Biology subjects

Treeck, M.

Publications and source records attributed to Treeck, M..

At least 19 recordsLinked to original sources

bioRxiv2026

FIKK1, a member of the FIKK kinase family, phosphorylates VAR2CSA and regulates adhesion of Plasmodium falciparum-infected erythrocytes to the placental receptor CSA

Plasmodium falciparum promotes the adhesion of infected erythrocytes (IEs) to host cells by extensively remodeling their surface. For this process, the parasite exports a large number of proteins to its host erythrocyte, including members of the P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1) adhesin family and members of the FIKK family. Several FIKK have been shown to play a role in P. falciparum virulence, notably affecting IES cell surface remodeling, rigidity and cytoadhesion. VAR2CSA, a member of the PfEMP1 adhesin family, is associated with IES sequestration in the placenta and has been shown to be phosphorylated. In view of the previously described importance of VAR2CSA phosphorylation, we investigated the role of FIKK1. We show that FIKK1 is capable of phosphorylating VAR2CSA in vitro, and that this phosphorylation increases the binding of recombinant VAR2CSA to the placental receptor chondroitin sulphate A (CSA). In an inducible transgenic cell line expressing HA-tagged FIKK1, immunofluorescence assays indicate that the kinase localizes to punctuated foci within Maurers Cleft, similarly to VAR2CSA. Rapamycin-induced knock out of FIKK1 reduces IEs cytoadhesion to CSA, even though levels of VAR2CSA are not affected. In vitro phosphorylation assays show that FIKK1 can phosphorylate recombinant DBL1-3 domains on several residues, including S429 and T934, previously implicated in in vitro binding and IES cytoadhesion to CSA. Taken together, these data support a model whereby FIKK1 contributes to placental malaria virulence through IEs sequestration mediated by VAR2CSA phosphorylation. Having no orthologs in mammals, this orphan kinase therefore represents an attractive target for the development of drugs against placental malaria. Author summarySequestration of Plasmodium falciparum infected erythrocytes (IEs) in the placenta is a hallmark of placental malaria and a major driver of adverse maternofetal outcomes. This process is mediated by VAR2CSA, a member of the P. falciparum Erythrocyte Membrane Protein (PfEMP1) family. VAR2CSA binds to chondroitin sulfate A (CSA) on the placental syncytium, facilitating IEs sequestration. In a previous study, we demonstrated that endogenous VAR2CSA is phosphorylated and identified specific phosphosites important for its cytoadhesive function. To elucidate the molecular mechanisms underlying these post-translational modifications, we examined the role of the P. falciparum FIKK1 kinase in VAR2CSA phosphorylation and its impact on IEs adhesion. Using a FIKK1::HA conditional knockout transgenic line, we found that FIKK1 deletion impairs IEs cytoadhesion, likely due to altered VAR2CSA phosphorylation. Importantly, both endogenous and recombinant FIKK1 interact with and phosphorylate the extracellular region of VAR2CSA. Furthermore, recombinant FIKK1 also enhances VAR2CSA binding to CSA in vitro and phosphorylates a residue previously identified as important for CSA binding and IEs adhesion. Collectively, these findings highlight a pivotal role for FIKK1 in placental adhesion and underscore the potential of targeting this kinase family for interventions against placental malaria.

biochemistry↗

bioRxiv2025

Physiological febrile heat stress increases cytoadhesion through increased protein trafficking of Plasmodium falciparum surface proteins into the red blood cell

