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.

記載されたプロトコルは、Tを分離するために、マウス骨髄由来マクロファージおよびフォワード遺伝学の活性化を使用して偏りのないアプローチを提供彼らの能力の欠陥とトキソプラズマ変異体は、感染したマクロファージの活性化を生き残るために。マクロファージ活性化に続いて突然変異体の表現型は、一般的に2つのカテゴリのいずれかに分類されます。1）寄生虫はその?...

トキソプラズマトキソプラズマ（トキソプラズマ）は偏性細胞内、原生動物病原体である。それは、トキソプラズマ症の原因物質、免疫無防備状態の個体における健康被害である。また、 クリプトスポリジウム及びシクロスポラ含むヒトに感染する他のアピコンプレクサの病原体についてのモデル系である。トキソプラズマ症は、最も一般的に寄生虫ブラディゾイトまたはオーシスト段階で汚染された食物または水の摂取を介して取得される。摂取時には、これらのステージは、宿主細胞内で複製し、全身に広める寄生虫のタキゾイトステージに変換する。 T細胞は、IFN-γ、および、より少ない程度に、窒素酸化物1-4は 、感染症の制御に重要であるが、タキゾイトの割合が組織嚢内で保護されブラディゾイト期に変換するように、疾患を排除することができない長命の慢性感染症になる。実際には、全く治療薬は慢性シストSに対して効果はありません病気の田下。重度のトキソプラズマ症は、プライマリおよび急性感染の急速複製タキゾイト段階特性に戻って変換する寄生虫のブラディゾイト期で、起因する持続感染の再活性化に最も頻繁にある。 先天性免疫応答の面で初期の生存は、寄生虫が十分な寄生虫数に到達するため、ならびに慢性感染症の確立を可能にするために、遠位部位に到達させることが重要である。T.トキソプラズマは、おそらく複製し、感染の早期普及する能力に貢献する宿主防御機構に対抗するための戦略を発展させてきました。まず、T。トキソプラズマは、他の細胞内病原体5-9と比べて、宿主細胞のエンドサイトーシスおよびエキソサイトーシスのプロセスから大幅に偏析する寄生虫の侵入時にユニークなPVを形成している。また、成功したすべての細胞内病原体のようなT.トキソプラズマは、許容される環境fを作成するために、その宿主細胞を修飾するまたは成長。これは、細胞活性化10-15を調節するための重要なものを含む宿主細胞の転写因子を変更することによって再プログラミング宿主細胞遺伝子発現を含む。 ROP16 16-19は 、GRA15 20、GRA16 21とGRA24 22はすべてT.感染した宿主細胞のシグナル伝達カスケード転写応答及び細胞の調節に重要であることが示されているトキソプラズマ 。明確な表現型を有する寄生虫株の間遺伝的交雑を用いた最近の研究では、免疫関連GTPアーゼ（IRGs）16,19,23-26の回避などの寄生虫の遺伝子型に依存する特性の基礎となる寄生虫の遺伝子の同定に生産性の高いされている。非常に毒性の強いタイプIの遺伝子型は、マウスIRGsを回避する機構を進化させてきたが、マウスでは、免疫関連GTPアーゼ（IRGs）は、寄生虫のタイプIIおよびIIIの遺伝子型の制御のために重要である。しかし、寄生虫抗菌メディアを回避する機構を進化させていることも明らかであるIRGsに加えて、これらのメカニズムのいくつかは、寄生虫の遺伝子型27,28を越えて保存されるよう役。また、非常に少ないが、Tに対する細胞自律的な免疫の重要なメディエーターについてはほとんど知られていない人間のトキソプラズマ症の間にトキソプラズマ 。細胞自律的な免疫のメディエーターへの抵抗のために重要な寄生虫の遺伝子はまた、宿主の免疫応答によってトリガすることが可能な変換をブラディゾイトするタキゾイト中に生存のために重要であるかもしれない。例えば、高いレベルでの一酸化窒素は、感染したマクロファージにおける寄生虫の複製を抑制することができるが、それはまた、嚢胞の生産30-32、結果の変換をブラディゾイトするタキゾイトを刺 ​​激することができる。 ToxoDBはT.のための機能的なゲノムデータベースですトキソプラズマ 、そのコミュニティの注釈を含む公開され、未発表のゲノムスケールのデータにシーケンス寄生虫ゲノムの情報およびアクセスを提供するという点でフィールドの重要なリソースとして機能し、遺伝子EXP<html> ressionとプロテオミクスデータ33。多くの原生動物の病原体と同様に、ゲノムの大半は彼らのポテンシャル関数への洞察を提供するために、遺伝子の相同性に基づいて、利用可能な情報がないとの仮定の遺伝子からなる。このように、フォワード遺伝学は免疫回避、嚢胞変換や寄生虫病因に重要な他の機能のためだけでなく、個別の発達段階との間の変換のための重要な新規寄生虫遺伝子を同定するための強力なツールです。フォワード遺伝学のさらなる長所は、免疫回避及び嚢胞形成を含む病因における特定のタスクのために重要な遺伝子のような寄生虫を調べるために、比較的偏りのないアプローチとして使用することができることである。突然変異プロファイリングのための次世代の配列決定における最近の改善は、化学的および挿入突然変異34-37の両方を用いた順遺伝学研究から責任寄生虫遺伝子を同定するための選択方法で行った。 