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.

Le protocole décrit fournit une approche non biaisée qui utilise l&#39;activation des macrophages dérivés de la moelle osseuse de souris et de la génétique à terme pour isoler T. gondii mutants avec un défaut dans leur capacité à survivre activation des macrophages infectés. Le phénotype des mutants activation des macrophages suivante tombe généralement dans l&#39;une des deux grandes catégories: 1) Les parasites apparaissent intacts mais ne parviennent pas à reproduire au-delà de 1 parasite pa...

Toxoplasma gondii (T. gondii) est un intracellulaire obligatoire, pathogène protozoaire. Il est l&#39;agent causal de la toxoplasmose, un danger pour la santé chez les personnes immunodéprimées. Ce est aussi le système de modèle pour d&#39;autres agents pathogènes qui infectent les humains apicomplexes y compris Cryptosporidium et Cyclospora. La toxoplasmose est le plus souvent acquise par l&#39;ingestion d&#39;aliments ou d&#39;eau contaminés par le stade du parasite bradyzoïte ou oocystes. Lors de l&#39;ingestion, ces étapes se convertir à l&#39;étape de tachyzoïtes du parasite qui se réplique à l&#39;intérieur des cellules hôtes et diffuse de manière systémique. Lymphocytes T, l&#39;IFN-γ et, dans une moindre mesure, de l&#39;oxyde nitrique 4/1, sont importants pour le contrôle de l&#39;infection mais ne sont pas capables d&#39;éliminer la maladie, dans une proportion de tachyzoïtes convertir à l&#39;étape de bradyzoite qui sont protégés à l&#39;intérieur de kystes tissulaires résultant en une infection chronique à long terme. En fait, il ne existe pas de thérapies efficaces contre le kyste chronique stage de la maladie. Toxoplasmose sévère est le plus souvent due à la réactivation d&#39;une infection persistante, avec le stade de bradyzoïte du parasite reconversion à la caractéristique de scène tachyzoïte répliquer rapidement de l&#39;infection primaire et aiguë. La survie au début de la face de la réponse immunitaire innée est important de permettre au parasite à atteindre nombre de parasites suffisantes, ainsi que pour atteindre les sites distales, pour permettre l&#39;établissement d&#39;une infection chronique. T. gondii a développé des stratégies pour lutter contre les mécanismes de défense de l&#39;hôte qui contribuent probablement sa capacité à reproduire et diffuser au début de l&#39;infection. Premièrement, T. gondii constitue un PV unique lors de l&#39;invasion parasitaire qui est en grande partie séparé des processus d&#39;endocytose et d&#39;exocytose de la cellule hôte par rapport à d&#39;autres agents pathogènes intracellulaires 5-9. En outre, comme tous les succès intracellulaire pathogènes T. gondii modifie sa cellule hôte pour créer un environnement permissif fou la croissance. Cela comprend la reprogrammation hôte d&#39;expression génique des cellules en modifiant les facteurs de transcription de la cellule hôte, y compris ceux qui sont importants pour la régulation de l&#39;activation des cellules 10-15. ROP16 16-19, GRA15 20, 21 et GRA16 GRA24 22 ont tous été montré pour être importante dans la régulation de la réponse transcriptionnelle de signalisation cellulaire et des cascades de cellules hôtes infectées avec T. gondii. Des études récentes utilisant des croisements génétiques entre les souches de parasites avec des phénotypes distincts ont été très productif dans l&#39;identification des gènes parasitaires qui sous-tendent traits parasites génotype dépendant notamment de fraude liées à l&#39;immunité GTPases (IRGS) 16,19,23-26. Chez la souris, l&#39;immunité liée GTPases (IRGS) sont essentiels pour le contrôle de type II et III génotypes du parasite tandis que le type très virulente je génotypes ont développé des mécanismes pour échapper aux IRGS murins. Cependant, il est également clair que le parasite a développé des mécanismes pour échapper médias antimicrobienteurs, en plus des IRGS et que certains de ces mécanismes peuvent être conservées à travers les génotypes de parasites 27,28. En outre, on sait très peu sur les médiateurs critiques de l&#39;immunité cellulaire contre autonome T. gondii pendant la toxoplasmose humaine. Parasite gènes importants pour la résistance aux médiateurs de l&#39;immunité cellulaire autonome peuvent également être importantes pour la survie au cours tachyzoïte à bradyzoïte conversion qui peut également être déclenché par les réponses immunitaires de l&#39;hôte. Par exemple, l&#39;oxyde nitrique à des niveaux élevés peut supprimer la réplication du parasite dans les macrophages infectés, mais il peut aussi stimuler tachyzoïte à bradyzoïte conversion résultant de la production de kystes 30-32. ToxoDB est une base de données de génomique fonctionnelle pour T. gondii qui fonctionne comme une ressource essentielle pour le domaine en termes de fournir des informations de séquence du génome du parasite et l&#39;accès à des données publiées et non publiées de l&#39;échelle du génome, y compris les annotations de la communauté, le gène exp<html> écrêtages et protéomique données 33. Comme pour de nombreux agents pathogènes protozoaires, la majorité du génome est constitué de gènes hypothétiques avec aucune information disponible sur la base d&#39;homologie de gène de donner un aperçu de leurs fonctions potentielles. Ainsi, la génétique à terme est un outil puissant pour identifier de nouveaux gènes importants pour échapper au système immunitaire, la conversion de kyste et d&#39;autres fonctions critiques pour la pathogenèse du parasite ainsi que pour la conversion entre les stades de développement distincts parasites. Une force supplémentaire vers l&#39;avant de la génétique est qu&#39;il peut être utilisé comme une approche relativement non biaisé pour interroger le parasite à des gènes qui sont importants pour des tâches spécifiques, y compris dans la pathogenèse évasion immunitaire et la formation de kystes. De récentes améliorations dans le séquençage de prochaine génération pour le profilage mutationnel ont fait une méthode de choix pour l&#39;identification des gènes responsables de parasites avant les études de génétique par mutagenèse chimique