细胞Rho和RacGTP酶在刚地弓形虫入侵宿主细胞过程中的作用研究

发布时间：2018-06-03 18:17

  本文选题：刚地弓形虫 + 纳虫泡膜　； 参考：《南方医科大学》2013年硕士论文

【摘要】：刚地弓形虫(Toxoplasma gondii)是细胞内寄生原虫,能感染多种有核细胞,全世界约有三分之一的人口隐性感染弓形虫。弓形虫感染人体后,在免疫正常人群可由急性感染快速地转为隐性感染,因而危害不明显；而在免疫抑制患者体内会广泛散播,寄生于有核细胞引起炎症,弓形虫主要损害脑、眼和淋巴结,甚至会引起死亡；在孕妇感染后,弓形虫可通过胎盘传播给胎儿,造成流产、死产、死胎、畸胎或新生儿弓形虫病。人类感染弓形虫的途径主要是通过误食未煮熟的中间宿主的肉或被终宿主(猫)排泄的卵囊污染的食物。 弓形虫感染宿主后会刺激机体产生免疫反应以抵抗弓形虫感染,在感染早期,细胞免疫反应在机体内发挥主要抗感染作用。免疫的形成虽可有效地限制感染的发展和新损伤的形成,但一般并不能消除弓形虫感染。多数研究者认为,弓形虫具有带虫免疫的特点。机体只在感染过程中才对再感染具有一定程度的抵抗力,而虫体清除后该种免疫力也消失。弓形虫可以在机体免疫反应环境中保持存活,主要是通过在弓形虫纳虫泡内保持良好增殖和干扰宿主细胞信号转导的方式进行的,但宿主免疫反应只能被部分削弱,而不是完全被消除。弓形虫通过在诱导和抑制宿主免疫反应过程中保持着精致平衡,来保证在宿主细胞内寄生、增殖并有机会传播。弓形虫入侵和寄生过程中有许多宿主细胞信号通路的参与并出现广泛变化,在弓形虫入侵、寄生、增殖及与弓形虫-宿主细胞相互关系中起着重要的作用。 弓形虫感染宿主细胞的机制已被广泛研究,在弓形虫入侵时,速殖子是弓形虫的主要致病虫期,其在细胞内寄生和迅速繁殖破坏宿主细胞,逸出后的裂殖子又侵袭邻近的细胞,如此反复,刺激淋巴细胞、巨噬细胞的浸润,导致组织的急性炎症和坏死。弓形虫侵入细胞的过程依次为：附着于宿主细胞；伸出类锥体；进入宿主细胞；虫体向后移动的运动连接(moving junction, MJ)形成；微线体的分泌作用；棒状体的分泌作用；虫体滑入纳虫空泡,在10s内完成整个侵入过程,虫体进入胞浆后运动较为缓慢,并且有向核移动的特性。在弓形虫侵入宿主细胞过程中会形成纳虫泡(parasitophorous vacuole, PV),在这个过程中,MJ会清除宿主细胞的一些组分(如跨膜蛋白),同时保留大部分的宿主细胞膜组分组成到纳虫泡膜(parasitophorous vacuole membrane, PVM)上,此外因宿主细胞膜中能与溶酶体和内涵体融合的组分已被MJ过滤掉,使PV成为一个非融合小室,有助于其内的速殖子抵抗宿主细胞内含体的融合及溶酶体的酸化作用。 GTP酶(GTPase)是能够与三磷酸鸟苷(guanine triphosphate, GTP)结合并在Mg2+作用下将其水解为二磷酸鸟苷(guanine diphosphate, GDP)的超蛋白家族。小分子GTP酶是相对分子质量为20kDa-40kDa的单体调控性GTP酶,至少由5个家族组成,分别是:Ras、Rho、Rab、Sar1/Arf和Ran。目前研究较多的为Ras和Rho家族。其中Rho族蛋白主要包括Rho、Rac和Cdc42三个亚家族,这些蛋白最主要的功能是调节细胞骨架和肌动蛋白重组,其他功能包括调控基因转录、细胞周期和膜泡运输等。 Rho GTP酶最主要的功能是调节肌动蛋白和细胞骨架的重组,从而调节细胞的形态变化和运动。该族蛋白的活化能促使细胞外基质的降解、破坏上皮层的结构和增加细胞的运动性。Rho族蛋白的活化和失活在真核细胞的许多基础性生命活动中都起着关键的作用。弓形虫入侵宿主细胞的过程伴随着宿主细胞结构的重组,包括宿主细胞中心粒及高尔基复合体定位在纳虫泡上,线粒体、微管及溶酶体被纳入了纳虫泡的边缘。 弓形虫感染宿主细胞时,宿主细胞由干扰素诱导产生的GTP酶(IFN-y-Inducible GTPase6, Irga6)和小分子GTP酶ARF6可聚集在弓形虫PVM上,Irga6可以抵抗小鼠被弓形虫感染,Irga6在未感染的细胞内主要是以GDP非活化结合状态存在,但当弓形虫感染细胞后Irga6会聚集在PVM上并且转化为GTP活化状态存在,Irga6的这种聚集最终导致PVM的破裂。ARF6通过激活PI3K、PIP2和PIP3一起参与纳虫泡的形成,并且是通过聚集在PVM上起重要作用的。GTP酶在弓形虫入侵宿主过程中起所起的重要作用促使我们进一步去探讨,是否其它小分子GTP酶也同样参与了弓形虫感染宿主细胞的过程。 目的： 1.分析在弓形虫感染宿主细胞时,宿主细胞的RhoA和Racl GTP酶是否组成到弓形虫的PVM上。 2.了解RhoA和Rac1GTP酶负显性突变体是否组成到弓形虫的PVM上。 3.分析弓形虫速殖子感染后宿主细胞内源性RhoA和Rac1GTP酶的活化状态。 4.