239 research outputs found
Identification of Toxoplasma gondii effector proteins by pooled CRISPR-Cas9 knockout screening
No full textToxoplasma gondii is an intracellular eukaryotic pathogen of global clinical significance. Secretion of effector proteins by T. gondii into a host cell is critical for infection. At least two hundred putative effector proteins are secreted into the host cell, of which the vast majority are uncharacterised. In this work, I used CRISPR-Cas9 knockout screening in T. gondii to identify effector proteins involved in parasite evasion of host immune responses.
From a screen of T. gondii mutants in a mouse model of infection, I identified genes that confer parasite fitness in vivo. I found that the first-identified rhoptry protein of T. gondii, ROP1, is necessary for parasite virulence and is important for evasion of the cell-autonomous, interferon-gamma-stimulated immune response in both murine and human macrophages.
Then, I adapted CRISPR-Cas9 tools in T. gondii to enable pooled knockout screening with a single-cell, dual host-parasite transcriptome readout. This method, which I term dual perturb-seq, enables identification of parasite effectors that modulate host cell transcription in a high-throughput and unbiased manner. Using dual perturb-seq and additional cellular assays, I show that the dense granule protein GRA59 contributes to export of other dense granule effectors from the parasitophorous vacuole to the host cell. I also identify a rhoptry protein, newly named TgSOS1, that is required for sustained parasite-induced STAT6 signalling in the host cell and M2 polarisation of infected macrophages.
Together, these genetic screens and the characterisation of newly identified effector proteins advance the understanding of how T. gondii manipulates the host cell and evades host immune responses.Open Acces
Functional characterisation of the FIKK kinase family of Plasmodium falciparum
Get PDFKey to P. falciparum virulence is its capacity to remodel the host erythrocyte. Infected erythrocytes become rigid and cytoadhere to the vascular endothelium leading to the disease symptoms and preventing their filtration by the spleen. Unlike other human- infecting Plasmodium species, P. falciparum exports a family of 18 FIKK kinases into the host cell.
Here, a conditional knockout strategy based on the DiCre/LoxPint technology was used to study 4 FIKK kinases (FIKK4.1, FIKK7.1, FIKK10.1 and FIKK11) and identify their potential targets by quantitative phosphoproteome analysis. The deletion of FIKK4.1 led to a significant reduction in the phosphorylation of host cytoskeletal proteins and parasite proteins involved in remodelling. The characterisation of FIKK4.1 KO parasites confirmed its role both in the rigidification of the infected erythrocytes and in the trafficking of the adherence-mediating virulence factor PfEMP1 to the host cell surface.
Additionally, recombinant versions of several FIKK kinase domains were used to identify potential pan-FIKK inhibitors. When tested in vitro, these compounds showed activity on both P. falciparum and P. knowlesi, raising concerns regarding their specificity. A whole genome sequencing on drug-resistant parasites did not allow to identify additional targets. Moreover, it was shown that the compounds were not active on the FIKK kinases in culture due to the high intra-erythrocytic ATP concentration.
Using the recombinant FIKK kinase domains it was also shown that FIKK kinases possess distinct substrate specificity. Whereas most of them conserved the ancestral basophilicity, some evolved to phosphorylate preferentially acidic motifs. Strikingly, FIKK13 was found to be a tyrosine kinase, a feature supposed to be absent in Plasmodium. Finally, by studying the FIKK kinases from another Plasmodium species closely related to P. falciparum, it was shown that FIKK kinases substrate specificity is conserved across species of the Laverania clade.Open Acces
Functional characterisation of cyclase-associated protein (CAP) in Toxoplasma gondii
Get PDFToxoplasma gondii progression through the lytic cycle depends on a divergent actin and an array of associated unconventional myosin motors. These acto-myosin systems power mechanical processes, such as active invasion and egress of host cells, cell division and organellar trafficking. How actin-associated proteins regulate the organisation and turnover of the actin filaments to support these diverse processes is poorly understood. Cyclase-associated protein (CAP) is an actin-binding protein conserved across eukaryotes. We show that Toxoplasma CAP has a unique extension that is not present in most other Apicomplexa, giving rise to two isoforms with distinct subcellular localisations: one is localised at the parasite apex while the other is cytosolic. Here we investigated the role of CAP in Toxoplasma biology using a new generation of RH ∆ku80∆hxgprt DiCre parasites where loss of DiCre activity is prevented. Conditional knockout of CAP led to significant defects in motility, invasion, active egress, dense granule trafficking, daughter cell orientation, juxtanuclear accumulation of actin and cell-cell communication but only modest defects in synchronicity of division and no defect in replication of the apicoplast. Despite displaying phenotypes closely resembling the actin knockout, CAP is dispensable for in vitro culture. Strikingly, CAP knockout in the type I RH parasite strain does not affect in vivo virulence but results in complete attenuation of the type II Pru strain. 3D electron microscopy reveals that loss of CAP results in a defect in formation of a normal central residual body, but parasites remain connected within the vacuole. This dissociates synchronicity of division and parasite rosetting and reveals that establishment and maintenance of the residual body may be more complex than previously thought. These results highlight the different spatial requirements for F-actin dynamics in Toxoplasma that depend, in part, on CAP function.Open Acces
Identification and characterisation of Toxoplasma gondii effectors mediating parasite virulence in human cells
No full textToxoplasma gondii is an intracellular eukaryotic parasite that chronically infects
approximately one third of the global human population. To establish an infection,
Toxoplasma must initially avoid clearance by disarming the human immune system.