et insertion 34-37. MÉNAGEMENT &quot;&gt; Il est important d&#39;identifier les vulnérabilités dans T. gondii qui peuvent être exploitées pour améliorer l&#39;efficacité des mécanismes immunitaires autonomes cellulaires contre le parasite en particulier ceux qui peuvent également être actif contre le stade de kyste résistant. Vers ce but, in vitro murin l&#39;infection des macrophages et le modèle d&#39;activation a été développé pour identifier des mutations dans le parasite qui altère spécifiquement T. gondii remise en forme suite à l&#39;activation des macrophages infectés, mais pas dans les macrophages naïfs. Cet écran de macrophages a été utilisé pour interroger une bibliothèque de T. gondii mutants d&#39;insertion pour finalement identifier les gènes de T. gondii importants pour la résistance à l&#39;oxyde nitrique 27,28. L&#39;isolement d&#39;un panel de mutants de T. gondii avec facultés affaiblies résistance à l&#39;activation des macrophages infectés, en particulier une sensibilité marquée à l&#39;oxyde nitrique, prouvé l&#39;utilité de l&#39;écran pour identifier des gènes pour la résistance parasites importantsde médiateurs de l&#39;immunité cellulaire autonome autre que les mécanismes de résistance décrites pour les IRGS 28 murins. Mutagenèse insertionnelle a des avantages sur mutagenèse chimique en termes de génération d&#39;un nombre limité de mutations aléatoires dans chaque clone parasitaire et, en théorie, une identification plus facile du site de mutation. Toutefois, l&#39;identification du site d&#39;insertion du génome du plasmide en T. mutants d&#39;insertion gondii, dans la pratique, a été étonnamment difficile dans de nombreux cas 37. L&#39;insertion d&#39;un gène dans un plasmide est également susceptible de perturber la fonction d&#39;un gène, contrairement à une mutagenèse chimique qui se traduit généralement par des changements d&#39;un seul nucleotide. Cependant, une mutagenèse chimique avec soit la N-éthyl-N-nitrosourée (ENU) ou un sulfonate d&#39;éthylméthane (EMS) peut offrir une plus grande capacité d&#39;analyser une plus grande portion du génome du parasite, par rapport à une mutagenèse insertionnelle, car il crée plusieurs polymorphismes nucléotidiques simples ( estimé à 10 -100) par mutant 34, 38. En outre, les progrès récents dans le profilage du génome entier a permis d&#39;utiliser le séquençage de prochaine génération pour identifier les gènes candidats les plus probables responsables du phénotype identifié d&#39;un parasite muté 34,38. Indépendamment de l&#39;approche de mutagenèse, la confirmation du rôle du gène parasite dans la résistance à l&#39;activation des macrophages nécessite finalement délétion du gène et la complémentation de remplir les postulats de Koch moléculaire. La capacité de disséquer la fonction d&#39;un gène par manipulation génétique à la fois du parasite et le macrophage est important car un grand nombre des gènes identifiés par l&#39;avant génétique dans T. gondii, ainsi que d&#39;autres agents pathogènes, se caractérisent encore en tant que gènes hypothétiques avec peu ou pas d&#39;homologie de séquence avec d&#39;autres protéines ayant des fonctions connues. Le présent document décrit une méthode générale qui peut être utilisée pour déterminer si le gène disrupté dans un mutant est important pour la résistance à un connue ouinconnue médiateur de l&#39;immunité cellulaire autonome. L&#39;analyse initiale des hôtes facteurs antimicrobiens est effectué en évaluant la survie de type sauvage et les parasites mutants dans les macrophages de souris de type sauvage par rapport à celles présentant des délétions de gènes spécifiques dans synthase inductible de l&#39;oxyde nitrique (iNOS), GP-91 phox (NADPH oxydase), et liées à l&#39;immunité GTPases spécifiques (IRGS). Cela permettra de déterminer si les gènes parasites identifiés sont importants pour la résistance à l&#39;oxyde nitrique, intermédiaires réactifs de l&#39;oxygène ou de l&#39;immunité liée GTPases 28 respectivement ou si un mécanisme immunitaire inconnu est impliqué. Activation des macrophages infectés à la fois avec l&#39;IFN-γ et LPS, décrites dans le protocole actuel, résulte principalement de l&#39;isolement de gènes importants pour la résistance du parasite à l&#39;oxyde nitrique 28. L&#39;utilisation d&#39;agents pharmacologiques qui induisent l&#39;oxyde nitrique en l&#39;absence d&#39;activation des macrophages (donneurs d&#39;oxyde nitrique) a confirmé que la majorité des gènes identifiés étaient importantes pour rerésistance à l&#39;oxyde nitrique plutôt que l&#39;oxyde nitrique de concert avec des médiateurs supplémentaires associés à l&#39;activation des macrophages 28. Étape un et deux décrivent un écran de génétique avant conçu pour isoler des mutants parasites avec un défaut de remise en forme suite à l&#39;activation des macrophages dérivés de la moelle osseuse infectée in vitro. Première étape décrit une analyse de titrage de dose pour déterminer empiriquement une dose de γ et LPS IFN à utiliser pour l&#39;activation des macrophages qui réduit la réplication du parasite mais ne inhibe pas complètement la réplication du type sauvage T. gondii souche parentale qui est utilisé pour la création de la banque de mutants de parasites. Deuxième étape décrit l&#39;écran vers l&#39;avant génétique des clones mutants dans des macrophages dans les plaques à 96 puits. Étape trois présente une approche pour confirmer le phénotype de chaque mutant identifié dans l&#39;écran des plaques à 96 puits et d&#39;évaluer si le défaut de chaque mutant affecte la survie du parasite, la réplication,ou la production de kyste en réponse à l&#39;activation des macrophages. Etape quatre décrit l&#39;utilisation de macrophages dérivés de la moelle osseuse provenant de souris avec des deletions dans les voies d&#39;antimicrobiens pour identifier les médiateurs immunitaires à laquelle le mutant est particulièrement sensible parasite. Cinquième étape décrit une méthode pour déterminer si un mutant de parasite est également compromise par pathogenèse in vivo tel qu&#39;évalué par la production de kystes dans les cerveaux de souris infectées.

