Parasite microtubule arrays.

Microtubules are a key component of eukaryotic cell architecture. Regulation of the dynamic growth and shrinkage of microtubules gives cells their shape, allows cells to swim, and drives the separation of chromosomes. Parasites have developed intriguingly divergent biology, seemingly expanding upon and reinventing microtubule use in fascinating ways. These organisms affect life on the planet at scales that are often overlooked: there are likely more parasitic than free-living organisms on Earth, and they have a sizeable influence across ecosystems. As parasites can cause devastating diseases, this in turn drives evolutionary adaptations and species diversity. Parasites are varied, living in all environments and at all scales - from the tiny 2 µm single-celled Plasmodium merozoite that invades red blood cells to the 40 m long Tetragonoporus, a large intestinal tapeworm of whales. To survive in their various niches, parasites have undergone striking adaptations and developed complex life cycles, often involving two or more host species. This diversity is reflected at the cellular level, where unique molecular mechanisms, cytoskeletal structures and organellar compositions are found. Hence, the study of parasite cell biology provides a biological playground for understanding diversity and species diversification. It also facilitates the identification of specific targets to develop urgently needed therapeutics: for example, drugs targeting microtubules are used at large scale to treat intestinal worms and parasites that form tissue cysts in our livers and brains. Here, we discuss some of the curious microtubule arrays found in a small, select number of human-infecting, single-celled parasites of medical importance (Table 1). Our aim is to put a spotlight on distinctive molecular features in a field that promises exciting cell-biological discoveries with the potential for therapeutic breakthroughs.

Ultrastructural characterization of the tegument in protoscoleces of Echinococcus ortleppi.

Cystic echinococcosis is a globally distributed zoonosis caused by cestodes of the Echinococcus granulosus sensu lato (s.l.) complex, with Echinococcus ortleppi mainly involved in cattle infection. Protoscoleces show high developmental plasticity, being able to differentiate into either adult worms or metacestodes within definitive or intermediate hosts, respectively. Their outermost cellular layer is called the tegument, which is important in determining the infection outcome through its immunomodulating activities. Herein, we report an in-depth characterization of the tegument of E. ortleppi protoscoleces performed through a combination of scanning and transmission electron microscopy techniques. Using electron tomography, a three-dimensional reconstruction of the tegumental cellular territories was obtained, revealing a novel structure termed the 'tegumental vesicular body' (TVB). Vesicle-like structures, possibly involved in endocytic/exocytic routes, were found within the TVB as well as in the parasite glycocalyx, distal cytoplasm and close inner structures. Furthermore, parasite antigens (GST-1 and AgB) were unevenly localised within tegumental structures, with both being detected in vesicles found within the TBV. Finally, the presence of host (bovine) IgG was also assessed, suggesting a possible endocytic route in protoscoleces. Our data forms the basis for a better understanding of E. ortleppi and E. granulosus s.l. structural biology.

Linking murine resistance to secondary cystic echinococcosis with antibody responses targeting Echinococcus granulosus tegumental antigens.

Successful establishment of a parasite infection depends partially on the host intrinsic susceptibility to the pathogen. In cystic echinococcosis (CE), a zoonotic disease caused by the cestode parasite Echinococcus granulosus, the infection outcome in the murine model of secondary CE varies according to the mouse strain used. In this regard, intrinsic differences in susceptibility to the infection were previously reported for Balb/c and C57Bl/6 mice, being C57Bl/6 animals less permissive to secondary CE. Induction of parasite-specific antibodies has been suggested to play relevant roles in such susceptibility/resistance phenomena. Here, we report an in deep comparison of antibody responses induced in both mouse strains. Firstly, only C57Bl/6 mice were shown to induce specific-antibodies with efficient anti-parasite activities during early secondary CE. Then, through ImmunoTEM and Serological Proteome Analysis (SERPA), an evaluation of specific antibody responses targeting parasite tegumental antigens was performed. Both strategies showed that infected C57Bl/6 mice -unlike Balb/c animals- narrowed their IgG recognition repertoire against tegumental antigens, targeting fewer but potentially more relevant parasite components. In this sense, tegumental antigens recognition between Balb/c and C57Bl/6 mice, either by natural and/or induced antibodies, was analyzed through SERPA and MALDI-TOF/TOF studies. A total of 13 differentially recognized proteins (DRPs) uniquely targeted by antibodies from C57Bl/6 mice were successfully identified, wherein a subset of 7 DRPs were only recognized by infection-induced antibodies, suggesting their potential as natural protective antigens. In this regard, immunoinformatic analyses showed that such DRPs exhibited higher numbers of possible T cell epitopes towards the H-2-IAb haplotype, which is present in C57Bl/6 mice but absent in Balb/c animals. In summary, our results showed that the genetic predisposition to generate better T-dependent antibody responses against particular tegumental antigens might be a key factor influencing host susceptibility in the murine model of secondary CE.

