JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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면역학 및 감염병학

正向遗传学屏风使用巨噬细胞识别<em>弓形虫</em>基因的重要的抗IFN-γ依赖性细胞免疫自主

Published: March 12, 2015
doi:
10.3791/52556
, , , , , , ,
1Department of Microbiology and Immunology,New York Medical College

Summary

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.

Abstract

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 naïve 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.

Introduction

弓形虫（弓形虫）是一种专性细胞内，原虫病的病原体。这是弓形体病的病原体，免疫功能低下的个体健康危险。这也是其他顶复门病原体感染人类，包括隐孢子虫和模型系统。弓形体病是通过食物或水沾染寄生虫的缓殖子或卵囊阶段摄入最常获得的。一旦摄入，这些阶段转换为复制的宿主细胞内，并系统地传播寄生虫的速殖子阶段。 T细胞，IFN-γ和，在较小程度上，一氧化氮1-4，是用于控制感染重要的，但不能够消除疾病，如速殖子的比例转换为被组织囊肿内保护缓殖子阶段产生一个长寿命的慢性感染。事实上，还没有治疗剂有效对抗慢性囊肿s踏歌疾病。严重弓形体病是最常见的是由于持续感染的再活化，与寄生虫转换回的初级和急性感染迅速复制速殖子阶段特性的缓殖子阶段。

早生存于先天免疫应答的面是非常重要的，以允许寄生虫达到足够寄生虫数目，以及到达末端位置，以使建立慢性感染的T。弓形虫已经发展战略，以抵消可能的复制和传播感染的早期能力有助于宿主的防御机制。首先，T.弓形虫形成寄生虫入侵期间的唯一的PV是从该宿主细胞的相比其他细胞内病原体5-9内吞和胞吐过程基本上隔离。此外，像所有成功的细胞内病原体T.弓形虫修改它的宿主细胞创造一个宽松的环境˚F或生长。这包括重编程的宿主细胞的基因表达，通过改变宿主细胞的转录因子包括那些用于调节细胞活化10-15重要。 ROP16 16-19 GRA15 20，GRA16 21和GRA24 22都被证明在调节转录反应和细胞信号感染T.宿主细胞的级联是重要弓形虫 。使用寄生虫株不同的表型之间的遗传杂交最近的研究已经查明背后的寄生虫基因相关的性状，包括免疫相关的GTP酶（IRGs）16,19,23-26逃避寄生虫基因高产。在小鼠中，免疫相关GTP酶（IRGs）是用于第二类型的控制和III基因型寄生虫的临界而很毒的I型基因型已经进化机制以逃避鼠IRGs。然而，同样明显的是寄生虫已经进化机制以逃避抗菌媒体除了​​IRGs和一些这些机制器可跨越寄生虫基因型27,28保守的。此外，很少有人知道的细胞自主免疫力T.关键调解人人类弓形体病弓形虫期间。寄生虫基因抗性的细胞中自主免疫介重要也可以是用于生存重要期间速殖子要缓殖子转换其也可以通过宿主的免疫反应引起的。例如，一氧化氮在高的水平，可以抑制在受感染的巨噬细胞寄生虫复制，但它也可以刺激速殖子至缓殖子转变导致囊肿生产30-32。

ToxoDB是一种功能性基因组数据库T.弓形虫充当对字段中提供的序列信息用于寄生虫的基因组，并获得公布和未公布的基因组尺度的数据包括社区注解方面的重要资源，基因EXP ression和蛋白质组学数据33。类似于许多原虫病原体，多数基因组由基于基因同源性为深入了解其潜在功能可以假设基因，没有信息。因此，正向遗传学是一种强大的工具，以确定新的寄生虫基因免疫逃避，囊肿转换等功能为寄生虫的发病机制，以及对不同的发育阶段之间的转换关键重要。正向遗传学的附加优点是，它可被用作一个相对无偏见的方式来询问寄生虫为那些对在发病特定的任务，包括免疫逃避和囊肿形成重要的基因。在新一代测序的突变分析近期的改进，使它的首选识别从正向遗传学研究的负责疟原虫基因用化学方法和插入突变34-37的方法。