ntent &quot;&gt;それは、寄生虫に対する細胞自律的な免疫機構にも耐性の嚢胞段階に対して活性であり得ることを特にの実効性を高めるために活用することができトキソプラズマの脆弱性を特定することが重要である。この目的に向けて、in vitroでネズミマクロファージ感染と活性化モデルは、具体的にナイーブマクロファージ内で感染したマクロファージの活性化に続いてではなく、 トキソプラズマの適応度を損なう寄生虫に変異を同定するために開発されました。このマクロファージ画面は、最終的にするために、 トキソプラズマ挿入変異体のライブラリーを調べるために使用された一酸化窒素27,28への抵抗のための重要なトキソプラズマ遺伝子を同定する。感染したマクロファージの活性化に損なわれた抵抗性を有するトキソプラズマ変異体のパネルの単離、一酸化窒素に特に顕著な感度は、識別するために、画面の有用性を証明した抵抗のための重要な寄生虫の遺伝子ネズミIRGs 28について記載した耐性機構以外の細胞自律的な免疫のメディエーターに。挿入変異誘発は、制限されたそれぞれの寄生虫クローンにおけるランダム変異の数と、理論的には、突然変異の部位をより容易に識別を生成するという点で化学的突然変異誘発を超える利点を有している。しかし、Tのプラスミド挿入のゲノム部位を特定するトキソプラズマ挿入変異体は、実際には、多くの場合、37で、驚くべきことには困難であった。遺伝子へのプラスミドの挿入は、典型的には、単一のヌクレオチド変化をもたらす化学的突然変異誘発とは対照的に、遺伝子の機能を破壊する可能性がある。それは、複数の一塩基多型を作成しかし、化学的突然変異誘発は、N-エチル-N-ニトロソウレア（ENU）、またはエチルメタンスルホン酸（EMS）のいずれかで（、挿入突然変異と比較して、寄生虫のゲノムの大部分を分析する能力の増加を提供することができる突然変異体34あたり10 -100）と見積もら、38。また、全ゲノムプロファイリングの最近の進歩により、変異した寄生虫34,38の識別された表現型に関与する可能性が最も高い候補遺伝子を同定するために、次世代シークエンシングを使用するようになった。かかわらず、変異誘発アプローチの、マクロファージ活性化に抵抗の寄生虫遺伝子の役割の確認は最終的に、分子コッホの仮説を満たすために遺伝子欠失および相補性を必要とします。 寄生虫とマクロファージの両方の遺伝子操作によって遺伝子の機能を分析する能力は、Tに前進遺伝学を経由して同定された遺伝子の多くが同様に重要であるトキソプラズマ原虫、ならびに他の病原体は、まだ既知の機能を有する他のタンパク質と全く配列相同性をほとんどと仮定的遺伝子として特徴づけられる。現在の紙は、変異体において破壊された遺伝子は、既知のまたはそれに抵抗するために重要であるかどうかを識別するために使用できる一般的なアプローチを概説細胞自律的な免疫の未知の仲介者。ホスト抗菌因子の初期分析は、野生型および誘導型一酸化窒素合成酵素（iNOS）のgp-91 phoxの（NADPHオキシダーゼ）における特定の遺伝子欠失を有するものに対して、野生型マウス由来のマクロファージにおける変異寄生虫の生存を評価することによって行われ、特異免疫関連分子量GTPase（IRGs）。識別された寄生虫の遺伝子は、一酸化窒素、反応性酸素中間体または免疫関連分子量GTPase 28それぞれまたは未知の免疫機構が関与している場合への抵抗のために重要である場合、これが決定されます。現在のプロトコルに記載されてIFN-γ及びLPSの両方に感染したマクロファージの活性化は、主に一酸化窒素28への抵抗のために重要な寄生虫遺伝子の単離をもたらす。マクロファージ活性化の非存在下で一酸化窒素を誘導する薬理学的薬剤の使用は、（一酸化窒素供与体）が同定された遺伝子の大部分は再ために重要であることが確認されたマクロファージ活性化28に関連する追加メディエーターと協調して一酸化窒素のではなく、一酸化窒素にsistance。 ステップ1と2は、in vitroで感染した骨髄由来マクロファージの活性化に続いてフィットネスの欠陥とは寄生虫の変異体を分離するために設計されたフォワード遺伝学の画面について説明します。第一段階は、寄生虫複製を減少させるが、完全に野生型Tの複製を阻害しないマクロファージの活性化のために使用することが経験的にIFN-γの用量を決定するために、用量滴定分析を説明し、LPS寄生虫の変異体のライブラリーを作成するために使用されるトキソプラズマ親株。ステップ2は、96ウェルプレート中のマクロファージにおける変異クローンのフォワード遺伝子スクリーニングを説明しています。ステップ3は、96ウェルプレートのスクリーニングで同定された各変異体の表現型を確認し、各変異体に欠陥が寄生虫の生存に影響を及ぼすかどうかを評価するために、複製アプローチを概説マクロファージ活性化に応答して、または嚢胞製造。ステップ4つの寄生虫変異体が特に影響を受けやすいれる免疫メディエーターを同定するための特定の抗菌経路の欠失を有するマウスからの骨髄由来マクロファージの使用を記載している。ステップ5は、感染したマウスの脳内シスト生産によって評価される寄生虫の変異体はまた、インビボ病因のために危険にさらされているかどうかを判断するためのアプローチの概要を説明します。