An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens.

Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming. Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were coupled to double-stranded DNA probes with defined force thresholds for DNA unzipping. Receptor-mediated forces in the pN range are thereby semi-quantitatively converted into fluorescence signals, which can be detected by standard fluorescence microscopy at the resolution limit (~0.2 µm). The modular design of the assay allows to vary the presented ligands and the mechanical strength of the DNA probes, which provides a number of possibilities to probe the adhesion of different eukaryotic cell types and pathogens and is exemplified here with osteosarcoma cells and Plasmodium berghei Sporozoites.

Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection.

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed "clean linear B cell epitopes," and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.

Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable.

BACKGROUND: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite. METHODS: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH). RESULTS: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite. CONCLUSIONS: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.

Inter-subunit interactions drive divergent dynamics in mammalian and Plasmodium actin filaments.

Cell motility is essential for protozoan and metazoan organisms and typically relies on the dynamic turnover of actin filaments. In metazoans, monomeric actin polymerises into usually long and stable filaments, while some protozoans form only short and highly dynamic actin filaments. These different dynamics are partly due to the different sets of actin regulatory proteins and partly due to the sequence of actin itself. Here we probe the interactions of actin subunits within divergent actin filaments using a comparative dynamic molecular model and explore their functions using Plasmodium, the protozoan causing malaria, and mouse melanoma derived B16-F1 cells as model systems. Parasite actin tagged to a fluorescent protein (FP) did not incorporate into mammalian actin filaments, and rabbit actin-FP did not incorporate into parasite actin filaments. However, exchanging the most divergent region of actin subdomain 3 allowed such reciprocal incorporation. The exchange of a single amino acid residue in subdomain 2 (N41H) of Plasmodium actin markedly improved incorporation into mammalian filaments. In the parasite, modification of most subunit-subunit interaction sites was lethal, whereas changes in actin subdomains 1 and 4 reduced efficient parasite motility and hence mosquito organ penetration. The strong penetration defects could be rescued by overexpression of the actin filament regulator coronin. Through these comparative approaches we identified an essential and common contributor, subdomain 3, which drives the differential dynamic behaviour of two highly divergent eukaryotic actins in motile cells.

Immunization efficacy of cryopreserved genetically attenuated Plasmodium berghei sporozoites.

Malaria is transmitted through the injection of Plasmodium sporozoites into the skin by Anopheles mosquitoes. The parasites first replicate within the liver before infecting red blood cells, which leads to the symptoms of the disease. Experimental immunization with attenuated sporozoites that arrest their development in the liver has been extensively investigated in rodent models and humans. Recent technological advances have included the capacity to cryopreserve sporozoites for injection, which has enabled a series of controlled studies on human infection with sporozoites. Here, we used a cryopreservation protocol to test the efficiency of genetically attenuated cryopreserved sporozoites for immunization of mice in comparison with freshly isolated controls. This showed that cryopreserved sporozoites are highly viable as judged by their capacity to migrate in vitro but show only 20% efficiency in liver infection, which impacts their capacity to generate protection of animals in immunization experiments.

Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow.

Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito.

Tailored environments to study motile cells and pathogens.

