Geometric constrains for detecting short actin filaments by cryogenic electron tomography

Polymerization of actin into filaments can push membranes forming extensions like filopodia or lamellipodia, which are important during processes such as cell motility and phagocytosis. Similarly, small organelles or pathogens can be moved by actin polymerization. Such actin filaments can be arranged in different patterns and are usually hundreds of nanometers in length as revealed by various electron microscopy approaches. Much shorter actin filaments are involved in the motility of apicomplexan parasites. However, these short filaments have to date not been visualized in intact cells. Here, we investigated Plasmodium sporozoites, the motile forms of the malaria parasite that are transmitted by the mosquito, using cryogenic electron tomography. We detected filopodia-like extensions of the plasma membrane and observed filamentous structures in the supra-alveolar space underneath the plasma membrane. However, these filaments could not be unambiguously assigned as actin filaments. In silico simulations of EM data collection and tomographic reconstruction identify the limits in revealing the filaments due to their length, concentration and orientation

Using green fluorescent malaria parasites to screen for permissive vector mosquitoes

BACKGROUND: The Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot. METHODS: P. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands. RESULTS: A single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection. CONCLUSION: Fluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed

Synergistic and Additive Effects of Epigallocatechin Gallate and Digitonin on Plasmodium Sporozoite Survival and Motility

BACKGROUND: Most medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to facilitate absorption of polar agents across cellular membranes. However, little is known about synergistic effects between such potential drug candidates and adjuvants. To probe for such effects, we tested the green tea compound epigallocatechin gallate (EGCG) and the membrane permeabilising digitonin on Plasmodium sporozoite motility and viability. METHODOLOGY/PRINCIPAL FINDINGS: Green fluorescent P. berghei sporozoites were imaged using a recently developed visual screening methodology. Motility and viability parameters were automatically analyzed and IC50 values were calculated, and the synergism of drug and adjuvant was assessed by the fractional inhibitory concentration index. Validating our visual screening procedure, we showed that sporozoite motility and liver cell infection is inhibited by EGCG at nontoxic concentrations. Digitonin synergistically increases the cytotoxicity of EGCG on sporozoite survival, but shows an additive effect on sporozoite motility. CONCLUSIONS/SIGNIFICANCE: We proved the feasibility of performing highly reliable visual screens for compounds against Plasmodium sporozoites. We thereby could show an advantage of administering mixtures of plant metabolites on inhibition of cell motility and survival. Although the effective concentration of both drugs is too high for use in malaria prophylaxis, the demonstration of a synergistic effect between two plant compounds could lead to new avenues in drug discovery

Gliding motility protein LIMP promotes optimal mosquito midgut traversal and infection by Plasmodium berghei

© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Substrate-dependent gliding motility is key to malaria transmission. It mediates host cell traversal, invasion and infection by Plasmodium and related apicomplexan parasites. The 110 amino acid-long cell surface protein LIMP is essential for P. berghei sporozoites where it is required for the invasion of the mosquito’s salivary glands and the liver cells of the rodent host. Here we define an additional role for LIMP during mosquito invasion by the ookinete. limp mRNA is provided as a translationally repressed mRNP (messenger ribonucleoprotein) by the female gametocyte and the protein translated in the ookinete. Parasites depleted of limp (Δlimp) develop ookinetes with apparent normal morphology and no defect during in vitro gliding motility, and yet display a pronounced reduction in oocyst numbers; compared to wildtype 82 % more Δlimp ookinetes remain within the mosquito blood meal explaining the decrease in oocysts. As in the sporozoite, LIMP exerts a profound role on ookinete infection of the mosquito.This study was supported by Fundação para a Ciência e a Tecnologia grants to GRM (PTDC/BIA-BCM/105610/2008 and PTDC/SAU-MIC/122082/2010) and JMS (SFRH/BD/63849/2009); the National Institutes of Allergy and Infectious Diseases to GRM (1R21AI139579-01A1); the Horizon 2020 Framework Programme Marie Sklodowska-Curie grant agreement No 660211 to SE; as well as a Human Frontier Science Program Grant (RGY/0071/2011) to FF. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The authors have no competing interests to declare.info:eu-repo/semantics/publishedVersio

Toolbox for in vivo imaging of host-parasite interactions at multiple scales

Animal models have for long been pivotal for parasitology research. Over the last few years, techniques such as intravital, optoacoustic and magnetic resonance imaging, optical projection tomography, and selective plane illumination microscopy developed promising potential for gaining insights into host-pathogen interactions by allowing different visualization forms in vivo and ex vivo. Advances including increased resolution, penetration depth, and acquisition speed, together with more complex image analysis methods, facilitate tackling biological problems previously impossible to study and/or quantify. Here we discuss advances and challenges in the in vivo imaging toolbox, which hold promising potential for the field of parasitology

