (PEXEL)/vacuolar transfer signal (VTS) that functions as a cleavable sorting signal permitting export to the host erythrocyte. However, there are some exported proteins, such as the skeleton-binding protein 1 (PfSBP1) that lack the PEXEL/VTS motif and that are not N-terminally processed, suggesting the presence of alternative sorting signals and/or mechanisms. In this study, we have investigated trafficking of PfSBP1 to the Maurer’s clefts. Our data show that the transmembrane domain of PfSBP1 functions as an internal signal sequence for entry into the parasite’s secretory pathway and for transport to the parasite plasma membrane. Trafficking beyond the parasite’s plasma membrane required additional N-terminal domains, which are characterized by a high negative net charge. Biochemical data indicate that these domains affect the solubility and extraction profile, the orientation of the protein within the membrane and the subcellular localization. Our findings suggest new principles of protein export in P. falciparum-infected erythrocytes. [ABSTRACT FROM AUTHOR]

Resistance to several anti-malarial drugs has been associated with polymorphisms within the P-glycoprotein homologue (Pgh-1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh-1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasite's digestive vacuole. How polymorphisms within this transporter mediate alter anti-malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh-1 transports vinblastine, an established substrate of mammalian MDR1, and the anti-malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh-1 alter the substrate specificity for the anti-malarial drugs. Wild-type Pgh-1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh-1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh-1. Our data are discussed in terms of the model that Pgh-1-mediates, in a variant-specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question. [ABSTRACT FROM AUTHOR]

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite's food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. [ABSTRACT FROM AUTHOR]

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed. [ABSTRACT FROM AUTHOR]

The human malarial parasite Plasmodium falciparum exports proteins to destinations within its host erythrocyte, including cytosol, surface and membranous profiles of parasite origin termed Maurer's clefts. Although several of these exported proteins are determinants of pathology and virulence, the mechanisms and trafficking signals underpinning protein export are largely uncharacterized—particularly for exported transmembrane proteins. Here, we have investigated the signals mediating trafficking of STEVOR, a family of transmembrane proteins located at the Maurer's clefts and believed to play a role in antigenic variation. Our data show that, apart from a signal sequence, a minimum of two addition signals are required. This includes a host cell targeting signal for export to the host erythrocyte and a transmembrane domain for final sorting to Maurer's clefts. Biochemical studies indicate that STEVOR traverses the secretory pathway as an integral membrane protein. Our data suggest general principles for transport of transmembrane proteins to the Maurer's clefts and provide new insights into protein sorting and trafficking processes in P. falciparum. [ABSTRACT FROM AUTHOR]

Aims: This study aims to set priorities for anti-disease malaria vaccines by determining community preference in a hyperendemic area. Methods: A bidding game technique was used to elucidate willingness to pay in rural Burkina Faso and 2,326 adults were interviewed. Results: It is shown that there are significant differences between community preference for an anti-disease vaccine aimed at reducing pathology in pregnant women, and for a vaccine directed against childhood malaria. While the target population was willing to pay CFAfr 2101 for a vaccine against maternal malaria, its members were prepared to pay only CFAfr 1433 for a vaccine against childhood malaria. Conclusions: Whilst it is increasingly likely that anti-disease malaria vaccines will become available in the foreseeable future, lessons from the past suggest that a lack of acceptance and support from the intended recipients may lead to less than optimal compliance, and hence efficacy. For the planning of vaccine development and application strategies, it is therefore highly important to take community views into account. Here it is argued that such information could help researchers and funding agencies to set priorities for future vaccine research. [ABSTRACT FROM AUTHOR]

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum remains controversial. By investigating the kinetics of chloroquine accumulation under varying-trans conditions, we recently presented evidence for a saturable and energy-dependent chloroquine efflux system present in chloroquine resistant P. falciparum strains. Here, we further characterize the putative chloroquine efflux system by investigating its substrate specificity using a broad range of different antimalarial drugs. Our data show that preloading cells with amodiaquine, primaquine, quinacrine, quinine, and quinidine stimulates labeled chloroquine accumulation under varying-trans conditions, while mefloquine, halofantrine, artemisinin, and pyrimethamine do not induce this effect. In the reverse of the varying-trans procedure, we show that preloaded cold chloroquine can stimulate quinine accumulation. On the basis of these findings, we propose that the putative chloroquine efflux system is capable of transporting, in addition to chloroquine, structurally related quinoline and methoxyacridine antimalarial drugs. Verapamil and the calcium/calmodulin antagonist W7 abrogate stimulated chloroquine accumulation and energy-dependent chloroquine extrusion. Our data are consistent with a substrate specific and inhibitible drug efflux system being present in chloroquine resistant P. falciparum strains. [ABSTRACT FROM AUTHOR]