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.

O protocolo descrito fornece uma abordagem não-tendenciosa que usa a ativação dos macrófagos derivadas da medula óssea de camundongos e genética para a frente para isolar T. gondii mutantes com um defeito na sua capacidade de sobreviver a activação de macrófagos infectados. O fenótipo dos mutantes após a ativação dos macrófagos normalmente cai em uma das duas grandes categorias: 1) Os parasitas parecem intactos, mas não conseguem replicar para além de 1 parasita por PV; 2) Os parasitas parecem d...

Toxoplasma gondii (T. gondii) é uma intracelular obrigatório, patógeno protozoários. É o agente causador da toxoplasmose, um perigo para a saúde em indivíduos imunocomprometidos. É também o sistema modelo para outros agentes patogénicos apicomplexan que infectam os seres humanos, incluindo o Cryptosporidium e Cyclospora. A toxoplasmose é mais comumente adquirida através da ingestão de alimentos ou água contaminados com a bradyzoite ou oocisto fase do parasita. Após a ingestão, estes estágios converter ao palco taquizoíto do parasita que se replica dentro das células hospedeiras e divulga sistemicamente. As células T, IFN-γ e, em menor grau, o óxido nítrico 1-4, são importantes para o controlo de infecção, mas não são capazes de eliminar a doença, como uma proporção de taquizoítos converter para a fase bradyzoite que estão protegidas no interior dos cistos de tecido resultando numa infecção crónica de longa duração. De facto, não há terapias eficazes contra o cisto s crónicatagem da doença. Toxoplasmose grave é na maioria das vezes, devido à reativação da infecção persistente, com o estágio bradyzoite do parasita converter de volta para o palco rapidamente replicar característica taquizoíto de infecção primária e aguda. Sobrevivência no início da face da resposta imune inata é importante para permitir que o parasita para alcançar números suficientes de parasitas, assim como para alcançar locais distais, para permitir o estabelecimento de infecção crónica. T. gondii evoluiu estratégias para combater os mecanismos de defesa do hospedeiro que provavelmente contribuem para a sua capacidade de se replicar e propagar no início da infecção. Em primeiro lugar, T. gondii constitui um PV original durante a invasão do parasita que é largamente segregado a partir dos processos de endocitose e exocíticas da célula hospedeira em comparação com outros patogénios intracelulares 5-9. Além disso, como todos bem sucedidos patógenos intracelulares T. gondii altera a sua célula hospedeira para criar um ambiente permissivo fou crescimento. Isso inclui a expressão do gene da célula hospedeira reprogramação alterando fatores de transcrição da célula hospedeira, incluindo os que são importantes para regular a ativação de células 10-15. ROP16 16-19, GRA15 20, 21 e GRA16 GRA24 22 tenham sido mostrados para ser importante na regulação da resposta de transcrição e de sinalização celular em cascata de células hospedeiras infectadas com T. gondii. Estudos recentes utilizando cruzamentos genéticos entre linhagens do parasita com fenótipos distintos têm sido altamente produtivo na identificação de genes do parasita que fundamentam traços genótipo-dependente de parasitas, incluindo a evasão de imunidade relacionada GTPases (IRGs) 16,19,23-26. Em ratinhos, relacionadas com a imunidade GTPases (IRGs) são críticos para o controlo do Tipo II e III genótipos do parasita enquanto o tipo I muito virulento genótipos desenvolveram mecanismos para escapar às IRGs murino. No entanto, também é claro que o parasita se desenvolveram mecanismos para escapar meios antimicrobianares em adição aos IRGs e que alguns destes mecanismos pode ser conservado entre os genótipos de parasitas 27,28. Além disso, muito pouco se sabe sobre os mediadores críticos da imunidade celular autónoma contra T. gondii durante toxoplasmose humana. Genes dos parasitas importantes para a resistência aos mediadores da imunidade celular autónoma pode também ser importante para a sobrevivência durante taquizoíto para bradyzoite conversão que também pode ser desencadeada por respostas imunitárias do hospedeiro. Por exemplo, o óxido nítrico em níveis elevados pode suprimir a replicação do parasita em macrófagos infectados, mas também pode estimular a taquizoíto bradyzoite conversão resultando na produção de cisto 30-32. ToxoDB é um banco de dados de genômica funcional para T. gondii, que funciona como um recurso crítico para o campo em termos de fornecimento de informações de seqüência do genoma do parasita e acesso a dados em larga escala genômica publicados e não publicados, incluindo anotações da comunidade, exp gene<html> dados ression e proteômica 33. Semelhante a muitos patogénios protozoários, a maior parte do genoma é constituído por genes hipotéticos sem informações disponíveis, com base na homologia de genes para fornecer informações sobre as suas funções potenciais. Assim, a genética para a frente é uma ferramenta poderosa para identificar genes do parasita novos importantes para a evasão imune, conversão de cisto e outras funções críticas para parasita patogenia, bem como para a conversão entre diferentes estágios de desenvolvimento. Uma resistência adicional da genética para a frente é que ele pode ser usado como uma abordagem relativamente não enviesada para interrogar o parasita como para os genes que são importantes para tarefas específicas em patogénese, incluindo a evasão imune e formação de quistos. As recentes melhorias na próxima geração de sequenciamento para perfilamento mutacional fizeram-lhe um método de escolha para identificar os genes do parasita responsáveis ​​de estudos de genética para a frente usando mutagênese tanto química