Eric Hanssen

Origin, composition, organization and function of the inner membrane complex of Plasmodium falciparum gametocytes

The most virulent of the human malaria parasites, Plasmodium falciparum, undergoes a remarkable morphological transformation as it prepares itself for sexual reproduction and transmission via mosquitoes. Indeed P. falciparum is named for the unique falciform or crescent shape of the mature sexual stages. Once the metamorphosis is completed, the mature gametocyte releases from sequestration sites and enters the circulation, thus making it accessible to feeding mosquitoes. Early ultrastructural studies showed that gametocyte elongation is driven by the assembly of a system of flattened cisternal membrane compartments underneath the parasite plasma membrane and a supporting network of microtubules. Here we describe the molecular composition and origin of the sub-pellicular membrane complex, and show that it is analogous to the inner membrane complex, an organelle with structural and motor functions that is well conserved across the apicomplexa. We identify novel crosslinking elements that might help stabilize the inner membrane complex during gametocyte development. We show that changes in gametocyte morphology are associated with an increase in cellular deformability and postulate that this enables the gametocytes to circulate in the bloodstream without being detected and removed by the mechanical filtering mechanisms in the spleen of the host.

A molecular nematic liquid crystalline material for high-performance organic photovoltaics

Solution-processed organic photovoltaic cells (OPVs) hold great promise to enable roll-to-roll printing of environmentally friendly, mechanically flexible and cost-effective photovoltaic devices. Nevertheless, many high-performing systems show best power conversion efficiencies (PCEs) with a thin active layer (thickness is ~100 nm) that is difficult to translate to roll-to-roll processing with high reproducibility. Here we report a new molecular donor, benzodithiophene terthiophene rhodanine (BTR), which exhibits good processability, nematic liquid crystalline behaviour and excellent optoelectronic properties. A maximum PCE of 9.3% is achieved under AM 1.5G solar irradiation, with fill factor reaching 77%, rarely achieved in solution-processed OPVs. Particularly promising is the fact that BTR-based devices with active layer thicknesses up to 400 nm can still afford high fill factor of ~70% and high PCE of ~8%. Together, the results suggest, with better device architectures for longer device lifetime, BTR is an ideal candidate for mass production of OPVs.

Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery. DOI: http://dx.doi.org/10.7554/eLife.03080.001

Prion-infected cells regulate the release of exosomes with distinct ultrastructural features

Exosomes are small membrane‐bound vesicles released from cells and found in vivo in most biological fluids. Functions reported for exosomes include cell–cell communication, roles in modulating immune responses, and roles in the transfer of pathogens such as prions. Here we investigated the molecular characteristics of the structure of exosomes that harbor prion infectivity to determine the native structure of exosomes and whether infected exosomes have a distinct structure. Cryo‐electron tomography revealed the previously unidentified ultrastructural detail of exosomes with high resolution. Exosomes were found to be naturally spherical in shape and to have a diverse population that varies in size and internal structure, such as differences in the number of membrane structures. Exosomes isolated from prion‐infected cells contained a significantly different population of exosomes with distinct structural features compared to control vesicles from mock‐infected cells. Exosomes are highly structured vesicles that can modify their structure on altering their protein cargo. This finding provides further insight into the role that the exosomal protein cargo plays on influencing the structure of the vesicles as well as highlighting the diversity of exosomes and their relationship to biological processes.—Coleman, B. M., Hanssen, E., Lawson, V. A., Hill, A. F. Prion‐infected cells regulate the release of exosomes with distinct ultrastructural features FASEB J. 26, 4160–4173 (2012). www.fasebj.org

Digestive-vacuole genesis and endocytic processes in the early intraerythrocytic stages of Plasmodium falciparum.

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of haemoglobin digestion and haem detoxification, and is the target of chloroquine and other antimalarials. The mechanisms for genesis of the digestive vacuole and transfer of haemoglobin from the host cytoplasm are still debated. Here, we use live-cell imaging and photobleaching to monitor the uptake of the pH-sensitive fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in ring-stage and trophozoite-stage parasites. We compare these results with electron tomography of serial sections of parasites at different stages of growth. We show that uptake of erythrocyte cytoplasm is initiated in mid-ring-stage parasites. The host cytoplasm is internalised via cytostome-derived invaginations and concentrated into several acidified peripheral structures. Haemoglobin digestion and haemozoin formation take place in these vesicles. The ring-stage parasites can adopt a deeply invaginated cup shape but do not take up haemoglobin via macropinocytosis. As the parasite matures, the haemozoin-containing compartments coalesce to form a single acidic digestive vacuole that is fed by haemoglobin-containing vesicles. There is also evidence for haemoglobin degradation in compartments outside the digestive vacuole. The work has implications for the stage specificity of quinoline and endoperoxide antimalarials.