To do this, Toxoplasma secretes over two hundred effector proteins, which
collectively reshape the intracellular environment and resist innate immune
responses. Previous efforts to characterise Toxoplasma secreted effectors largely
centered on their role in the murine host. However, innate immune control of
Toxoplasma has significantly diverged between humans and mice, therefore
effector functions are not always conserved between the two hosts. Relatively
little is known about which secreted effectors are required for Toxoplasma to
evade the human immune response.
In this work I used targeted CRISPR screening of Toxoplasma secreted effectors to
identify which are required for survival in interferon-gamma-activated human cells.
CRISPR screens were carried out using two Toxoplasma strains that differ in
virulence in mice. From these screens I found that a complex of secreted dense
granule proteins (GRA57, GRA70, and GRA71) resists interferon-gamma-induced
parasite clearance in human fibroblasts. This complex was conversely dispensable
for parasite survival in mouse fibroblasts, suggesting it may have species-specific
functions. Additionally, I found that multiple components of the Toxoplasma
effector export machinery are required for immune resistance in human and mouse
fibroblasts. I next investigated the function of the GRA57, GRA70, and GRA71
complex, and ruled out roles in effector export, transcriptional modulation,
tryptophan acquisition or formation of the parasite vacuole. I instead found that
GRA57, GRA70, and GRA71 knockout parasite vacuoles are less ubiquitinated by
the host cell, but this is uncoupled from their function in immune resistance.
Together these results advance our previously limited understanding of which
Toxoplasma effector proteins are required for the parasite to establish human
infections.Open Acces
Functional characterisation of a putative amino acid transporter in toxoplasma gondii
Get PDFTo establish infection and disseminate within its host, the obligate intracellular parasite,
Toxoplasma gondii, must undergo cycles of invasion, replication, and egress from the host
cell. These recurring cycles rely upon a network of finely tuned signalling pathways that
allow the parasite to sense and respond to its environment. Phosphoproteomic analysis
of Calcium Dependent Protein Kinase 3 (TgCDPK3), a key moderator of these processes,
has allowed for the study of the proteins acting downstream of this kinase. Intriguingly, the
phosphoproteome revealed several proteins involved in metabolism, including the
branched chain amino acid dehydrogenase (BCKDH). This revealed a potential link
between TgCDPK3 and metabolic processes. Three candidates identified from the
TgCDPK3 phosphoproteome analysis were chosen for functional characterisation. These
were a lipid-binding protein (phosphorylated lipid binding protein; PLBP), a hypothetical
protein (putative TgCDPK3-phosphorylated protein; PCPP), and a putative branched
chain amino acid (BCAA) transporter (ApiAT5-3; Apicomplexan amino acid transporter 5-
3). As TgCDPK3 was hypothesised to mediate BCKDH phosphorylation, potentially
regulating BCAA metabolism, the ApiAT5-3 became the main focus of this study. Ectopictagging
demonstrated that all three candidates localise to the parasite periphery, the same
subcellular location as TgCDPK3. Upon conditional knockout (cKO) of ApiAT5-3, growth
was completely ablated. Complementation of these cKOs with an ectopic copy of the WT
gene rescued this phenotype. A combination of ectopic expression in Xenopus laevis and
metabolomic analysis of the cKOs revealed this protein to be a transporter of the essential
amino acid tyrosine, and not BCAAs. Mutation of the TgCDPK3-dependent
phosphorylation site, serine 56, revealed that it is important for parasite fitness. However,
phosphorylation of this site alone is not essential for tyrosine import into the parasite.