Fever is a hallmark of malaria. Several studies have linked febrile temperatures to reduced parasite viability, but also to increased cytoadhesion, a key driver of pathology. However, different mechanisms have been proposed to cause changes in cytoadhesion and parasite sensitivity to heat. Here, we demonstrate that exposure of Plasmodium falciparum-infected red blood cells (iRBCs) to physiologically relevant febrile heat stress (39 {degrees}C), derived from patient data, enhances cytoadhesion through increased trafficking of the major virulence factor PfEMP1 to the iRBC surface. This phenomenon is not limited to PfEMP1 and common laboratory strains, as it extends to the surface nutrient channel PSAC in four clinical isolates of diverse geographic origin. The increased surface protein display occurs without changes in overall protein expression or parasite developmental progression. Using phosphoproteomics and proximity labelling, we find that elevated temperature also increases trafficking and phosphorylation of exported proteins into the RBC. Enhanced export is likely reliant on the presence of a transmembrane domain as shown by NanoLuc reporter assays. Collectively, our results indicate that febrile temperatures commonly experienced during infection can accelerate protein export, likely at the parasitophorous vacuole. This enhanced export following heat stress is relevant because increased cytoadhesion could influence disease severity through earlier iRBC sequestration and elevated bound parasite mass.

cell biology↗

bioRxiv2024

In vivo CRISPR screens identify GRA12 as a transcendent secreted virulence factor across Toxoplasma gondii strains and mouse subspecies

Toxoplasma gondii parasites exhibit extraordinary host promiscuity owing to over 250 putative secreted proteins that disrupt host cell functions, enabling parasite persistence. However, most of the known effector proteins are specific to Toxoplasma genotypes or hosts. To identify virulence factors that function across different parasite isolates and mouse strains that differ in susceptibility to infection, we performed systematic pooled in vivo CRISPR-Cas9 screens targeting the Toxoplasma secretome. We identified several proteins required for infection across parasite strains and mouse species, of which the dense granule protein 12 (GRA12) emerged as the most important effector protein during acute infection. GRA12 deletion in IFN{gamma}-activated macrophages results in collapsed parasitophorous vacuoles and increased host cell necrosis, which is partially rescued by inhibiting early parasite egress. GRA12 orthologues from related coccidian parasites, including Neospora caninum and Hammondia hammondi, complement Tg{Delta}GRA12 in vitro, suggesting a common mechanism of protection from immune clearance by their hosts.

microbiology↗

bioRxiv2024

Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-{gamma}-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While {Delta}MYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues {Delta}MYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell. SIGNIFICANCE STATEMENTPooled CRISPR screens are powerful tools to interrogate gene function in a high-throughput manner. Genes conferring fitness advantages or disadvantages upon disruption can be identified by sequencing. However, in Toxoplasma gondii pooled CRISPR screens in mice, fitness defects for some selected mutants drastically diverge from those observed in single-strain infections. Here, we show that a growth defect of a single Toxoplasma gene deletion mutant is rescued if co-infected with wildtype parasites. These results shine light on Toxoplasmas ability to subvert the host response beyond the infected cell, and highlight an important limitation of pooled CRISPR screens in mice. This limitation is probably encountered in CRISPR screens in general where paracrine effects occur.

microbiology↗

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

bioRxiv2023

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but not linked to sexual commitment

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines the variable proportions of MSPDBL2 positivity in schizonts does not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous HA-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different to those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection.

microbiology↗

bioRxiv2023

The substrate quality of CK2 target sites has a determinant role on their function and evolution

Most biological processes are regulated by peptide-recognition modules (PRMs) that bind to short linear motifs (SLiMs). Such interactions are rapidly reversible and often occur at low affinity. The protein kinase domain represents one such binding module, and known substrates may have full or only partial matches to the kinase recognition motif, a property known as substrate quality. However, it is not yet clear whether differences in substrate quality represent neutral variation along the phosphosite sequence or if these differences have functional consequences that are subject to selection. We explore this question in detail for the acidophilic kinase CK2. CK2 is well-characterised, clinically important, and a fundamental enzyme for many aspects of cell biology. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions whereas minimal substrates are phosphorylated at lower stoichiometries, are more dynamic during the cell cycle, and have regulatory functions. Optimal CK2 sites also tend to be older and more conserved than minimal sites, and evolutionary simulations indicate that the substrate quality of CK2 phosphosites is often tuned by selection. For intermediate target sites, increases or decreases to substrate quality may be deleterious, which we demonstrate experimentally for a CK2 substrate at the kinetochore. The results together suggest that minimal and optimal phosphosites are strongly differentiated in terms of their functional and evolutionary properties.

