Cytoskeleton Organization in Formation and Motility of Apicomplexan Parasites.

Apicomplexan parasites are a group of eukaryotic protozoans with diverse biology that have affected human health like no other group of parasites. These obligate intracellular parasites rely on their cytoskeletal structures for giving them form, enabling them to replicate in unique ways and to migrate across tissue barriers. Recent progress in transgenesis and imaging tools allowed detailed insights into the components making up and regulating the actin and microtubule cytoskeleton as well as the alveolate-specific intermediate filament-like cytoskeletal network. These studies revealed interesting details that deviate from the cell biology of canonical model organisms. Here we review the latest developments in the field and point to a number of open questions covering the most experimentally tractable parasites: Plasmodium, the causative agent of malaria; Toxoplasma gondii, the causative agent of toxoplasmosis; and Cryptosporidium, a major cause of diarrhea.

Overcoming the egress block of Plasmodium sporozoites expressing fluorescently tagged circumsporozoite protein.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

Putative prefoldin complex subunit 5 of Plasmodium berghei is crucial for microtubule formation and parasite development in the mosquito.

Plasmodium sporozoite development in and egress from oocysts in the Anopheles mosquito remains largely enigmatic. In a previously performed high-throughput knockout screen, the putative subunit 5 of the prefoldin complex (PbPCS5, PBANKA_0920100) was identified as essential for parasite development during mosquito and liver stage development. Here we generated and analyzed a PbPCS5 knockout parasite line during its development in the mosquito. Interestingly, PbPCS5 deletion does not significantly affect oocyst formation but leads to a growth defect resulting in aberrantly shaped sporozoites. Sporozoites produced in the absence of PbPCS5 were thinner, markedly elongated, and did, in most cases, not contain a nucleus. Sporozoites contained fewer subpellicular microtubules, which reached deep into the sporoblast during sporogony where they contacted and indented nuclei. These aberrantly shaped sporozoites did not reach the salivary glands, and we, therefore, conclude that PbPCS5 is essential for sporogony and the life cycle progression of the parasite during its mosquito stage.

Clearing of hemozoin crystals in malaria parasites enables whole-cell STED microscopy.

Malaria is a devastating mosquito-borne parasitic disease that manifests when Plasmodium parasites replicate within red blood cells. During the development within the red blood cell, the parasite digests hemoglobin and crystalizes the otherwise toxic heme. The resulting hemozoin crystals limit imaging by STED nanoscopy owing to their high light-absorbing capacity, which leads to immediate cell destruction upon contact with the laser. Here, we establish CUBIC-P-based clearing of hemozoin crystals, enabling whole-cell STED nanoscopy of parasites within red blood cells. Hemozoin-cleared infected red blood cells could reliably be stained with antibodies, and hence proteins in the hemozoin-containing digestive vacuole membrane, as well as in secretory vesicles of gametocytes, could be imaged at high resolution. Thus, this process is a valuable tool to study and understand parasite biology and the potential molecular mechanisms mediating drug resistance. This article has an associated First Person interview with the first author of the paper.

Conformational change of Plasmodium TRAP is essential for sporozoite migration and transmission.

Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.

Divergent Plasmodium actin residues are essential for filament localization, mosquito salivary gland invasion and malaria transmission.

Actin is one of the most conserved and ubiquitous proteins in eukaryotes. Its sequence has been highly conserved for its monomers to self-assemble into filaments that mediate essential cell functions such as trafficking, cell shape and motility. The malaria-causing parasite, Plasmodium, expresses a highly sequence divergent actin that is critical for its rapid motility at different stages within its mammalian and mosquito hosts. Each of Plasmodium actin's four subdomains have divergent regions compared to canonical vertebrate actins. We previously identified subdomains 2 and 3 as providing critical contributions for parasite actin function as these regions could not be replaced by subdomains of vertebrate actins. Here we probed the contributions of individual divergent amino acid residues in these subdomains on parasite motility and progression. Non-lethal changes in these subdomains did not affect parasite development in the mammalian host but strongly affected progression through the mosquito with striking differences in transmission to and through the insect. Live visualization of actin filaments showed that divergent amino acid residues in subdomains 2 and 4 enhanced localization associated with filaments, while those in subdomain 3 negatively affected actin filaments. This suggests that finely tuned actin dynamics are essential for efficient organ entry in the mosquito vector affecting malaria transmission. This work provides residue level insight on the fundamental requirements of actin in highly motile cells.