Together this study advances our understanding of the TgCDPK3 signalling pathway and
the way in which T. gondii scavenges nutrients from its host.Open Acces
Transport and function of adhesive proteins of the malaria pathogen plasmodium falciparum (Welch, 1897)
No full textMalaria ist eine oft tödlich verlaufende Erkrankung, die durch einen Einzeller der Gattung Plasmodium hervorgerufen wird. Die Pathologie im Menschen wird hauptsächlich durch die intrazelluläre Lebensweise des Erregers in den roten Blutkörperchen, den Erythrozyten, hervorgerufen. Die invasive Form des Parasiten, der Merozoit, invadiert den Erythrozyten und bildet bis zu 32 Tochterzellen. Diese werden durch Ruptur der Wirtszelle entlassen und können neue Erythrozyten invadieren. Im apikalen Bereich der Merozoiten befinden sich sekretorische Organellen (Rhoptrien und Mikronemen), die während der Invasion Proteine auf die Merozoitenoberfläche oder in den interzellulären Spalt zwischen Erreger und Wirtszelle sekretieren. Wie diese, für die Invasion wichtigen Proteine, in die sekretorischen Organellen transportiert werden ist nicht bekannt.
Im Rahmen dieser Arbeit sollte erforscht werden, welchen Einfluss die zytoplasmatischen Domänen von ausgesuchten Typ I Transmembranproteinen der Mikronemen auf den zielgerichteten Transport in die Organellen und für die Invasion von Erythrozyten hat.
Durch Deletionen des kodierenden Bereiches der zytoplasmatischen Domänen einiger Mikronemenproteine konnte festgestellt werden, dass weder die Proteine der „Erythrocyte Binding Antigen“ Familie (EBA-175, EBA-140, EBA-181), noch das „Apical Membrane Antigen-1“ (AMA-1) von zytoplasmatischen Sequenzmotiven zielgerichtet in die Mikronemen dirigiert werden. Vielmehr konnte anhand von EBA-175 gezeigt werden, dass der Transport über eine konservierte, Cystein-reiche, luminale Domäne vermittelt wird. Dies wurde durch die Verwendung von episomal exprimierten Minigenen ermittelt, die unterschiedliche Domänenkombinationen von EBA-175 als GFP-Fusionsprotein im Parasiten exprimieren. Zusätzlich konnte mit verschiedenen Promotoren gezeigt werden, dass eine zeitlich korrekte Expression der Mikronemenproteine innerhalb der asexuellen Blutphase eine Voraussetzung für den korrekten Transport darstellt.
Das für die Invasion essentielle AMA-1 wurde als GFP-Fusionsprotein in Parasiten exprimiert, um dessen Lokalisation, Dynamik und Funktion im Invasionsprozess zu analysieren. AMA-1 wird kurz vor der Invasion auf die Oberfläche von Merozoiten verteilt, wobei eine apikale Konzentration beobachtet werden kann. Wie diese beiden Populationen physisch und funktionell im Zusammenhang stehen ist nicht bekannt. Mit Hilfe von FLIP (Fluorescence Loss in Photobleaching) und FRAP (Fluorescence Recovery After Photobleaching) Analysen konnten zwei AMA-1 Populationen auf der Oberfläche der Merozoiten unterschieden werden: i) Eine periphere, mobile Fraktion, die an der Reorientierung des Merozoiten beteiligt sein könnte und ii) eine apikale, weniger mobile Fraktion, die an der Bildung einer intimen Zell-Zellverbindung (moving junction) beteiligt sein könnte. Durch ein synthetisches Peptid, welches spezifisch an eine hydrophobe Tasche in der AMA-1 Ektodomäne bestimmter Parasiten-Isolate wie 3D7 (nicht aber an W2mef) bindet und die Reinvasion von 3D7-Parasiten in Erythrozyten spezifisch inhibiert, konnte durch Lebendvideomikroskopie die Inhibition der Invasion in Echtzeit dargestellt werden. Dabei zeigte sich, dass die Blockierung der hydrophoben Tasche von AMA-1 nicht die initiale Bindung oder Reorientierung an den Erythrozyten beeinflusst, aber das aktive Eindringen in die Wirtszelle verhindert, was auf eine Funktion bei der „moving junction“ Ausbildung kurz vor der Invasion hindeutet.