探讨RhoA GTP酶在弓形虫PVM上聚集时哪些结构域是必须的。 5.分析聚集在PVM上的RhoA和Rac1GTP酶的活性状态是否依赖其本身的GTP酶活性。 6.探讨细胞RhoA和Rac1GTP酶的缺失是否影响弓形虫速殖子入侵宿主细胞效率。 方法： 1.转染pECFP-RhoA-WT和pECFP-Rac1-WT质粒于COS-7细胞,48hr后细胞分别用弓形虫RH株和Pru株速殖子侵染2hr,4%多聚甲醛固定细胞30min后,用DAPI室温下染色10mmin,荧光显微镜下观察CFP标签的RhoA和Rac1是否被组成到纳虫泡膜上。 2.弓形虫RH速殖子感染16HBE细胞后,用间接免疫荧光法对内源性RhoA和Rac1进行定位。 3.弓形虫速殖子感染转染了pECFP-RhoA-WT于COS-7细胞后,在感染5min后用共聚焦显微法每10min实时观察宿主细胞RhoA GTP酶在PVM上的聚集情况。 4.分别转染RhoA和Racl负显性突变体(pECFP-RhoA-N19和pECFP-Racl-N17)于COS-7细胞中,48hr后用速殖子侵染2hr,4%多聚甲醛固定细胞30mmin后,用DAPI室温下染色10min,荧光显微镜下观察非活化的RhoA和Rac1是否会聚集在纳虫泡膜上。 5.16HBE细胞用RH株速殖子感染后收集细胞,用RIPA试剂裂解,120ug总蛋白用于Rho GST pull down,检测弓形虫入侵是否激活RhoA和Rac1GTP酶。 6.以pECFP-RhoA-WT为亲本质粒,构建RhoA每10个氨基酸缺失的突变体19个即M1-M19。将M1-M19转化入COS-7细胞,48hr后用RH株速殖子侵染2hr,4%多聚甲醛固定细胞30min后,用DAPI室温下染色10min,荧光显微镜下观察各突变体RhoA在纳虫泡膜上的聚集情况。 7.COS-7细胞转染pECFP-RhoA-WT后速殖子侵染2hr,100nM EGF加于六孔板一角,激活5min后用多聚甲醛固定细胞,DAPI室温下染色,荧光显微镜下观察RhoA的转位情况。 8.宿主细胞RhoA和Racl的活性及存在与弓形虫感染率关系的研究： a.正常COS-7细胞COS-7细胞分别转染pECFP-RhoA-WI、pECFP-RhoA-N19和pECFP-Racl-WT、pECFP-Rael-N17,48hr后速殖子侵染2hr,多聚甲醛固定细胞,Giemsa染色,计算细胞感染率并进行统计学分析,比较正常细胞与转染细胞的感染率是否有统计学差异。 b.正常16HBE细胞及单独转染RhoA-siRNA、Rac1-siRNA和共同RhoA-siRNA和Racl-siRNA转染组的16HBE细胞,用速殖子侵染2hr,多聚甲醛固定细胞,Giemsa染色,计算细胞感染率并进行统计学分析,比较正常细胞与转染细胞的感染率是否有统计学差异。 结果： 1.小分子GTP酶-RhoA和Racl在弓形虫感染宿主细胞时会在PVM上聚集。 2. RhoA和Racl GTP酶在PVM上的聚集以GTP酶活性状态存在。 3.弓形虫速殖子的感染可以激活宿主细胞内源性RhoA和Racl GTP酶。 4. RhoA GTP酶依赖不同的RhoA结构域在弓形虫PVM上聚集。 5.细胞RhoA和Racl GTP酶为弓形虫速殖子有效入侵宿主细胞所必需。 结论： 1.无论弓形虫弱毒株还是强毒株速殖子,宿主细胞RhoA和Racl GTP酶在弓形虫速殖子入侵时都会聚集在PVM上,而且这种聚集依赖其完整的GTP酶活性。 2.弓形虫速殖子感染细胞可以激活内源性RhoA和Racl GTP酶,并且在弓形虫刚开始入侵时RhoA和Racl GTP酶就会在PVM上聚集,聚集的RhoA和Racl GTP酶来自细胞膜或者细胞质。 3.决定RhoA在PVM上聚集的结构域有:GTP/Mg2+结合位点、Mdia效应器作用位点、G1box、G2box和G5box。 4.表皮生长因子(EGF)刺激感染的宿主细胞后,RhoA和Racl GTP酶在PVM上并没有发生移位现象,这显示RhoA和Racl可能以GTP活化形式存在于PVM上。 5.细胞过表达pECFP-RhoA-WT、pECFP-Rac1-WT、pECFP-RhoA-N19、 pECFP-Racl-N17和将细胞内RhoA和Racl GTP酶基因沉默后对细胞感染率的统计数据显示RhoA和Racl GTP酶为弓形虫有效入侵所必需,RhoA和Racl GTP酶在PVM上聚集及其必须的生物活性都显示RhoA和Racl GTP酶参与并作用于弓形虫速殖子感染宿主细胞这一过程。