ntent“>重要的是要确定在弓形虫漏洞可以被利用以提高的细胞中自主免疫机制对寄生虫特别是那些也可以有效对抗耐药囊肿阶段。为了实现这一目的， 在体外鼠的效力是很重要的巨噬细胞的感染和激活模型的开发是为了确定特异性削弱弓形虫健身以下活化感染的巨噬细胞，但不是在幼稚的巨噬细胞的寄生虫的突变。该巨噬细胞的屏幕被用于询问弓形虫插入突变体文库，以最终识别弓形虫基因抗性很重要的一氧化氮27,28。 弓形虫突变体活化感染的巨噬细胞，特别是一个显着的敏感性，一氧化氮的受损性的面板的分离，证实在屏幕的效用，以确定寄生虫基因性重要到的细胞中自主免疫比鼠IRGs 28中描述的抗性机制的其他介质。插入突变有超过化学诱变优点在产生的随机突变在每个寄生虫克隆和，在理论上，便于识别突变位点的数量有限的条款。然而，确定质粒插入的T.的基因组位点弓形虫插入突变体，在实践中，已经令人惊奇地难以在许多情况下，37。一个质粒导入的基因的插入也有可能打乱一个基因的功能在对比化学诱变通常导致单核苷酸变化。然而，化学诱变是N-乙基-N-亚硝基脲（ENU）或甲磺酸乙酯（EMS），可以提供分析寄生虫基因组的较大部分增加的能力，相对于插入诱变，因为它创建多个单核苷酸多态性（每突变体34估计为10 -100）38。此外，在全基因组谱的最新进展使得有可能使用下一代测序，以确定负责突变寄生虫34,38的所识别的表型是最有可能的候选基因。不管诱变方法，确认该寄生虫基因抗性巨噬细胞活化中的作用的最终需要的基因缺失和互补履行分子柯赫氏法则。

解剖的基因通过两个寄生虫和巨噬细胞的遗传操作的功能的能力是重要的，因为许多经由正向遗传学T中鉴定的基因的弓形虫，以及其它病原体，仍然表征为假想的基因几乎没有序列同源性的其他蛋白与已知功能。当前文件概述了可被用于识别在一个突变体被破坏的基因是否是用于抵抗重要到一个已知的或一般的做法未知调解员的细胞自主的免疫力。通过评估野生型和从野生型小鼠与那些与诱导型一氧化氮合酶（iNOS），GP-91 PHOX（NADPH氧化酶）特异性基因缺失突变体的巨噬细胞的寄生虫的存活进行的宿主抗微生物因素最初的分析，并特异性免疫相关的GTP酶（IRGs）。这将确定是否所识别的寄生虫基因是抗一氧化氮，活性氧中间体或分别或者如果未知免疫机制参与免疫相关GTP酶28重要。受感染的巨噬细胞与这两种IFN-γ和LPS，在当前协议中描述的活化，导致主要在寄生虫基因抗性很重要的一氧化氮28的隔离。采用药理学试剂诱导的一氧化氮在没有巨噬细胞活化的（一氧化氮供体）证实，大多数鉴定的基因的人重新重要sistance一氧化氮，而不是一氧化氮的音乐会与巨噬细胞活化28相关的其他调解员。

步骤1和2描述了一个向前遗传学屏幕旨在隔离寄生虫突变体与健身缺陷以下体外活化感染的骨髓衍生巨噬细胞。步骤1描述了一个剂量滴定分析来凭经验确定IFN-γ和LPS的剂量用于巨噬细胞活化，减少寄生虫复制，但不能完全抑制野生型T的复制弓形虫父母菌株用于创建寄 ​​生虫突变体库。步骤2描述了突变体克隆在96孔板的巨噬细胞的前向遗传屏幕。步骤3概述的方法来确认鉴定在96孔板的屏幕，并评估在每个突变体中的缺陷是否影响寄生虫存活，复制每个突变体的表型，或囊肿生产响应于巨噬细胞的活化。步骤4描述了使用骨髓衍生的巨噬细胞的小鼠与特定抗微生物途径缺失来识别免疫介质到的寄生虫突变体是特别敏感的。步骤5概述的方法，以确定是否一个寄生虫突变体也遭到了破坏用于体内发病如在感染小鼠的脑中评价由囊肿的生产。

Protocol

注：包括使用动物的所有协议均按照规定由纽约医学院的动物护理和使用委员会的指导原则和规定执行。 注：详细有限稀释38，小鼠骨髓巨噬细胞39的隔离，T.增长协议化学诱变38，寄生虫孤立弓形虫在人包皮成纤维细胞（HFF）细胞和囊肿生产的巨噬细胞和基本免疫荧光分析（IFA）32顷引用。进行，在37℃的所有细胞培养在5％CO 2在D10培养基…

Representative Results

弓形虫自由复制在幼稚的巨噬细胞，并具有6-12小时取决于该寄生虫的应变之间的倍增时间。 图1示出了在相对 ​​于幼稚激活骨髓衍生的巨噬细胞代表的寄生虫。 图2示出了寄生在HFF的一般形态宿主细胞在2，4，8，16和32个寄生虫/ PV。在当前的协议，该寄生虫可以侵入幼稚的巨噬细胞，并建立一个新生虫空泡（PV）前的强效激活刺激，LPS和IFN-的输送，对受感染的巨…