A putative small solute transporter is responsible for the secretion of G377 and TRAP-containing secretory vesicles during Plasmodium gamete egress and sporozoite motility

Regulated protein secretion is required for malaria parasite life cycle progression and transmission between the mammalian host and mosquito vector. During transmission from the host to the vector, exocytosis of highly specialised secretory vesicles, such as osmiophilic bodies, is key to the dissolution of the red blood cell and parasitophorous vacuole membranes enabling gamete egress. The positioning of adhesins from the TRAP family, from micronemes to the sporozoite surface, is essential for gliding motility of the parasite and transmission from mosquito to mammalian host. Here we identify a conserved role for the putative pantothenate transporter PAT in Plasmodium berghei in vesicle fusion of two distinct classes of vesicles in gametocytes and sporozoites. PAT is a membrane component of osmiophilic bodies in gametocytes and micronemes in sporozoites. Despite normal formation and trafficking of osmiophilic bodies to the cell surface upon activation, PAT-deficient gametes fail to discharge their contents, remain intraerythrocytic and unavailable for fertilisation and further development in the mosquito. Sporozoites lacking PAT fail to secrete TRAP, are immotile and thus unable to infect the subsequent rodent host. Thus, P. berghei PAT appears to regulate exocytosis in two distinct populations of vesicles in two different life cycle forms rather than acting as pantothenic transporter during parasite transmission

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

Copyright © 2015, The Author(s). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/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.FF was supported by European Research Council (ERC-SG 281719) and the EU FP7 research network EVIMalar; JMS by a PhD fellowship grant SFRH/BD/63849/2009 from Fundação para a Ciência e a Tecnologia (FCT) and GRM by FCT grants PTDC/SAU-MIC/122082/2010 and PTDC/BIA-BCM/105610/2008.info:eu-repo/semantics/publishedVersio

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

Microstructured blood vessel surrogates reveal structural tropism of motile malaria parasites

Plasmodium sporozoites, the highly motile forms of the malaria parasite, are transmitted naturally by mosquitoes and traverse the skin to find, associate with, and enter blood capillaries. Research aimed at understanding how sporozoites select blood vessels is hampered by the lack of a suitable experimental system. Arrays of uniform cylindrical pillars can be used to study small cells moving in controlled environments. Here, an array system displaying a variety of pillars with different diameters and shapes is developed in order to investigate how Plasmodium sporozoites associate to the pillars as blood vessel surrogates. Investigating the association of sporozoites to pillars in arrays displaying pillars of different diameters reveals that the crescent-shaped parasites prefer to associate with and migrate around pillars with a similar curvature. This suggests that after transmission by a mosquito, malaria parasites may use a structural tropism to recognize blood capillaries in the dermis in order to gain access to the blood stream

Screening for potential prophylactics targeting sporozoite motility through the skin

Background: Anti-malarial compounds have not yet been identified that target the first obligatory step of infection in humans: the migration of Plasmodium sporozoites in the host dermis. This movement is essential to find and invade a blood vessel in order to be passively transported to the liver. Here, an imaging screening pipeline was established to screen for compounds capable of inhibiting extracellular sporozoites. Methods: Sporozoites expressing the green fluorescent protein were isolated from infected Anopheles mosquitoes, incubated with compounds from two libraries (MMV Malaria Box and a FDA-approved library) and imaged. Effects on in vitro motility or morphology were scored. In vivo efficacy of a candidate drug was investigated by treating mice ears with a gel prior to infectious mosquito bites. Motility was analysed by in vivo imaging and the progress of infection was monitored by daily blood smears. Results: Several compounds had a pronounced effect on in vitro sporozoite gliding or morphology. Notably, monensin sodium potently affected sporozoite movement while gramicidin S resulted in rounding up of sporozoites. However, pre-treatment of mice with a topical gel containing gramicidin did not reduce sporozoite motility and infection. Conclusions: This approach shows that it is possible to screen libraries for inhibitors of sporozoite motility and highlighted the paucity of compounds in currently available libraries that inhibit this initial step of a malaria infection. Screening of diverse libraries is suggested to identify more compounds that could serve as leads in developing ‘skin-based’ malaria prophylactics. Further, strategies need to be developed that will allow compounds to effectively penetrate the dermis and thereby prevent exit of sporozoites from the skin