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			<td height="21" style="height:21px;">Vectashield mounting media</td>
			<td>Vector Labs</td>
			<td>H1200</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-conjugated dolichos</td>
			<td>Vector Labs</td>
			<td>FL-1031</td>
			<td>https://www.vectorlabs.com/catalog.aspx?prodID=188</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody to LAMP1</td>
			<td colspan="2">Developmental Studies Hybridoma Bank</td>
			<td>http://dshb.biology.uiowa.edu/LAMP-1</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL6 mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">664</td>
			<td>http://jaxmice.jax.org/strain/000664.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gp91 phox knock out mice</td>
			<td>Jackson Labaoratories</td>
			<td align="right">2365</td>
			<td>http://jaxmice.jax.org/strain/002365.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iNOS knock out mice</td>
			<td>Jackson Laboratories</td>
			<td align="right">2609</td>
			<td>http://jaxmice.jax.org/strain/002609.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium nitroprusside</td>
			<td>ACROS Organics</td>
			<td>AC21164-0250&#160;</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2627727&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DETA NONOate</td>
			<td>ACROS Organics</td>
			<td>AC32865-0250</td>
			<td>https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&#38;langId=-1&#38;catalog 
			Id=29104&#38;productId=2252389&#38;dis 
			type=0&#38;highlightProductsItemsFlag 
			=Y&#38;fromSearch=1&#38;searchType= 
			PROD&#38;hasPromo=1</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Monoclonal mouse anti-Toxoplasma gondii Ab</td>
			<td></td>
			<td>10T19A</td>
			<td>http://1degreebio.org/reagents/product/1069274/?qid=652947</td>
		</tr>
	</tbody>
</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.

トキソプラズマはナイーブマクロファージ内で自由に複製し、寄生虫の株に依存6-12時間の間の倍加時間を持っています。 図1活性化した骨髄由来マクロファージに対してナイーブで代表的な寄生虫を示している。 図2は、HFFで寄生虫の一般的な形態を示す/ PV 2、4、8、16、32寄生虫で宿主細胞。現在のプロトコルでは、寄生虫はナイーブマクロファージに侵入し...

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.

フォワード遺伝学は、識別するためのマクロファージを使用した画面<em&gt;トキソプラズマ</emIFN-γ依存性細胞自律イミュニティへの抵抗のための重要な&gt;遺伝子

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 ビュー.  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. 

ビデオ: 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

高純度の取得トキソプラズマ</em不連続塩化セシウム勾配別>オーシスト

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

免疫・感染学

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.

活性骨髄由来マクロファージとBradyzoite条件におけるトキソプラズマのシスト壁形成

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.

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

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.

の選択熱帯熱マラリア原虫</em人間の脳内皮細胞へのCytoadhesion用>寄生虫

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.

分離するために遺伝学的スクリーニングトキソプラズマホストセル出口変異体

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.

の遺伝的操作<em&gt;Δku80</emの機能ゲノム解析のための&gt;株<em&gt;トキソプラズマ</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.

フォワード遺伝的アプローチで<em&gt;クラミジアトラコマチス</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.

非宿主抵抗性に関与する遺伝子の同定のためのVIGS媒介フォワード遺伝スクリーニング


	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.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

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.