Special thanks to Dr. Peter Bradley for the antibody to detect the T. gondii mitochondria. The work was supported by National Institute of Health Grants AI072028 and AI107431 to D.G.M and a generous donation to New York Medical College for the study of tropical medicine.

The authors have no competing financial interests.

Die beschriebenen Protokoll stellt eine nicht voreingenommen Ansatz, der die Aktivierung von Maus-Knochenmark stammenden Makrophagen und zukunfts Genetik T. isolieren verwendet gondii-Mutanten mit Defekt in der Fähigkeit, die Aktivierung von infizierten Makrophagen zu überleben. Der Phänotyp der Mutanten nach Makrophagenaktivierung fällt in der Regel in eine von zwei Kategorien: 1) Die Parasiten scheinen intakt, aber nicht über den 1. Parasiten pro PV replizieren; 2) Die Parasiten erscheinen abgeb...

Toxoplasma gondii (T. gondii) ist ein obligat intrazelluläre, Protozoen-Erreger. Es ist der Erreger der Toxoplasmose, ein Gesundheitsrisiko bei immungeschwächten Individuen. Es ist auch das Modellsystem für andere apicomplexan Krankheitserreger, die Menschen, einschließlich Cryptosporidium und Cyclospora infizieren. Toxoplasmose ist am häufigsten durch den Verzehr von Nahrung oder Wasser mit dem bradyzoite oder Oozysten Stadium des Parasiten verunreinigt erworben. Nach der Einnahme konvertieren diese Stufen auf die Tachyzoiten Stadium des Parasiten, die in Wirtszellen repliziert und verbreitet systemisch. T-Zellen, IFN-γ und, in geringerem Maße, Stickstoffmonoxid 1-4, sind wichtig für die Kontrolle der Infektion jedoch nicht zur Eliminierung der Erkrankung, als Anteil der Tachyzoiten umwandeln zum bradyzoite Stufe, die innerhalb Gewebezysten geschützt was zu einem langlebigen chronischen Infektion. In der Tat gibt es keine wirksame Therapie gegen die chronische Zyste sTage der Erkrankung. Schwere Toxoplasmose ist meist durch die Reaktivierung der persistierenden Infektion mit dem bradyzoite Stadium des Parasiten Konvertierung zurück in die schnell zu replizieren Tachyzoiten Phase charakteristisch für primäre und akute Infektion. Frühe Überleben angesichts der angeborenen Immunantwort ist wichtig, damit der Parasit, genügend Parasitenzahlen zu erreichen, sowie distalen Stellen zu erreichen, für die Niederlassung einer chronischen Infektion zu ermöglichen. T. gondii hat Strategien zur Wirtsabwehrmechanismen, die wahrscheinlich ihre Fähigkeit zu replizieren und Verbreitung der Infektion früh beitragen entgegenzuwirken entwickelt. Zuerst T. gondii bildet eine einzigartige PV bei Parasitenbefall weitgehend getrennt von den endozytischen und Exozytose-Prozesse der Wirtszelle im Vergleich zu anderen intrazellulären Pathogenen 5-9 ist. Auch, wie alle erfolgreichen intrazelluläre Erreger T. gondii modifiziert seine Wirtszelle, um einen permissiven Umgebung f erstellenoder Wachstum. Dies beinhaltet eine Anpassung Wirtszelle Genexpression durch Veränderung Wirtszelle Transkriptionsfaktoren einschließlich der wichtig für die Regulierung Zellaktivierung 10-15. ROP16 16-19 GRA15 20. GRA16 21 und GRA24 22 erwiesen sich alle als bei der Regulierung der Transkriptionsreaktion und Zellsignalkaskaden von Wirtszellen mit T. infiziert wichtig gondii. Jüngste Studien mit genetischen Kreuzungen zwischen Parasitenstämme mit unterschiedlichen Phänotypen sehr produktiv bei der Identifizierung von Genen, die Parasiten Parasiten Genotyp-abhängigen Eigenschaften einschließlich Umgehung der Immunität GTPasen (IRGs) 16,19,23-26 zugrunde liegen gewesen. In Mäusen sind Immunität GTPasen (IRGs) kritisch für die Steuerung der Typ II und III Genotypen des Parasiten während der sehr virulenten Typ I Genotypen Mechanismen entwickelt, um die murinen IRGs entziehen. Es ist aber auch klar, dass der Parasit hat Mechanismen entwickelt, um antimikrobielle Medien umgehenren neben den IRGs und dass einige