e insercional 34-37. ntent &quot;&gt; É importante identificar vulnerabilidades no T. gondii que podem ser explorados para reforçar a eficácia das células imunitárias mecanismos autónomos contra o parasita particularmente aqueles que possam também ser activos contra o estágio de cisto resistente. Com este objectivo, in vitro murino infecção de macrófagos e modelo de ativação foi desenvolvido para identificar mutações no parasita que prejudicar especificamente T. gondii aptidão após a ativação dos macrófagos infectados, mas não em macrófagos ingênuos. Essa tela de macrófagos foi usado para interrogar uma biblioteca de T. gondii mutantes de inserção, a fim de, em última instância identificar genes de T. gondii importantes para a resistência ao óxido nítrico 27,28. O isolamento de um painel de mutantes de T. gondii com resistência diminuída à activação de macrófagos infectados, particularmente uma sensibilidade marcada ao óxido nítrico, comprovou a utilidade da tela para identificar genes do parasita importantes para a resistênciaa mediadores da imunidade celular autónoma do que os outros mecanismos de resistência descritos para os 28 IRGs murino. Mutagénese de inserção tem vantagens em relação a mutagénese química em termos de geração de um número limitado de mutações aleatórias em cada clone parasita e, em teoria, mais fácil identificação do local da mutação. No entanto, a identificação do sítio genómico de inserção no plasmídeo T. Os mutantes de inserção gondii, na prática, tem sido surpreendentemente difícil em muitos casos 37. A inserção de um plasmídeo em um gene é também susceptível de perturbar a função de um gene, em contraste com a mutagénese química que tipicamente resulta em alterações de um só nucleótido. No entanto, a mutagénese química com ou N-etil-N-nitrosoureia (ENU) ou sulfonato ethylmethane (EMS) pode oferecer uma maior capacidade de analisar um maior parte do genoma do parasita, em comparação com mutagénese de inserção, uma vez que cria vários polimorfismos de um único nucleótido ( estimado em 10 -100) por 34 mutante, De 38 anos. Além disso, avanços recentes na caracterização do genoma inteiro tornou possível a utilização de sequenciação próxima geração para identificar os genes candidatos mais prováveis ​​responsáveis ​​pelo fenótipo identificado de um parasita mutado 34,38. Independentemente da abordagem de mutagénese, a confirmação da função do gene parasita na resistência a activação de macrófagos em última análise requer a deleção do gene de complementação e para cumprir os postulados de Koch molecular. A capacidade para dissecar a função de um gene através da manipulação genética de tanto o parasita e o macrófago é importante como muitos dos genes identificados através da genética para a frente em T. gondii, bem como outros agentes patogénicos, são ainda caracterizadas como genes hipotéticos com pouca ou nenhuma homologia de sequência com outras proteínas com funções conhecidas. O papel actual descreve uma abordagem geral que pode ser usado para identificar se o gene interrompido num mutante é importante para a resistência a um ou conhecidadesconhecido mediador da imunidade autônomo celular. A análise inicial do hospedeiro factores antimicrobianos é realizada através da avaliação da sobrevivência de tipo selvagem e mutantes de parasitas em macrófagos dos murganhos do tipo selvagem versus pacientes com deleções de genes específicos em indutível da sintase do óxido nítrico (iNOS), gp-91 phox (NADPH-oxidase), e GTPases específicas relacionadas com a imunidade (IRGs). Isso vai determinar se os genes do parasita identificados são importantes para a resistência ao óxido nítrico, intermediários reativos de oxigênio ou relacionadas com a imunidade GTPases 28, respectivamente, ou se um mecanismo imunológico desconhecido está envolvido. A activação de macrófagos infectados com tanto IFN-γ e LPS, descritos no protocolo de corrente, resulta primeiramente no isolamento de genes de parasitas importantes para a resistência ao óxido nítrico 28. A utilização de agentes farmacológicos que induzem o óxido nítrico na ausência de activação de macrófagos (dadores de óxido nítrico) confirmou que a maioria dos genes identificados, foram importantes para a resistência ao óxido nítrico em vez de óxido nítrico em combinação com mediadores adicionais associados com a activação de macrófagos 28. Passo um e dois descrevem uma tela genética para a frente destinada a isolar mutantes do parasita com um defeito de fitness a seguir à activação de macrófagos derivados da medula óssea infectadas in vitro. O primeiro passo descreve uma análise de titulação da dose para determinar empiricamente uma dose de LPS e IFN-γ a ser usado para a activação de macrófagos, que reduz a replicação do parasita, mas não inibe completamente a replicação do tipo selvagem T. gondii estirpe parental que é usada para a criação da biblioteca de mutantes do parasita. Passo dois descreve o rastreio genético para a frente dos clones mutantes em macrófagos em placas de 96 poços. Passo três descreve uma abordagem para confirmar o fenótipo de cada mutante identificado na triagem das placas de 96 poços e para avaliar se o defeito em cada mutante afecta a sobrevivência do parasita, replicação,ou a produção de cisto em resposta à activação de macrófagos. Passo quatro descreve a utilização de macrófagos derivados da medula óssea de ratinhos com deleções em vias antimicrobianos específicos para identificar os mediadores imunes ao qual o parasita mutante é especificamente susceptíveis. Passo cinco descreve uma abordagem para determinar se um parasita mutante é também comprometida para patogénese in vivo como avaliado pela produção de quisto nos cérebros de ratos infectados.