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a parasitic protozoa in cells and can infect a variety of nucleated cells. About 1/3 of the population in the world recessive infection of Toxoplasma gondii. After Toxoplasma infection, the immune normal population can be rapidly transformed from acute infection to recessive infection, and the harm is not obvious. "Spread, parasitic on nuclear cells causing inflammation, and Toxoplasma mainly damage the brain, eyes and lymph nodes and even cause death; after infection, Toxoplasma can be transmitted to the fetus through the placenta, causing abortion, stillbirth, stillbirth, Teratosis, or neonatal toxoplasmosis. Human infection of Toxoplasma gondii is mainly through misfeeding of uncooked middle Lodge." Food contaminated by the main oocysts or the excretory oocysts of the final host (CAT).
When Toxoplasma infects the host, it can stimulate the body to produce immune response to resist Toxoplasma infection. In the early stage of the infection, the cellular immune response plays a major anti infection role in the body. Although the formation of immune formation can effectively restrict the development of infection and the formation of new injuries, the infection can not be eliminated. Most researchers believe that the toxoplasmosis is arcuate. The insect has the characteristics of immunization with insects. The body has a certain degree of resistance to reinfection only in the process of infection, and the immunity of the species is disappearing after the insect body is cleared. Toxoplasma can remain alive in the immune response environment of the body, mainly by maintaining good proliferation and interfering with the signal transduction of host cells in the Toxoplasma Nana vesicles. The host immune response can only be partially weakened, rather than completely eliminated. Toxoplasma maintains a delicate balance in the induction and suppression of host immune responses to ensure parasitism in the host cell, proliferate and spread organically. In the process of Toxoplasma invasion and parasitism, there are many host cell signaling pathways. And extensive changes play an important role in the invasion, parasitism, proliferation and Toxoplasma gondii host cell interactions.