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			<td>Life Technologies</td>
			<td>A20181</td>
			<td>http://www.lifetechnologies.com/order/catalog/product/A10235</td>
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			<td>MP Biomedicals</td>
			<td>ICN19135680</td>
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			<td height="21" style="height:21px;">LysoTracker</td>
			<td>Life Technologies</td>
			<td>L-7526</td>
			<td>https://www.lifetechnologies.com/order/catalog/product/L7526?ICID=search-product</td>
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			<td>Jackson Laboratories</td>
			<td align="right">664</td>
			<td>http://jaxmice.jax.org/strain/000664.html</td>
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			<td>Jackson Laboratories</td>
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			<td height="21" style="height:21px;">sodium nitroprusside</td>
			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
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			<td height="21" style="height:21px;">DETA NONOate</td>
			<td>ACROS Organics</td>
			<td>AC32865-0250</td>
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			<td>10T19A</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 reproduit librement dans les macrophages naïfs et a un temps de doublement entre 6-12 h en fonction de la souche du parasite. La figure 1 montre parasites représentatifs de naïf par rapport macrophages dérivés de la moelle osseuse activées. La figure 2 montre la morphologie générale de parasites dans HFF des cellules hôtes à 2, 4, 8, 16 et 32 ​​parasites / PV. Dans le protocole actuel, le parasite est autorisé à envahir macrophages naïfs et d&...

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.

Forward Génétique écrans à l&#39;aide pour identifier les macrophages<em&gt; Toxoplasma gondii</em&gt; gènes importants pour la résistance à l&#39;IFN-γ-cellulaire dépendant immunité autonome

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 vues.  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. 

Vidéo: 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

Obtention hautement purifiée Toxoplasma gondii Les oocystes par un gradient de chlorure de césium discontinu

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

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Immunologie et infection

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 Formation paroi du kyste dans la moelle osseuse Activé macrophages dérivés et Conditions Bradyzoite

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.

Souris Transnuclear avec des spécificités T pré-définis Cell Receptor contre Toxoplasma gondii Obtenues par TNCS

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.

Sélection des Plasmodium falciparum Parasites des Cytoadhesion aux cellules du cerveau endothéliales humaines

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.

Un crible génétique pour isoler Toxoplasma gondii Mutants Egress de la cellule hôte

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.

Manipulation génétique<em&gt; Δku80</em&gt; Les souches pour l&#39;analyse génomique fonctionnelle de<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.

Approches avant génétiques dans<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-Mediated dépistage génétique avant pour l&#39;identification des gènes impliqués dans la résistance non hôte


	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.

En utilisant l&#39;ARN interférence pour étudier la réponse immunitaire innée dans les macrophages de souris

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.