Apicomplexans: A conoid ring unites them all.

Apicomplexan parasites are defined by complex apical structures, which are necessary for interaction with incredibly diverse host cells. Two studies now amend a long-standing paradigm by showing conservation of an essential ring structure in the entire phylum.

Phosphorylation of myosin A regulates gliding motility and is essential for Plasmodium transmission.

Malaria-causing parasites rely on an actin-myosin-based motor for the invasion of different host cells and tissue traversal in mosquitoes and vertebrates. The unusual myosin A of Plasmodium spp. has a unique N-terminal extension, which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19. Here, using the rodent-infecting P. berghei we show that phosphorylation of serine 19 increases ookinete but not sporozoite motility and is essential for efficient transmission of Plasmodium by mosquitoes as S19A mutants show defects in mosquito salivary gland entry. S19A along with E6R mutations slow ookinetes and salivary gland sporozoites in both 2D and 3D environments. In contrast to data from purified proteins, both E6R and S19D mutations lower force generation by sporozoites. Our data show that the phosphorylation cycle of S19 influences parasite migration and force generation and is critical for optimal migration of parasites during transmission from and to the mosquito.

Plasmodium sporozoite disintegration during skin passage limits malaria parasite transmission.

During transmission of malaria-causing parasites from mosquitoes to mammals, Plasmodium sporozoites migrate rapidly in the skin to search for a blood vessel. The high migratory speed and narrow passages taken by the parasites suggest considerable strain on the sporozoites to maintain their shape. Here, we show that the membrane-associated protein, concavin, is important for the maintenance of the Plasmodium sporozoite shape inside salivary glands of mosquitoes and during migration in the skin. Concavin-GFP localizes at the cytoplasmic periphery and concavin(-) sporozoites progressively round up upon entry of salivary glands. Rounded concavin(-) sporozoites fail to pass through the narrow salivary ducts and are rarely ejected by mosquitoes, while normally shaped concavin(-) sporozoites are transmitted. Strikingly, motile concavin(-) sporozoites disintegrate while migrating through the skin leading to parasite arrest or death and decreased transmission efficiency. Collectively, we suggest that concavin contributes to cell shape maintenance by riveting the plasma membrane to the subtending inner membrane complex. Interfering with cell shape maintenance pathways might hence provide a new strategy to prevent a malaria infection.

Proximity-dependent biotinylation approaches to study apicomplexan biology.

In the last 10 years, proximity-dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from specific protein complexes to entire compartments. This is achieved by using enzymes such as BirA* and APEX that are fused to proteins of interest and biotinylate proteins in their proximity. PDB techniques are now also increasingly used in apicomplexan parasites. In this review, we first give an overview of the main PDB approaches and how they compare with other techniques that address similar questions. PDB is particularly valuable to detect weak or transient protein associations under physiological conditions and to study cellular structures that are difficult to purify or have a poorly understood protein composition. We also highlight new developments such as novel smaller or faster-acting enzyme variants and conditional PDB approaches, providing improvements in both temporal and spatial resolution which may offer broader application possibilities useful in apicomplexan research. In the second part, we review work using PDB techniques in apicomplexan parasites and how this expanded our knowledge about these medically important parasites.

Microtubule number and length determine cellular shape and function in Plasmodium.

Microtubules are cytoskeletal filaments essential for many cellular processes, including establishment and maintenance of polarity, intracellular transport, division and migration. In most metazoan cells, the number and length of microtubules are highly variable, while they can be precisely defined in some protozoan organisms. However, in either case the significance of these two key parameters for cells is not known. Here, we quantitatively studied the impact of modulating microtubule number and length in Plasmodium, the protozoan parasite causing malaria. Using a gene deletion and replacement strategy targeting one out of two α-tubulin genes, we show that chromosome segregation proceeds in the oocysts even in the absence of microtubules. However, fewer and shorter microtubules severely impaired the formation, motility and infectivity of Plasmodium sporozoites, the forms transmitted by the mosquito, which usually contain 16 microtubules. We found that α-tubulin expression levels directly determined the number of microtubules, suggesting a high nucleation barrier as supported by a mathematical model. Infectious sporozoites were only formed in parasite lines featuring at least 10 microtubules, while parasites with 9 or fewer microtubules failed to transmit.

