No abstract

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next generation anti-malarial agents.

Exported proteins of the malaria parasite Plasmodium falciparum interact with proteins of the erythrocyte membrane and induce substantial changes in the morphology, physiology and function of the host cell. These changes underlie the pathology that is responsible for the deaths of 1-2 million children every year due to malaria infections. The advent of molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, has increased our understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided us with insights into the functions of the parasite protein exportome. We review these developments, focusing on parasite proteins that interact with the erythrocyte membrane skeleton or that promote delivery of the major virulence protein, PfEMP1, to the erythrocyte membrane.

Background: The carbon metabolism of the blood stages of Plasmodium falciparum, comprising rapidly dividing asexual stages and non-dividing gametocytes, is thought to be highly streamlined, with glycolysis providing most of the cellular ATP. However, these parasitic stages express all the enzymes needed for a canonical mitochondrial tricarboxylic acid (TCA) cycle, and it was recently proposed that they may catabolize glutamine via an atypical branched TCA cycle. Whether these stages catabolize glucose in the TCA cycle and what is the functional significance of mitochondrial metabolism remains unresolved. Results: We reassessed the central carbon metabolism of P. falciparum asexual and sexual blood stages, by metabolically labeling each stage with 13 C-glucose and 13 C-glutamine, and analyzing isotopic enrichment in key pathways using mass spectrometry. In contrast to previous findings, we found that carbon skeletons derived from both glucose and glutamine are catabolized in a canonical oxidative TCA cycle in both the asexual and sexual blood stages. Flux of glucose carbon skeletons into the TCA cycle is low in the asexual blood stages, with glutamine providing most of the carbon skeletons, but increases dramatically in the gametocyte stages. Increased glucose catabolism in the gametocyte TCA cycle was associated with increased glucose uptake, suggesting that the energy requirements of this stage are high. Significantly, whereas chemical inhibition of the TCA cycle had little effect on the growth or viability of asexual stages, inhibition of the gametocyte TCA cycle led to arrested development and death.

Artemisinin and its derivatives (collectively referred to as ARTs) rapidly reduce the parasite burden in Plasmodium falciparum infections, and antimalarial control is highly dependent on ART combination therapies (ACTs). Decreased sensitivity to ARTs is emerging, making it critically important to understand the mechanism of action of ARTs. Here we demonstrate that dihydroartemisinin (DHA), the clinically relevant ART, kills parasites via a two-pronged mechanism, causing protein damage, and compromising parasite proteasome function. The consequent accumulation of proteasome substrates, i.e., unfolded/damaged and polyubiquitinated proteins, activates the ER stress response and underpins DHA-mediated killing. Specific inhibitors of the proteasome cause a similar build-up of polyubiquitinated proteins, leading to parasite killing. Blocking protein synthesis with a translation inhibitor or inhibiting the ubiquitin-activating enzyme, E1, reduces the level of damaged, polyubiquitinated proteins, alleviates the stress response, and dramatically antagonizes DHA activity.

SummaryThe Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of antigenically diverse proteins is expressed on the surface of human erythrocytes infected with the malaria parasite P. falciparum , and mediates cytoadherence to the host vascular endothelium. In this report, we show that export of PfEMP1 is slow and inefficient as it takes several hours to traffic newly synthesized proteins to the erythrocyte membrane. Upon removal by trypsin treatment, the surface-exposed population of PfEMP1 is not replenished during subsequent culture indicating that there is no cycling of PfEMP1 between the erythrocyte surface and an intracellular compartment. The role of Maurer's clefts as an intermediate sorting compartment in trafficking of PfEMP1 was investigated using immunoelectron microscopy and proteolytic digestion of streptolysin O-permeabilized parasitized erythrocytes. We show that PfEMP1 is inserted into the Maurer's cleft membrane with the Cterminal domain exposed to the erythrocyte cytoplasm, whereas the N-terminal domain is buried inside the cleft. Transfer of PfEMP1 to the erythrocyte surface appears to involve electron-lucent extensions of the Maurer's clefts. Thus, we have delineated some important aspects of the unusual trafficking mechanism for delivery of this critical parasite virulence factor to the erythrocyte surface.

Enabling patients to deal with their spiritual needs through affirmative relationships with health professionals may improve quality of life and reduce use of health resources. Further research to explore the relationship between spiritual distress and health service utilization is indicated.

SummaryThe malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite-infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/ phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di-and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono-, di-and triacylglycerol suspensions promote b b b b -haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol-rich and cholesterol-poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.