MAHRP-1, a novel Plasmodium falciparum histidine-rich protein,binds ferriprotoporphyrin IX and localizes to the Maurer's clefts

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.     

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.     

Solution behavior of hematin under acidic conditions and implications for its interactions with chloroquine

During the intraerythrocytic stage of its lifecycle, the malaria parasite digests host erythrocyte hemoglobin, producing free ferriprotoporhyrin IX (FP). Crystallization of FP into hemozoin is essential for its detoxification and is the target of quinoline antimalarials. To gain further insight into the mechanism of hemozoin formation and quinoline action we have studied the behavior of FP and related derivatives in 40% methanol in water at different concentrations across a broad pH range (2–12). The complex behavior of FP can be modeled by incorporating a pH-dependent dimerization constant that reflects the influence of the ionization state of the propionate groups on the level of self-association. The analysis reveals that aqua-ligated FP has a low propensity to self-associate and that the predominant self-associated species are homodimeric hydroxide-ligated FP and heterodimeric aqua/hydroxide-ligated FP. The latter is predicted to be the main self-associated species at the pH of the parasite digestive vacuole. The state of FP also affects its interaction with chloroquine, with maximum affinity under neutral conditions and a more than 1,000-fold decrease in affinity under acidic (pH 2) and basic (pH 12) conditions. First-derivative absorption spectra of the chloroquine–FP complex indicate that the high-affinity interaction requires the chloroquine ring in its neutral aminoquinoline form and this in turn requires at least one of the FP species in the complex to be aqua-ligated.     

Illuminating Plasmodium falciparum-infected red blood cells

The malaria parasite undergoes a remarkable series of morphological transformations, which underpin its life in both human and mosquito hosts. The advent of molecular transfection technology coupled with the ability to introduce fluorescent reporter proteins that faithfully track and expose the activities of parasite proteins has revolutionized our view of parasite cell biology. The greatest insights have been realized in the erythrocyte stages of Plasmodium falciparum. P. falciparum invades and remodels the human erythrocyte: it feeds on haemoglobin, grows and divides, and subverts the physiology of its hapless host. Fluorescent proteins have been employed to track and dissect each of these processes and have revealed details and exposed new paradigms.     

Cryo transmission X-ray imaging of the malaria parasite,P. falciparum

Cryo transmission X-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin.     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

Evaluation of an intact,an ACL-deficient,and a reconstructed human knee joint finite element model

The human knee joint has a three-dimensional geometry with multiple body articulations that produce complex mechanical responses under loads that occur in everyday life and sports activities. Understanding the complex mechanical interactions of these load-bearing structures is of use when the treatment of relevant diseases is evaluated and assisting devices are designed. The anterior cruciate ligament (ACL) in the knee is one of four main ligaments that connects the femur to the tibia and is often torn during sudden twisting motions, resulting in knee instability. The objective of this work is to study the mechanical behavior of the human knee joint and evaluate the differences in its response for three different states, i.e., intact, ACL-deficient, and surgically treated (reconstructed) knee. The finite element models corresponding to these states were developed. For the reconstructed model, a novel repair device was developed and patented by the author in previous work. Static load cases were applied, as have already been presented in a previous work, in order to compare the calculated results produced by the two models the ACL-deficient and the surgically reconstructed knee joint, under the exact same loading conditions. Displacements were calculated in different directions for the load cases studied and were found to be very close to those from previous modeling work and were in good agreement with experimental data presented in literature. The developed finite element model for both the intact and the ACL-deficient human knee joint is a reliable tool to study the kinematics of the human knee, as results of this study show. In addition, the reconstructed human knee joint model had kinematic behavior similar to the intact knee joint, showing that such reconstruction devices can restore human knee stability to an adequate extent.     

Whole cell imaging reveals novel modular features of the exomembrane system of the malaria parasite, Plasmodium falciparum

During its intra-erythrocytic development Plasmodium falciparum establishes a membrane network beyond its own limiting membrane in the cytoplasm of its host. These membrane structures play an important role in the trafficking of virulence proteins to the erythrocyte surface, however their ultrastructure is only partly defined and there is on-going debate regarding their origin, organisation and connectivity. We have used two whole cell imaging modalities to explore the topography of parasitised erythrocytes. Three-dimensional structured illumination microscopy provides resolution beyond the optical diffraction limit and permits analysis of fluorescently labelled whole cells. Immunoelectron tomography offers the possibility of high resolution imaging of individual ultrastructural features in a cellular context. Combined with serial sectioning and immunogold labelling, this technique permits precise mapping of whole cell architecture. We show that the P. falciparum exported secretory system comprises a series of modular units, comprising flattened cisternae, known as Maurer’s clefts, tubular connecting elements, two different vesicle populations and electron-dense structures that have fused with the erythrocyte membrane. The membrane network is not continuous, pointing to an important role for vesicle-mediated transport in the delivery of cargo to different destinations in the host cell.