Artemisinin and a Series of Novel Endoperoxide Antimalarials Exert Early Effects on Digestive Vacuole Morphology

ABSTRACT Artermisinin and its derivatives are now the mainstays of antimalarial treatment; however, their mechanism of action is only poorly understood. We report on the synthesis of a novel series of epoxy-endoperoxides that can be prepared in high yields from simple starting materials. Endoperoxides that are disubstituted with alkyl or benzyl side chains show efficient inhibition of the growth of both chloroquine-sensitive and -resistant strains of Plasmodium falciparum. A trans-epoxide with respect to the peroxide linkage increases the activity compared to that of its cis-epoxy counterpart or the parent endoperoxide. The novel endoperoxides do not show a strong interaction with artemisinin. We have compared the mechanism of action of the novel endoperoxides with that of artemisinin. Electron microscopy reveals that the novel endoperoxides cause the early accumulation of endocytic vesicles, while artemisinin causes the disruption of the digestive vacuole membrane. At longer incubation times artemisinin causes extensive loss of organellar structures, while the novel endoperoxides cause myelin body formation as well as the accumulation of endocytic vesicles. An early event following endoperoxide treatment is the redistribution of the pH-sensitive probe LysoSensor Blue from the digestive vacuole to punctate structures. By contrast, neither artemisinin nor the novel endoperoxides caused alterations in the morphology of the endoplasmic reticulum nor showed antagonistic antimalarial activity when they were used with thapsigargin. Analysis of rhodamine 123 uptake by P. falciparum suggests that disruption of the mitochondrial membrane potential occurs as a downstream effect rather than as an initiator of parasite killing. The data suggest that the digestive vacuole is an important initial site of endoperoxide antimalarial activity.

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum.

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer-Lamberts law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.

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 Maurers clefts, outside the confines of its own plasma membrane. The Maurers 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 Maurers 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 ∼25 nm. Individual 25 nm vesicle‐like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurers 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 Maurers clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurers clefts and parasite membranes and the host plasma membrane.

The role of solvent vapor annealing in highly efficient air-processed small molecule solar cells

We demonstrate highly-efficient, solution-processed small molecule solar cells with the best power conversion efficiency (PCE) of more than 5%. The active layer consists of a diketopyrrolopyrrole-based donor molecule (DPP(TBFu)2) and a fullerene derivative (PC71BM) that is spin cast and subsequently treated with solvent vapor annealing (SVA) in air. We find not all solvent vapors lead to the best PCE. Solvents of high vapor pressures and medium donor solubilities, such as tetrahydrofuran or carbon disulfide, are most suitable for SVA in the context of organic solar cell application. On the other hand, acceptor solubility plays an insignificant role in such a treatment. An active layer treated with ideal solvent vapors develops desirable phase separation in both lateral and vertical directions, as revealed by AFM, TEM and TEM tomography. The SVA also leads to enhanced hole mobility. We believe the fast SVA treatment performed in air is a viable way to tune the active layer morphology for printed solar cells.

The Maurer's cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes

During the intra‐erythrocytic development of Plasmodium falciparum, the parasite modifies the host cell surface by exporting proteins that interact with or insert into the erythrocyte membrane. These proteins include the principal mediator of cytoadherence, P. falciparum erythrocyte membrane protein 1 (PfEMP1). To implement these changes, the parasite establishes a protein‐trafficking system beyond its confines. Membrane‐bound structures called Maurers clefts are intermediate trafficking compartments for proteins destined for the host cell membrane. We disrupted the gene for the membrane‐associated histidine‐rich protein 1 (MAHRP1). MAHRP1 is not essential for parasite viability or Maurers cleft formation; however, in its absence, these organelles become disorganized in permeabilized cells. Maurers cleft‐resident proteins and transit cargo are exported normally in the absence of MAHRP1; however, the virulence determinant, PfEMP1, accumulates within the parasite, is depleted from the Maurers clefts and is not presented at the red blood cell surface. Complementation of the mutant parasites with mahrp1 led to the reappearance of PfEMP1 on the infected red blood cell surface, and binding studies show that PfEMP1‐mediated binding to CD36 is restored. These data suggest an important role of MAHRP1 in the translocation of PfEMP1 from the parasite to the host cell membrane.