dieser Mechanismen können für Parasiten Genotypen 27,28 konserviert werden. Zudem ist sehr wenig über den kritischen Mediatoren der Zell autonome Immunität gegen T. bekannt gondii während menschliche Toxoplasmose. Parasite Gene wichtig für die Resistenz gegen Vermittler von Zell autonome Immunität kann auch überlebenswichtig sein, während Tachyzoiten Umwandlung, die auch von Wirtsimmunreaktionen ausgelöst werden können, um bradyzoite. So kann beispielsweise Stickstoffmonoxid auf hohem Niveau Parasitenreplikation in infizierten Makrophagen unterdrücken, aber es kann auch dazu anregen Tachyzoiten zu Umrechnung in Zyste Produktion 30-32 bradyzoite. ToxoDB ist eine funktionelle Genomdatenbank für T. gondii, der als wichtige Ressource für das Feld in Bezug auf die Bereitstellung Sequenzinformation für die Parasitengenom und den Zugang zu veröffentlichten und nicht veröffentlichten genomischen Maßstab Daten einschließlich Community Anmerkungen Gen exp<html> ression und Proteomik-Daten 33. Ähnlich wie bei vielen Protozoen- Pathogene, der Großteil des Genoms besteht aus hypothetischen Gene ohne von Informationen aus Genhomologie Einblick in ihre möglichen Funktionen bereitzustellen. So ist nach vorn Genetik ein leistungsfähiges Werkzeug, um neue Parasiten Gene wichtig für Immunevasion, Zyste Umwandlung und andere Funktionen entscheidend für Parasiten Pathogenese als auch für die Konvertierung zwischen verschiedenen Entwicklungsstadien zu identifizieren. Eine weitere Stärke der Vorwärtsgenetik ist, dass es als ein relativ nicht-vorgespannten Ansatz verwendet, um den Parasit zu den Genen, die für spezifische Aufgaben in Pathogenese, einschließlich Immunumgehung und Zystenbildung sind abzufragen. Die jüngsten Verbesserungen in Next Generation Sequencing für Mutationsprofiling haben sie eine Methode der Wahl für die Identifizierung der verantwortlichen Parasiten Gene von vorn Genetik Studien unter Verwendung von chemischen und Insertionsmutagenese 34-37 hergestellt. ntent &quot;&gt; Es ist wichtig, um Schwachstellen in T. gondii, die ausgenutzt werden, um die Wirksamkeit der Zell autonomen Immunmechanismen gegen den Parasiten insbesondere solche, die auch gegen die resistenten Zyste Bühne aktiv sein können. Um dieses Ziel zu, ein in vitro-Mäuse zu verbessern identifizieren Makrophageninfektion und Aktivierungsmodell wurde entwickelt, um Mutationen in die Parasiten, insbesondere T. gondii Fitness nach Aktivierung der infizierten Makrophagen, aber nicht in naiven Makrophagen limitieren. Dieses Makrophagen Bildschirm verwendet, um eine Bibliothek von T. gondii Insertionsmutanten um abzufragen letztendlich identifizieren T. gondii wichtige Gene für die Resistenz gegen Stickoxid 27,28. Die Isolierung von einem Panel von T. gondii-Mutanten mit verminderter Widerstandsfähigkeit gegenüber der Aktivierung der infizierten Makrophagen, insbesondere eine ausgeprägte Empfindlichkeit gegenüber Stickstoffmonoxid, erwies sich die Nützlichkeit des Screening zur Identifizierung Parasiten Gene wichtig für WiderstandMediatoren der Zell autonome Immunität mit Ausnahme der für die murine IRGs 28 beschriebenen Resistenzmechanismen. Insertionsmutagenese hat Vorteile gegenüber chemischen Mutagenese im Hinblick auf die Erzeugung einer begrenzten Anzahl von zufälligen Mutationen in jeder Parasit Klons und theoretisch eine leichtere Identifizierung der Mutationsstelle. Die Identifizierung der Genom-Stelle des Plasmids Insertion in T. gondii Insertionsmutanten in der Praxis hat sich überraschend schwierig, in vielen Fällen 37. Einführen eines Plasmids in einem Gen ist auch geeignet, die Funktion eines Gens, das im Gegensatz zur chemischen Mutagenese, die typischerweise zu einzelnen Nucleotidänderungen stören. Die chemische Mutagenese mit entweder N-Ethyl-N-nitrosoharnstoff (ENU) oder Ethylmethansulfonat (EMS) zu einer