Neurofilament light chain plasma levels are associated with area of brain damage in experimental cerebral malaria.

Neurofilament light chain (NfL), released during central nervous injury, has evolved as a powerful serum marker of disease severity in many neurological disorders, including infectious diseases. So far NfL has not been assessed in cerebral malaria in human or its rodent model experimental cerebral malaria (ECM), a disease that can lead to fatal brain edema or reversible brain edema. In this study we assessed if NfL serum levels can also grade disease severity in an ECM mouse model with reversible (n = 11) and irreversible edema (n = 10). Blood-brain-barrier disruption and brain volume were determined by magnetic resonance imaging. Neurofilament density volume as well as structural integrity were examined by electron microscopy in regions of most severe brain damage (olfactory bulb (OB), cortex and brainstem). NfL plasma levels in mice with irreversible edema (317.0 ± 45.01 pg/ml) or reversible edema (528.3 ± 125.4 pg/ml) were significantly increased compared to controls (103.4 ± 25.78 pg/ml) by three to five fold, but did not differ significantly in mice with reversible or irreversible edema. In both reversible and irreversible edema, the brain region most affected was the OB with highest level of blood-brain-barrier disruption and most pronounced decrease in neurofilament density volume, which correlated with NfL plasma levels (r = - 0.68, p = 0.045). In cortical and brainstem regions neurofilament density was only decreased in mice with irreversible edema and strongest in the brainstem. In reversible edema NfL plasma levels, MRI findings and neurofilament volume density normalized at 3 months' follow-up. In conclusion, NfL plasma levels are elevated during ECM confirming brain damage. However, NfL plasma levels fail short on reliably indicating on the final outcomes in the acute disease stage that could be either fatal or reversible. Increased levels of plasma NfL during the acute disease stage are thus likely driven by the anatomical location of brain damage, the olfactory bulb, a region that serves as cerebral draining pathway into the nasal lymphatics.

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.

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

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.

Evolutionarily distant I domains can functionally replace the essential ligand-binding domain of Plasmodium TRAP.

Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αß heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in Plasmodium and MIC2 in Toxoplasma. Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.

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 Sporozoite Motility on Flat Substrates.

Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

A suggested vital function for eIF-5A and dhs genes during murine malaria blood-stage infection.

The biological function of the post-translational modification hypusine in the eukaryotic initiation factor 5A (EIF-5A) in eukaryotes is still not understood. Hypusine is formed by two sequential enzymatic steps at a specific lysine residue in the precursor protein EIF-5A. One important biological function of EIF-5A which was recently identified is the translation of polyproline-rich mRNA, suggesting its biological relevance in a variety of biological processes. Hypusinated eIF-5A controls the proliferation of cancer cells and inflammatory processes in malaria. It was shown that pharmacological inhibition of the enzymes involved in this pathway, deoxyhypusine synthase (DHS) and the deoxyhypusine hydroxylase (DOHH), arrested the growth of malaria parasites. Down-regulation of both the malarial eIF-5A and dhs genes by in vitro and in vivo silencing led to decreased transcript levels and protein expression and, in turn, to low parasitemia, confirming a critical role of hypusination in eIF-5A for proliferation in Plasmodium. To further investigate whether eIF-5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei. Full disruption of both genes was not successful; instead parasites harboring the episome for eIF-5A and dhs genes were obtained, suggesting that these genes may perform vital functions during the pathogenic blood cell stage. Next, a knock-in strategy was pursued for both endogenous genes eIF-5A and dhs from P. berghei. The latter resulted in viable recombinant parasites, strengthening the observation that they might be essential for proliferation during asexual development of the malaria parasite.

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.

Protective efficacy and safety of liver stage attenuated malaria parasites.

During the clinically silent liver stage of a Plasmodium infection the parasite replicates from a single sporozoite into thousands of merozoites. Infection of humans and rodents with large numbers of sporozoites that arrest their development within the liver can cause sterile protection from subsequent infections. Disruption of genes essential for liver stage development of rodent malaria parasites has yielded a number of attenuated parasite strains. A key question to this end is how increased attenuation relates to vaccine efficacy. Here, we generated rodent malaria parasite lines that arrest during liver stage development and probed the impact of multiple gene deletions on attenuation and protective efficacy. In contrast to P. berghei strain ANKA LISP2(-) or uis3(-) single knockout parasites, which occasionally caused breakthrough infections, the double mutant lacking both genes was completely attenuated even when high numbers of sporozoites were administered. However, different vaccination protocols showed that LISP2(-) parasites protected better than uis3(-) and double mutants. Hence, deletion of several genes can yield increased safety but might come at the cost of protective efficacy.

Chemical attenuation of Plasmodium in the liver modulates severe malaria disease progression.

Cerebral malaria is one of the most severe complications of malaria disease, attributed to a complicated series of immune reactions in the host. The syndrome is marked by inflammatory immune responses, margination of leukocytes, and parasitized erythrocytes in cerebral vessels leading to breakdown of the blood-brain barrier. We show that chemical attenuation of the parasite at the very early, clinically silent liver stage suppresses parasite development, delays the time until parasites establish blood-stage infection, and provokes an altered host immune response, modifying immunopathogenesis and protecting from cerebral disease. The early response is proinflammatory and cell mediated, with increased T cell activation in the liver and spleen, and greater numbers of effector T cells, cytokine-secreting T cells, and proliferating, proinflammatory cytokine-producing T cells. Dendritic cell numbers, T cell activation, and infiltration of CD8(+) T cells to the brain are decreased later in infection, possibly mediated by the anti-inflammatory cytokine IL-10. Strikingly, protection can be transferred to naive animals by adoptive transfer of lymphocytes from the spleen at very early times of infection. Our data suggest that a subpopulation belonging to CD8(+) T cells as early as day 2 postinfection is responsible for protection. These data indicate that liver stage-directed early immune responses can moderate the overall downstream host immune response and modulate severe malaria outcome.