Discussion

所描述的协议提供了使用活化的鼠骨髓衍生巨噬细胞和向前遗传学分离T的非偏置的方式弓形虫突变体在自己的能力有缺陷的生存激活的巨噬细胞感染的。突变体以下的巨噬细胞的活化的表型通常分为两大类：1）的寄生虫出现完整的，但无法复制超过每光伏1寄生虫; 2）寄生虫出现退化并可能宽敞，无定形的PV。该突变体具有类似于在没有活化的幼稚的巨噬细胞的野生型寄生虫的表型?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Name of the material Company Catalog number Comments
DMEM Hyclone SH3008101 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29101&productId=3255471&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=0
Hyclone FBS Thermo SH3091003 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=11737973&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=0
Hyclone DPBS Thermo SH3002802  https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=2434305&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType
=PROD&hasPromo=0
Hyclone L-glutamine Thermo SH3003401  https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=3311957&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType
=PROD&hasPromo=0
Hyclone Pen strep Thermo SV30010  https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&productId=1309668
6&catalogId=29104&matchedCat
No=SV30010&fromSearch=1&
searchKey=SV30010&highlightPro
ductsItemsFlag=Y&endecaSearch
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&searchType=PROD&hasPromo=0
Hyclone Hanks BSS Thermo SH3003002 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=3064595&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=0
LPS LIST biologicals 201 http://www.listlabs.com/products-tech.php?cat_id=4&product_id=81&keywords
=LPS_from_%3Cem%3EEscherichia_coli%3C/em%3E_O111:B4
IFN-g Pepro Tech Inc 50-813-664 https://www.fishersci.com/ecomm/servlet/itemdetail?itemdetail='item'&storeId=10652&
productId=2988494&catalogId=29
104&matchedCatNo=50813664&
fromSearch=1&searchKey=murine+ifn+pepro+tech&highlightProductsItemsFlag
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=0.0&xrefEvent=1407778210608_
12&searchType=PROD&hasPromo
=0
Chamber slides Thermo 177402 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=2164545&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=0
96-well optical plates Thermo 165306 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=3010670&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
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96-well tissue culture plates 353072 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=3158736&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=0
Tissue culture flast T25 156367 https://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&productId=127039
67&catalogId=29104&matchedCat
No=12565351&fromSearch=1&
searchKey=156367&highlightProdu
ctsItemsFlag=Y&endecaSearchQu
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Ted Pella EM grade formaldehyde 18505 http://www.tedpella.com/chemical_html/chem3.htm#anchor267712
Triton X-100 Fisher BP151 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=3425922&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=1
Alexa 488 – protein conjugation kit Life Technologies A20181 http://www.lifetechnologies.com/order/catalog/product/A10235
goat serum MP Biomedicals ICN19135680 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=2133236&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&crossRefData
=ICN19135680=2&searchType
=PROD&hasPromo=0
Vectashield mounting media Vector Labs H1200 https://www.vectorlabs.com/catalog.aspx?prodID=428
FITC-conjugated dolichos Vector Labs FL-1031 https://www.vectorlabs.com/catalog.aspx?prodID=188
Antibody to LAMP1 Developmental Studies Hybridoma Bank http://dshb.biology.uiowa.edu/LAMP-1
LysoTracker Life Technologies L-7526 https://www.lifetechnologies.com/order/catalog/product/L7526?ICID=search-product
C57BL6 mice Jackson Laboratories 664 http://jaxmice.jax.org/strain/000664.html
gp91 phox knock out mice Jackson Labaoratories 2365 http://jaxmice.jax.org/strain/002365.html
iNOS knock out mice Jackson Laboratories 2609 http://jaxmice.jax.org/strain/002609.html
sodium nitroprusside ACROS Organics AC21164-0250  https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=2627727&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=1
DETA NONOate ACROS Organics AC32865-0250 https://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&langId=-1&catalog
Id=29104&productId=2252389&dis
type=0&highlightProductsItemsFlag
=Y&fromSearch=1&searchType=
PROD&hasPromo=1
Monoclonal mouse anti-Toxoplasma gondii Ab 10T19A http://1degreebio.org/reagents/product/1069274/?qid=652947
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Toxoplasma GondiiMacrophagesForward Genetics ScreenIFN-γCell Autonomous ImmunityParasite MutantsParasite GenesChronic InfectionTachyzoiteBradyzoite
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Walwyn, O., Skariah, S., Lynch, B., Kim, N., Ueda, Y., Vohora, N., Choe, J., Mordue, D. G. Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity. J. Vis. Exp. (97), e52556, doi:10.3791/52556 (2015).
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