erhöhten Fähigkeit, einen größeren Teil des Parasitengenoms zu analysieren bieten, im Vergleich zu Insertionsmutagenese, da sie mehrfache Punktmutationen erzeugt ( geschätzt bei 10 -100) pro Mutante 34, 38. Außerdem jüngsten Fortschritte in der gesamten Genoms Profilierung hat es möglich gemacht, um der nächsten Generation Sequenzierung verwenden, um die wahrscheinlichsten Kandidaten-Gene für die identifizierte Phänotyp eines mutierten Parasiten 34,38 verantwortlich sind. Unabhängig von der Mutagenese-Ansatz, Bestätigung der Rolle von dem Parasiten Gen der Resistenz gegen Makrophagenaktivierung erfordert letztendlich Gendeletion und Komplementation zu molekularer Koch-Postulate zu erfüllen. Die Fähigkeit, die Funktion eines Gens durch genetische Manipulation sowohl des Parasiten und dem Makrophagen zergliedern ist wichtig, da viele der über Vorwärts Genetik in T. identifizierten Gene gondii, wie auch andere Krankheitserreger, werden noch als hypothetische Gene mit wenig bis keine Sequenzhomologie zu anderen Proteinen mit bekannter Funktion aufweist. Die aktuelle Papier einen allgemeinen Ansatz, der verwendet werden kann, um zu ermitteln, ob die unterbrochene Gen in einer Mutante ist wichtig für den Widerstand gegen eine bekannte oder umreißtunbekannt Vermittler von Zell autonome Immunität. Die erste Analyse der Host antimikrobielle Faktoren wird durch die Auswertung der Überlebensrate von Wildtyp und Mutante Parasiten in Makrophagen, die von Wildtyp-Mäusen im Vergleich zu denen mit spezifischen Gendeletionen in induzierbaren Stickstoffmonoxid-Synthase (iNOS), gp-91 phox (NADPH-Oxidase) durchgeführt wird, und spezifische Immunität GTPasen (IRGs). Damit wird festgelegt, ob die identifizierten Parasiten Gene sind wichtig für die Resistenz gegen Stickstoffmonoxid, reaktive Sauerstoffzwischenprodukte oder Immunität GTPasen bzw. 28 oder wenn ein unbekannter Immunsystems beteiligt ist. Aktivierung der infizierten Makrophagen sowohl IFN-γ und LPS in der aktuellen Protokoll beschrieben, resultiert in erster Linie bei der Isolierung von Parasitengene wichtig für die Resistenz gegen Stickstoffoxid 28. Die Verwendung von pharmakologischen Mitteln, die Stickstoffmonoxid in Abwesenheit von Makrophagen-Aktivierung zu induzieren (Stickoxiddonoren) bestätigte, dass die Mehrheit der identifizierten Gene, die für Wieder warenWiderstand gegen Stickstoffmonoxid anstatt Stickoxid zusammen mit weiteren Mediatoren mit Makrophagenaktivierung 28 verbunden. Schritt eins und zwei beschreiben eine Vorwärtsgenetik Bildschirm entworfen, um Parasiten Mutanten mit einem Fitness-Defekt nach der Aktivierung von infizierten Knochenmark-Makrophagen in vitro zu isolieren. Schritt eins beschreibt eine Dosistitration Analyse empirisch bestimmen eine Dosis von IFN-γ und LPS für Makrophagenaktivierung, die Parasiten Replikation reduziert, aber nicht vollständig hemmen die Replikation von Wildtyp T. verwenden gondii Elternstamm, die für die Erstellung der Bibliothek des Parasiten Mutanten verwendet wird. Schritt zwei wird die Vorwärts genetischen Screening der Mutanten-Klone in Makrophagen in 96-Well-Platten. Schritt drei beschreibt einen Ansatz, um den Phänotyp jeder Mutante in dem Bildschirm der 96-Well-Platten identifiziert und zu bewerten, ob der Fehler in jeder Mutante wirkt das Überleben der Parasiten, die Replikation zu bestätigen,oder Zysten Produktion als Antwort auf die Makrophagenaktivierung. Schritt vier wird die Verwendung von Knochenmark-Makrophagen von Mäusen mit Deletionen in spezifischen antimikrobiellen Wege um die Immunmediatoren, auf die der Parasit Mutante spezifisch empfindlich identifizieren. Schritt fünf beschreibt einen Ansatz, um zu bestimmen, ob ein Parasit Mutante ist auch für in vivo Pathogenese beeinträchtigt, wie durch Zystenproduktion in den Gehirnen von infizierten Mäusen ausgewertet.