evolutionary biology↗

bioRxiv2023

Mapping host-microbe transcriptional interactions by dual perturb-seq

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonisation. Here, we combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA-sequencing to identify the molecular mediators of these transcriptional interactions, a method we term dual perturb-seq. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterised effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify a novel effector, TgSOS1, that is necessary for sustained host STAT6 signalling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a novel tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.

molecular biology↗

bioRxiv2023

PerTurboID: A targeted in situ method to measure changes in a local protein environment reveals the impact of kinase deletion on cytoadhesion in malaria causing parasites

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

cell biology↗

bioRxiv2023

Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma

Apicomplexan parasites use Ca2+-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca2+-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired microneme protein secretion, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle.

microbiology↗

bioRxiv2022

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma stimulated human cells

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFN{gamma}-activated human cells. Independent screens were carried out using two Toxoplasma strains which differ in virulence in mice, leading to the identification of effectors required for survival in IFN{gamma}-activated human cells. We identify the secreted protein GRA57 and two other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFN{gamma}-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFN{gamma} in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70 and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that deletion of this trimeric complex renders parasites hypersensitive to remaining ubiquitination, resulting in increased parasite clearance.

microbiology↗

bioRxiv2022

PfHDAC1 is an essential regulator of parasite asexual growth with its altered genomic occupancy and activity associated with artemisinin drug resistance in Plasmodium falciparum

Plasmodium falciparum is a deadly protozoan parasite and the causative agent of malaria, which accounts for close to 200 million cases and 400,000 deaths every year. It has been identified to possess a tightly regulated gene expression profile that is integrally linked to its timely development during the intraerythrocytic stage. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasites transcriptome. In this study, we characterize the solitary class I histone deacetylase PfHDAC1 and demonstrate that phosphorylation is required for its catalytic activity. PfHDAC1 binds to and regulates parasite genes responsible for housekeeping and stress-responsive functions. We show that PfHDAC1 activity in parasites is crucial for normal intraerythrocytic development and that its cellular abundance is correlated with parasitemia progression. We further show that PfHDAC1 has differential abundance and genomic occupancy in artemisinin drug-resistant vs sensitive parasites and that inhibition of its deacetylase activity can modulate the sensitivity of parasites to the drug. We also identify that artemisinin exposure can interfere with PfHDAC1 phosphorylation and its genomic occupancy. Collectively, our results demonstrate PfHDAC1 to be an important regulator of basic biological functions in parasites while also deterministic of responses to environmental stresses such as antimalarial drugs.

microbiology↗

bioRxiv2022

Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.

microbiology↗

bioRxiv2021

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in Toxoplasma gondii

Fundamental processes of obligate intracellular parasites, such as Toxoplasma gondii and Plasmodium falciparum, are controlled by a set of plant-like calcium dependent kinases (CDPKs), the conserved cAMP- and cGMP-dependent protein kinases (PKA and PKG), secondary messengers and lipid signalling. While some major components of the signalling networks have been identified, how these are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels to activate PKG or CDPKs, respectively. We show that both these inducers trigger near identical signalling pathways and provide evidence for a positive feedback loop involving CDPK3. We measure phospho- and lipid signalling in parasites treated with the Ca2+ ionophore A23187 in a sub-minute timecourse and show CDPK3-dependent regulation of diacylglycerol levels and increased phosphorylation of four phosphodiesterases (PDEs), suggesting their function in the feedback loop. Disruption of CDPK3 leads to elevated cAMP levels and inhibition of PKA signalling rescues the egress defect of {Delta}CDPK3 parasites treated with A23187. Biochemical analysis of the four PDEs identifies PDE2 as the only cAMP-specific PDE among these candidates, while the other PDEs are cGMP specific, two of which are inhibited by the predicted PDE inhibitor BIPPO. Conditional deletion of the four PDEs supports an important, but non-essential role for PDE1 and PDE2 in growth, with PDE2 controlling A23187-mediated egress. In summary we uncover a positive feedback loop that enhances signalling during egress and links several signalling pathways together.