In vitro activity of wALADin benzimidazoles against different life cycle stages of Plasmodium parasites.

wALADin1 benzimidazoles are specific inhibitors of δ-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 µM, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates.

AAV8-mediated in vivo overexpression of miR-155 enhances the protective capacity of genetically attenuated malarial parasites.

Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.

Novel type II fatty acid biosynthesis (FAS II) inhibitors as multistage antimalarial agents.

Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood- and pre-erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver-stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood-stage parasite growth with IC(50) values of 1.7 and 3.0 µM and lead to a more prominent developmental attenuation of liver-stage parasites than the gold-standard drug, primaquine.

Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.

Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.

Actin regulation in the malaria parasite.

Many intracellular pathogens hijack host cell actin or its regulators for cell-to-cell spreading. In marked contrast, apicomplexan parasites, obligate intracellular, single cell eukaryotes that are phylogenetically older than the last common ancestor of animals and plants, employ their own actin cytoskeleton for active motility through tissues and invasion of host cells. A hallmark of actin-based motility of the malaria parasite is a minimal set of proteins that potentially regulate microfilament dynamics. An intriguing feature of the Plasmodium motor machinery is the virtual absence of elongated filamentous actin in vivo. Despite this unusual actin regulation sporozoites, the transmission stages that are injected into the mammalian host by Anopheles mosquitoes, display fast (1-3 µm/s) extracellular motility. Experimental genetics and analysis of recombinant proteins have recently contributed to clarify some of the cellular roles of apicomplexan actin monomer- and filament-binding proteins in parasite life cycle progression. These studies established that the malaria parasite employs multiple proteins that bind actin to form pools of readily polymerizable monomers, a prerequisite for fast formation of actin polymers. The motile extracellular stages of Plasmodium parasites are an excellent in vivo model system for functional characterization of actin regulation in single cell eukaryotes.

Crystallization and preliminary structural characterization of the two actin-depolymerization factors of the malaria parasite.

The malaria parasite Plasmodium depends on its actin-based motor system for motility and host-cell invasion. Actin-depolymerization factors are important regulatory proteins that affect the rate of actin turnover. Plasmodium has two actin-depolymerization factors which seem to have different functions and display low sequence homology to the higher eukaryotic family members. Plasmodium actin-depolymerization factors 1 and 2 have been crystallized. The crystals diffracted X-rays to maximum resolutions of 2.0 and 2.1 A and belonged to space groups P3(1)21 or P3(2)21, with unit-cell parameters a = b = 68.8, c = 76.0 A, and P2(1)2(1)2, with unit-cell parameters a = 111.6, b = 57.9, c = 40.5 A, respectively, indicating the presence of one or two molecules per asymmetric unit in both cases.

Structure and function of a G-actin sequestering protein with a vital role in malaria oocyst development inside the mosquito vector.

Cyclase-associated proteins (CAPs) are evolutionary conserved G-actin-binding proteins that regulate microfilament turnover. CAPs have a modular structure consisting of an N-terminal adenylate cyclase binding domain, a central proline-rich segment, and a C-terminal actin binding domain. Protozoan parasites of the phylum Apicomplexa, such as Cryptosporidium and the malaria parasite Plasmodium, express small CAP orthologs with homology to the C-terminal actin binding domain (C-CAP). Here, we demonstrate by reverse genetics that C-CAP is dispensable for the pathogenic Plasmodium blood stages. However, c-cap(-) parasites display a complete defect in oocyst development in the insect vector. By trans-species complementation we show that the Cryptosporidium parvum ortholog complements the Plasmodium gene functions. Purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization. The crystal structure of C. parvum C-CAP shows two monomers with a right-handed beta-helical fold intercalated at their C termini to form the putative physiological dimer. Our results reveal a specific vital role for an apicomplexan G-actin-binding protein during sporogony, the parasite replication phase that precedes formation of malaria transmission stages. This study also exemplifies how Plasmodium reverse genetics combined with biochemical and structural analyses of orthologous proteins can offer a fast track toward systematic gene characterization in apicomplexan parasites.

Identification of a phosphotransferase system of Escherichia coli required for growth on N-acetylmuramic acid.

We report here that wild-type Escherichia coli grows on N-acetylmuramic acid (MurNAc) as the sole source of carbon and energy. Analysis of mutants defective in N-acetylglucosamine (GlcNAc) catabolism revealed that the catabolic pathway for MurNAc merges into the GlcNAc pathway on the level of GlcNAc 6-phosphate. Furthermore, analysis of mutants defective in components of the phosphotransferase system (PTS) revealed that a PTS is essential for growth on MurNAc. However, neither the glucose-, mannose/glucosamine-, nor GlcNAc-specific PTS (PtsG, ManXYZ, and NagE, respectively) was found to be necessary. Instead, we identified a gene at 55 min on the E. coli chromosome that is responsible for MurNAc uptake and growth. It encodes a single polypeptide consisting of the EIIB and C domains of a so-far-uncharacterized PTS that was named murP. MurP lacks an EIIA domain and was found to require the activity of the crr-encoded enzyme IIA-glucose (EIIA(Glc)), a component of the major glucose transport system for growth on MurNAc. murP deletion mutants were unable to grow on MurNAc as the sole source of carbon; however, growth was rescued by providing murP in trans expressed from an isopropylthiogalactopyranoside-inducible plasmid. A functional His(6) fusion of MurP was constructed, isolated from membranes, and identified as a polypeptide with an apparent molecular mass of 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Close homologs of MurP were identified in the genome of several bacteria, and we believe that these organisms might also be able to utilize MurNAc.