Auf dieser Basis wurde ein Komplementationsassay etabliert, bei dem in 3D7-Parasiten episomal W2mef-AMA-1 exprimiert wurde. Das ermöglichte erstmals die funktionelle Analyse von AMA-1 auf molekularer Basis, da episomal exprimiertes W2mef-AMA-1 die Funktion von endogenem 3D7-AMA-1 komplementieren kann. Dies konnte in Reinvasionsassays quantifiziert werden. Durch die Einführung von gezielten Mutationen in die zytoplasmatische Domäne des episomalen W2mef-AMA-1 konnten direkt die Auswirkungen der Mutationen an der Komplementationsfähigkeit gemessen werden. So führte die Deletion sowie die Substitution verschiedener konservierter Aminosäuren inklusive 6 putativer Phosphorylierungsstellen in der zytoplasmatischen Domäne zur kompletten, funktionellen Inaktivierung von AMA-1, obwohl der Transport in die Mikronemen nicht beeinträchtigt war. Die Phosphorylierung des essentiellen AMA-1 Proteins könnte sich als eine Achillesferse des Parasiten herausstellen. Die Identifikation und Charakterisierung der verantwortlichen Kinase(n) eröffnet damit die Möglichkeit einer neuen, innovativen Strategie zur Bekämpfung der Malaria
Functional analysis of kinases in the malaria parasite Plasmodium falciparum
Get PDFDespite massive control efforts and extensive successes in reducing the malaria burden in the past decades, malaria still causes more than 400’000 deaths worldwide each year with the highest toll on African children under the age of five. The protozoan parasite Plasmodium falciparum is responsible for the majority of disease burden and deaths and its complex life cycle represents a challenge in disease control and treatment. During the intraerythrocytic asexual cycle, a subset of parasites undergoes sexual commitment and their progeny develop into gametocytes, the parasite form transmissible from humans to Anopheles vectors. Extensive research has identified that a drop in the level of the host lipid lysophosphatidylcholine is perceived by the parasite and induces sexual commitment by activating the expression of the transcription factor PfAP2-G, the master regulator of sexual conversion.
In this PhD project, I aimed at elucidating a potential upstream molecular pathway that induces expression of PfAP2-G. In more detail, using a PfAP2-G reporter cell line and other techniques to study sexual commitment, we investigated the involvement of nine P. falciparum kinases in this putative signalling pathway. We therefore generated a variety of transgenic parasite lines including kinase knockout as well as conditional knockdown, overexpression and inducible knockout mutants. However, investigating the effect of increased and decreased kinase expression levels did not conclusively identify any of these kinases as pathway component involved in sexual commitment signalling.
Nevertheless, having generated a variety of transgenic cell lines, I further studied the effect of changes in kinase expression levels on asexual and sexual intraerythrocytic parasite development. Interestingly, I identified that while the mitogen-activated protein kinase (MAPK) PfMAP-2 is dispensable for asexual parasite development, it is essential for male gametogenesis. Furthermore, we confirmed the essential role of the casein kinase 2 catalytic subunit PfCK2α in erythrocyte invasion by merozoites and likely also asexual parasite development. In addition, we discovered that PfCK2α is indispensable for sexual development and to my knowledge, this is the first kinase identified as being essential for gametocyte maturation. Finally, I studied the function of PfPKAc, the catalytic subunit of the cAMP-dependent protein kinase A. I confirmed that PfPKAc is required for asexual parasite growth due to its importance in erythrocyte invasion. I further showed that conditional overexpression of PfPKAc is lethal for intraerythrocytic asexual parasite development. Interestingly, however, selection of parasites tolerant to PfPKAc overexpression was possible. All six independently obtained survivor populations, but none of the unselected overexpression-sensitive mother clones, carried mutations in the putative serine/threonine kinase Pf3D7_1121900. We identified Pf3D7_1121900 as the P. falciparum orthologue of the 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). In model eukaryotes, the PDK1 kinase is known to phosphorylate and activate various AGC kinases including PKA. Using targeted mutagenesis, I was able to show the essential role for PfPDK1-dependent phosphorylation of PfPKAc. This phosphorylation seems to be crucial for PfPKAc activity in P. falciparum.
The present PhD thesis describes new insights into the function of three essential kinases in asexual and sexual development of P. falciparum. Hence, this work broadens our knowledge on kinase function and regulation in malaria parasites in general and provides new potential antimalarial drug targets. Finally, the tools developed during this work will provide important resources for future research on sexual commitment und protein function in P. falciparum
Label-Based Mass Spectrometry Approaches for Robust Quantification of the Phosphoproteome and Total Proteome in Toxoplasma gondii
No full text
Reconsidering the Investment-Profit Nexus in Finance-Led Economies: an ARDL-Based Approach
Get PDFA simple Post Keynesian growth model is developed, in which financial variables are explicitly taken into account. Different possible accumulation regimes are derived with respect to changes of these variables. Several variants of an investment function are estimated econometrically. The ARDL-based approach proposed by Pesaran et al. (2001) is argued to be superior for this purpose to the traditional cointegration approach. The econometric results are discussed with respect to a remarkable phenomenon that can be observed for some important OECD countries since the early 1980s: accumulation has generally been declining while profit rates have shown a tendency to rise. The author concentrates on one potential explanation of this phenomenon which is particularly relevant for the USA and relies on the hypothesis of a high propensity to consume out of capital income. The paper also gives an alternative explanation of the so-called "New Economy boom" in the USA at the end of the 1990s.Investment, Profitability, Financialisation, Time Series Econometrics.
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