A function of profilin in force generation during malaria parasite motility that is independent of actin binding.

During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.

SPOT: a web-tool enabling swift profiling of transcriptomes.

The increasing number of single cell and bulk RNAseq datasets describing complex gene expression profiles in different organisms, organs or cell types calls for an intuitive tool allowing rapid comparative analysis. Here, we present Swift Profiling Of Transcriptomes (SPOT) as a web tool that allows not only differential expression analysis but also fast ranking of genes fitting transcription profiles of interest. Based on a heuristic approach the spot algorithm ranks the genes according to their proximity to the user-defined gene expression profile of interest. The best hits are visualized as a table, bar chart or dot plot and can be exported as an Excel file. While the tool is generally applicable, we tested it on RNAseq data from malaria parasites that undergo multiple stage transformations during their complex life cycle as well as on data from multiple human organs during development and cell lines infected by SARS-CoV-2. SPOT should enable non-bioinformaticians to easily analyse their own and any available dataset. AVAILABILITY AND IMPLEMENTATION: SPOT is freely available for (academic) use at: https://frischknechtlab.shinyapps.io/SPOT/ and https://github.com/EliasFarr/SPOT. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

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.

Limited Plasmodium sporozoite gliding motility in the absence of TRAP family adhesins.

BACKGROUND: Plasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated. METHODS: Four transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive-negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice. RESULTS: The major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement. CONCLUSIONS: The individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility.

A synthetic promoter for multi-stage expression to probe complementary functions of Plasmodium adhesins.

Gene expression of malaria parasites is mediated by the apicomplexan Apetala2 (ApiAP2) transcription factor family. Different ApiAP2s control gene expression at distinct stages in the complex life cycle of the parasite, ensuring timely expression of stage-specific genes. ApiAP2s recognize short cis-regulatory elements that are enriched in the upstream/promoter region of their target genes. This should, in principle, allow the generation of 'synthetic' promoters that drive gene expression at desired stages of the Plasmodium life cycle. Here we test this concept by combining cis-regulatory elements of two genes expressed successively within the mosquito part of the life cycle. Our tailored 'synthetic' promoters, named Spooki 1.0 and Spooki 2.0, activate gene expression in early and late mosquito stages, as shown by the expression of a fluorescent reporter. We used these promoters to address the specific functionality of two related adhesins that are exclusively expressed either during the early or late mosquito stage. By modifying the expression profile of both adhesins in absence of their counterpart we were able to test for complementary functions in gliding and invasion. We discuss the possible advantages and drawbacks of our approach.This article has an associated First Person interview with the first author of the paper.

Intravital microscopy: Imaging host-parasite interactions in the brain.

Intravital fluorescence microscopy (IVM) is a powerful technique for imaging multiple organs, including the brain of living mice and rats. It enables the direct visualisation of cells in situ providing a real-life view of biological processes that in vitro systems cannot. In addition, to the technological advances in microscopy over the last decade, there have been supporting innovations in data storage and analytical packages that enable the visualisation and analysis of large data sets. Here, we review the advantages and limitations of techniques predominantly used for brain IVM, including thinned skull windows, open skull cortical windows, and a miniaturised optical system based on microendoscopic probes that can be inserted into deep tissues. Further, we explore the relevance of these techniques for the field of parasitology. Several protozoan infections are associated with neurological symptoms including Plasmodium spp., Toxoplasma spp., and Trypanosoma spp. IVM has led to crucial findings on these parasite species, which are discussed in detail in this review.

Intravital imaging of host-parasite interactions in skin and adipose tissues.

Intravital microscopy allows the visualisation of how pathogens interact with host cells and tissues in living animals in real time. This method has enabled key advances in our understanding of host-parasite interactions under physiological conditions. A combination of genetics, microscopy techniques, and image analysis have recently facilitated the understanding of biological phenomena in living animals at cellular and subcellular resolution. In this review, we summarise findings achieved by intravital microscopy of the skin and adipose tissues upon infection with various parasites, and we present a view into possible future applications of this method.