Motile cells and pathogens migrate in complex environments and yet are mostly studied on simple 2D substrates. In order to mimic the diverse environments of motile cells, a set of assays including substrates of defined elasticity, microfluidics, micropatterns, organotypic cultures, and 3D gels have been developed. We briefly introduce these and then focus on the use of micropatterned pillar arrays, which help to bridge the gap between 2D and 3D. These structures are made from polydimethylsiloxane, a moldable plastic, and their use has revealed new insights into mechanoperception in Caenorhabditis elegans, gliding motility of Plasmodium, swimming of trypanosomes, and nuclear stability in cancer cells. These studies contributed to our understanding of how the environment influences the respective cell and inform on how the cells adapt to their natural surroundings on a cellular and molecular level.

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites.

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.

The Plasmodium palmitoyl-S-acyl-transferase DHHC2 is essential for ookinete morphogenesis and malaria transmission.

The post-translational addition of C-16 long chain fatty acids to protein cysteine residues is catalysed by palmitoyl-S-acyl-transferases (PAT) and affects the affinity of a modified protein for membranes and therefore its subcellular localisation. In apicomplexan parasites this reversible protein modification regulates numerous biological processes and specifically affects cell motility, and invasion of host cells by Plasmodium falciparum merozoites and Toxoplasma gondii tachyzoites. Using inhibitor studies we show here that palmitoylation is key to transformation of zygotes into ookinetes during initial mosquito infection with P. berghei. We identify DHHC2 as a unique PAT mediating ookinete formation and morphogenesis. Essential for life cycle progression in asexual blood stage parasites and thus refractory to gene deletion analyses, we used promoter swap (ps) methodology to maintain dhhc2 expression in asexual blood stages but down regulate expression in sexual stage parasites and during post-fertilization development of the zygote. The ps mutant showed normal gamete formation, fertilisation and DNA replication to tetraploid cells, but was characterised by a complete block in post-fertilisation development and ookinete formation. Our report highlights the crucial nature of the DHHC2 palmitoyl-S-acyltransferase for transmission of the malaria parasite to the mosquito vector through its essential role for ookinete morphogenesis.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development.

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

The alveolin IMC1h is required for normal ookinete and sporozoite motility behaviour and host colonisation in Plasmodium berghei.

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host.

Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes.

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites.

Microtubules are dynamic cytoskeletal structures important for cell division, polarity, and motility and are therefore major targets for anticancer and antiparasite drugs. In the invasive forms of apicomplexan parasites, which are highly polarized and often motile cells, exceptionally stable subpellicular microtubules determine the shape of the parasite, and serve as tracks for vesicle transport. We used cryoelectron tomography to image cytoplasmic structures in three dimensions within intact, rapidly frozen Plasmodium sporozoites. This approach revealed microtubule walls that are extended at the luminal side by an additional 3 nm compared to microtubules of mammalian cells. Fourier analysis revealed an 8-nm longitudinal periodicity of the luminal constituent, suggesting the presence of a molecule interacting with tubulin dimers. In silico generation and analysis of microtubule models confirmed this unexpected topology. Microtubules from extracted sporozoites and Toxoplasma gondii tachyzoites showed a similar density distribution, suggesting that the putative protein is conserved among Apicomplexa and serves to stabilize microtubules.

Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.

Local activation of Src at the plasma membrane by extracellular vaccinia virus results in a signalling cascade that acts to stimulate actin polymerization beneath the virus to enhance its cell-to-cell spread. Initiation of this signalling cascade involves Src-mediated phosphorylation of tyrosine 112 and 132 of the viral membrane protein A36R. Here we show that recruitment of Src is dependent on its myristoylation and an interaction with A36R upstream of tyrosine 112 and 132. We further show that Src, Fyn and Yes have unique specificities towards these tyrosine residues. Using cell lines deficient in Src, Fyn and Yes, we demonstrate that multiple Src family members can stimulate vaccinia-induced actin polymerization and also uncover a role for Abl family kinases. Additionally, Abl and Arg are able to phosphorylate A36R in vitro and are recruited to vaccinia-induced actin tails. The ability of multiple families of tyrosine kinases to directly phosphorylate A36R ensures robust cell-to-cell spread of vaccinia virus will occur under a variety of cellular conditions.