microbiology↗

bioRxiv2020

GDV1 C-terminal truncation of 39 amino acids disrupts sexual commitment in Plasmodium falciparum

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasites life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase KO lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but both lines harbour the same mutation that leads to a truncation in the extreme C-terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line we show that the truncation in the GDV1 C-terminus does neither interfere with the nuclear import of GDV1 nor its interaction with HP1 in vitro, but appears important to sustain GDV1 protein levels and thereby sexual commitment. ImportanceTransmission of malaria causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood, to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the lifecycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C-terminus of the Gametocyte Development 1 protein (GDV1), which abrogates sexual development. The mutation destabilises the protein but not its interaction with its cognate binding partner HP1. This suggest an important role for the GDV1 C-terminus beyond trafficking and protein stability.

microbiology↗

bioRxiv2020

Gliding motility of Plasmodium merozoites

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate-dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which in turn propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, P. falciparum and P. knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a new model to study the glideosome and may facilitate the pursuit of new targets for malaria treatment. Significance statementPlasmodium malaria parasites use a unique substrate-dependent locomotion termed gliding motility to translocate through tissues and invade cells. Dogma has suggested that the small labile invasive stages that invade erythrocytes, merozoites, use this motility solely to penetrate target erythrocytes. Here we reveal that merozoites use gliding motility for translocation across host cells prior to invasion. This forms an important pre-invasion step that is powered by a conserved actomyosin motor and is regulated by a complex signaling pathway. This work fundamentally changes our understanding of the role of gliding motility and invasion in the blood and will have a significant impact on our understanding of blood stage host-pathogen interactions, parasite biology, and could have implications for vaccine development.

microbiology↗

bioRxiv2020

Differential protein phosphorylation affects the localisation of two secreted Toxoplasma proteins and is widespread during stage conversion

The intracellular parasite Toxoplasma gondii resides within a membrane bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma both secretes kinases and that numerous proteins are phosphorylated after secretion. Here we assess the role of phosphorylation of SFP1 and the related GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. GRA29 likely acts as a seed for SFP1 strand formation, and these structures can form independently of other Toxoplasma secreted proteins. We show that an unstructured region at the C-terminus of SFP1 and GRA29 is required for the formation of strands and that mimicking phosphorylation of this domain negatively regulates strand development. When tachyzoites convert to chronic stage bradyzoites, both proteins show a dispersed localisation throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analysis comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. These data support a model in which secreted kinases and phosphatases are important to dynamically regulate parasite secreted proteins during stage conversion. IMPORTANCEToxoplasma gondii is a common parasite that infects up to one third of the human population. Initially the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages the parasite lives within a membrane bound vacuole within the host cell, but in the chronic stage a durable cyst wall is synthesized that provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that phosphorylation plays an important role in their regulation. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

microbiology↗

bioRxiv2019

Global profiling of myristoylation in Toxoplasma gondii reveals key roles for lipidation in CDPK1 and MIC7 function

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase has been proposed as an attractive drug target in several pathogens. Functionally the myristate often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence also suggests additional regulatory roles for myristoylation on proteins. Here we describe the first global chemoproteomic screening of protein myristoylation in Toxoplasma gondii. Through quantitative mass spectrometry coupled with validated chemoproteomic tools, we identify 65 myristoylated proteins. We report functionally important myristoylation on the key signalling protein CDPK1 and, surprisingly, myristoylation of the microneme protein 7 (MIC7), a predicted type-I-transmembrane protein. We demonstrate that myristoylation of MIC7 is not important for the trafficking to micronemes, but appears to play a role in host cell invasion. This dataset represents a large fraction of the parasites myristoylated proteome and a prerequisite to investigate this modification in Toxoplasma.

microbiology↗