The mechanism of Toxoplasma infection in host cells has been widely studied. At the time of the invasion of Toxoplasma gondii, tachyonus is the main pathogenic stage of Toxoplasma gondii. The parasitic and rapid propagation of the parasitic and rapid propagation of the host cells, the escape of the merozoites and the invasion of adjacent cells, so repeated, stimulating gonadal cells, macrophage infiltration, causing acute inflammation of the tissue. Disease and necrosis. The process of toxoplasmosis invades the cells in sequence: attaching to the host cell; extending the cones; entering the host cell; the movement of the moving junction, MJ; the secretory action of the micro body; the secretory effect of the rod; the insect body slips into the vacuoles of the NETA, and completes the whole invasion process within 10s. When the body enters the cytoplasm, it moves slowly and has the characteristics of moving to the nucleus. In the process of Toxoplasma invasion, parasitophorous vacuole (PV) can be formed. In this process, MJ will remove some components of the host cell (such as transmembrane protein), and retain most of the host cell membrane components to form the nanofilm membrane (PA Rasitophorous vacuole membrane, PVM), in addition, the components that can be fused with lysosomes and endosomes in the host cell membrane have been filtered out by MJ, making PV a non fusion chamber, helping the tachyonus to resist the fusion of the host cell and the acidification of the lysosome.
GTP enzyme (GTPase) is a superprotein family that combines with guanine triphosphate (guanine triphosphate, GTP) and hydrolyze it into guanosine two phosphate (guanine diphosphate, GDP) under the action of Mg2+. The small molecule GTP enzyme is a monomeric regulative GTP enzyme with relative molecular mass as 20kDa-40kDa, at least 5 families. Ras and Rho families, which are currently studied by Ran., are mainly composed of three subfamilies of Rho, Rac and Cdc42. The main functions of these proteins are the regulation of cytoskeleton and actin, and other functions include regulation of gene transcription, cell cycle and vesicle transport.
The main function of Rho GTP enzyme is to regulate the reorganization of actin and cytoskeleton, thereby regulating the morphological changes and movement of cells. The activation of this protein can induce the degradation of the extracellular matrix, destroy the structure of the epithelial layer and increase the activation and inactivation of the motile.Rho protein of the cell in many basic life activities of the eukaryotic cells. The process of the invasion of the host cell of Toxoplasma gondii is accompanied by the reorganization of the host cell structure, including the host cell centroid and the Golgi complex located on the nanoscale vesicles, and the mitochondria, microtubules and lysosomes are incorporated into the edge of the nanoscale vesicles.
When Toxoplasma infects host cells, the GTP enzyme (IFN-y-Inducible GTPase6, Irga6) and small molecule GTP enzyme ARF6 induced by interferon can accumulate on the Toxoplasma PVM. Irga6 can resist the infection of mice by Toxoplasma gondii. Irga6 is mainly in the non activated binding state of GDP in the uninfected cells, but when Toxoplasma infected cells are infected. Irga6 will accumulate on PVM and convert to GTP activation state. This aggregation of Irga6 eventually leads to the disruption of PVM by.ARF6 by activating PI3K, PIP2 and PIP3 to participate in the formation of Nana vesicles, and is an important role of the.GTP enzyme that plays an important role in the PVM and plays an important role in the invasion of Toxoplasma gondii. One step is to explore whether other small GTP enzymes are also involved in the process of Toxoplasma infection in host cells.
Objective:
1. to analyze whether the RhoA and Racl GTP enzymes of the host cells are on the PVM of Toxoplasma gondii.
2. understand whether RhoA and Rac1GTP negative dominant mutants are on the PVM of Toxoplasma gondii.
3. to analyze the activation of endogenous RhoA and Rac1GTP enzymes in Toxoplasma gondii infected tachyhost.
4. to explore which domains are necessary for RhoA GTP enzyme to accumulate on PVM of Toxoplasma gondii.
5. analyze whether the activity of RhoA and Rac1GTP enzymes aggregated on PVM depends on their GTP enzyme activity.
6. to investigate whether the deletion of RhoA and Rac1GTP enzymes affects the efficiency of Toxoplasma gondii in the invasion of host cells.
Method锛，

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