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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 height="21" style="height:21px;">goat serum</td>
			<td>MP Biomedicals</td>
			<td>ICN19135680</td>
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			<td>FL-1031</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>
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			<td>Jackson Labaoratories</td>
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			<td>Jackson Laboratories</td>
			<td align="right">2609</td>
			<td>http://jaxmice.jax.org/strain/002609.html</td>
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			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
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			<td>ACROS Organics</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 replica livremente em macrófagos naive e tem um tempo de duplicação entre 6-12 horas, dependendo da estirpe do parasita. A Figura 1 mostra parasitas representativos nos ingénuos contra macrófagos derivados da medula óssea activados. A Figura 2 mostra a morfologia geral de parasitas em HFF células hospedeiras em 2, 4, 8, 16 e 32 parasitas / PV. No protocolo atual, o parasita é permitido invadir macrófagos ingênuos e estabelecer um vacúolo parasitófo...

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.

Atacante Genetics telas usando macrófagos para Identificar<em&gt; Toxoplasma gondii</em&gt; genes importantes celular para Resistance to-IFN-γ Dependente Immunity Autónoma

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 visualizações.  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. 

Vídeo: 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

Obtenção altamente purificada Toxoplasma gondii Oocistos por um gradiente de cloreto de césio descontínuos

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

Imunologia e Infecção

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 Formação parede do cisto no Activated derivadas da medula óssea macrófagos e Condições 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.

Ratos Transnuclear com pré-definidos T Especificidades receptor da célula contra a Toxoplasma gondii Obtidos Via 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.

Seleção de Plasmodium falciparum Parasitas para Cytoadhesion para células do cérebro humano endotelial

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.

A tela de genética para isolar Toxoplasma gondii Host de células mutantes egresso

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.

Manipulação genética em<em&gt; Δku80</em&gt; Cepas para Análise Genômica Funcional 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.

Abordagens para a frente genéticos em<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 Mediada frente Triagem Genética para identificação de genes envolvidos na resistência não hospedeiras


	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.

Usando RNA-interferência para investigar a imunitário inatas de resposta em macrófagos

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.