Possible roles of amyloids in malaria pathophysiology

Abstract

The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist Plasmodium spp., and of designing vaccines against chosen antigens found on the parasite's surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles' heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood-brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.

Keywords: amyloids; intrinsically unstructured proteins; malaria; prions.

Figure 1.. Electron micrographs of the fibrils formed by (A) MSP2 8–15 peptide, (B) MSP2 1–25 peptide and (C) recombinant full-length FC27 MSP2.

Peptides and protein were incubated in PBS at 1 mg/ml. Samples were negatively stained and viewed using transmission electron microscopy. The scale bar represents 100 nm. Reproduced with permission from [6] © European Peptide Society and John Wiley & Sons, Ltd. (2007).

Figure 2.. Longitudinal section of a Plasmodium knowlesi merozoite showing the bristly appearance of the cell coat which covers the entire surface.

In this example, the parasite is attached apically to a red cell by a few long thread-like projections. Image reprinted with kind permission from [10]. Springer Science+Business Media B.V.

Figure 3.. Prediction of amyloid-forming sequences in the MSP2 isoforms 3D7 and FC27. Highlighted in yellow are the sequences with strong consensus prediction according to FoldAmyloid, Tango, Waltz, Aggrescan and Zhang's method algorithms.

The arrowheads indicate trypsin cleaving sites.

Figure 4.. Atomic force microscope images of aggregates of MSP2-derived peptides (digested or not with trypsin) formed after incubation in PBS at 37°C for 2–3 weeks.

The images were obtained using a Dimension 3100 atomic force microscope (Veeco Instruments, Inc., CA, USA) and NP-S probes (Veeco) with nominal spring constants of 0.06 N/m to scan the samples in tapping mode in liquid at 0.5–1.0 Hz scan rates. Highly oriented pyrolytic graphite (NT-MDT Co., Moscow, Russia) was used as a substrate.

Figure 5.. Circular dichroism analysis of MSP2 (A) signal and (B) GPI anchor peptides, before and after trypsin digestion.

Figure 6.. Adhesion of parasitized red blood cells and of free Plasmodium falciparum parasites to an in vitro blood–brain barrier model.

HBMECs were initially expanded in a 75-cm² flask precoated with fibronectin. Plasmodium falciparum strain E8B was grown in vitro in group B human erythrocytes using previously described conditions [26]. For the selection of cytoadherent parasites, pRBCs were passed over human umbilical vein endothelial cells (HUVECs) in two selection rounds. Late trophozoite- and schizont-containing pRBCs were selected in 70% Percoll and seeded at approximately 2 × 10⁷ pRBCs/cm² on an HUVEC monolayer. The co-culture model of the BBB was adapted from a previously described protocol [27]. Briefly, rat astrocytes (RA) obtained from 2-day newborn rats were initially seeded at 60,000 cells/well onto a poly-D-lysine-coated plate. After 4 days, HBMECs were seeded at 16,500 cells/well onto collagen-coated 0.4 µm pore membrane inserts. This EC monolayer separated the system into an apical (bloodstream) and basolateral (brain tissue) compartments. The growth medium was changed every 3 days until cells reached confluence and tight junctions appeared. On the 9th HBMECs-RA co-culture day, pRBCs were purified in 70% Percoll, stained with wheat germ agglutinin-tetramethylrhodamine conjugate, and seeded at 1 × 10⁶ cells/well onto membrane inserts (apical compartment of the BBB model containing the EC layer). The system was co-cultured overnight at 37°C in a humidified atmosphere with 5% CO₂ (protocol adapted from [28]). The day after, HBMECs and pRBCs were fixed and stained for the analysis by confocal microscopy of the tight junction marker protein zonula occludens-1 (ZO-1) [29]. Cell nuclei were stained with DAPI, and pRBCs were specifically detected with a monoclonal antibody against schizont-infected RBCs [26]. Arrowheads in the Merge panel indicate individual P. falciparum merozoites.