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			<td>ACROS Organics</td>
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</table>

forward genetics screens using macrophages to identify toxoplasma gondii genes important for resistance to ifn-&#947;-dependent cell autonomous immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Toxoplasma gondii repliziert frei in naiven Makrophagen und weist eine Verdopplungszeit von 6-12 h in Abhängigkeit von der Belastung des Parasiten. Abbildung 1 zeigt repräsentative Parasiten in naiven Vergleich aktiviert Knochenmark stammenden Makrophagen. Abbildung 2 zeigt die allgemeine Morphologie des Parasiten in HFF Wirtszellen, die nach 2, 4, 8, 16 und 32 Parasiten / PV. In der gegenwärtigen Protokoll wird der Parasit darf naiv Makrophagen eindringen und stellen Sie ei...

Watch this Scientific Journal Video about Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-Î³-Dependent Cell Autonomous Immunity at JoVE

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ Dependent Cell Autonomous Immunity | Immunology and Infection | Video

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Zukunfts Genetics Screens Mit Makrophagen zu identifizieren<em&gt; Toxoplasma gondii</em&gt; Gene wichtig für die Resistenz gegenüber IFN-γ-abhängige Zell autonome Immunität

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

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Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Unit 2

Population Genetics

Analyzing Gene Expression and Function

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11.0K Aufrufe.  New York Medical College. Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion. 

Serie: Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Gewinnung von hochgradig gereinigter Toxoplasma gondii Oocysten durch eine diskontinuierliche Cäsiumchloridgradienten

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Forschung

Immunologie und Infektion

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii Zystenwand Formation in Activated Knochenmark abgeleiteten Makrophagen und Bradyzoite AGB

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear Mäuse mit vordefinierten T-Zell-Rezeptor Spezifitäten gegen Toxoplasma gondii Via SCNT Erhalten

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Auswahl der Plasmodium falciparum Parasiten für Cytoadhesion zu Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Ein genetischer Screen zu isolieren Toxoplasma gondii Host-Zelle Egress Mutanten

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Genetische Manipulation in<em&gt; Δku80</em&gt; Die Stämme für Funktionelle Genomanalyse<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Vorwärts Genetische Ansätze in<em&gt; Chlamydia trachomatis</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

VIGS-vermittelte Stürmer Genetics Screening zur Identifizierung von Genen in Nichtwirtresistenz Beteiligte


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.