Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴宿主-寄生虫相互作用建模

• 批准号: RGPIN-2014-04023
• 负责人: Chadee, Khrisendath
• 金额: $ 2.19万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=587559
• 关键词: Modeling; Entamoeba; histolytica; host; parasite

Entamoeba histolytica (Eh) is a parasite that either harmlessly colonizes the mucus layer or trigger disease when it invades the colon. Upon sensing parasites, innate immune cells must determine the level of danger to the host and initiate appropriate responses. One way to discriminate the degree of parasite threat is to distinguish between direct contact with parasite surfaces vs components that are secreted/released at a distance. The sensors and cellular responses selectively activated by direct contact with innate immune cells are unknown for ameba. Direct contact of live Eh with host tissue/cells elicits a response that is different in both magnitude and quality compared to responses elicited by released Eh components. We recently identified Eh activates the NLRP3 inflammasome through a contact-dependent event to regulate release of bioactive IL-1ß, which correlates in vivo where IL-1ß is selectively released in response to invading parasites. This indicates NLRP3 inflammasome 1), senses direct binding of Eh to innate immune cells and 2), is a central pathway for distinguishing intact invasive parasites to focus certain host defenses only when/where they are required. Studies in animal models of disease have demonstrated the major Eh surface adhesin, the Gal-lectin, is critical for activating anti-Eh defenses. The Gal-lectin mediates Eh attachment to MUC2 mucin during colonization and host cells during invasion by binding Gal/GalNAc residues on mucin and unknown host cell receptors, respectively. We identified NLRP3 activation requires Eh binding via surface-associated Gal-lectin. Native soluble Gal-lectin is immunostimulatory and induces robust TNF-a but it does not activate the NLRP3 inflammasome. Thus, signals conveyed by specific parasite molecules acting upon the host cell deliver critical information about the nature of the parasite encounter, which are distinguished and translated into responses that match the type of parasite threat. Our data suggest contact directly provokes immune activation via an adherence mechanism involving the Gal-lectin and hypothesize that a contact-dependent signaling synapse containing the Gal-lectin engages a signaling cascade leading to NLRP3 inflammasome activation. The study aima are 1, to determine innate immune cell responses that are contact-dependent vs contact-independent, define those that are mediated by surface-associated Gal-lectin vs native soluble Gal-lectin, and which ones require activation of the NLRP3 inflammasome and, 2 to determine the molecular mechanism of the contact-dependent-Gal-lectin-signaling synapse that activates NLRP3 inflammasome. For aim 1 we will use multiplex cytokine array to assess secretion of up to 60 infection-associated cytokines to define innate immune cell signatures reflecting the quality/magnitude of contact vs. non-contact modes. For aim 2 we have already identified a5ß1 integrin is a surface receptor for Eh-induced NLRP3 activation that requires binding and activation by EhCP5. However, despite binding to a5ß1 integrin, purified EhCP5 induces weak activation and alone does not trigger NLRP3. Full a5ß1 integrin activation and NLRP3 induction simultaneously requires surface bound Gal-lectin. Confocal microscopy revealed a5ß1 integrin, p-paxillin (active integrin signaling) and NLRP3 rapidly localize to the site of Eh contact, suggesting Gal-lectin regulates a5ß1 integrin recruitment. We will test if Gal-lectin immobilize on the surface of Eh recruits a5ß1 integrin into an active signaling complex containing Gal-lectin, EhCP5, a5ß1 integrin and NLRP3. We will determine whether NLRP3 is recruited following a5ß1 integrin activation and whether a5ß1 integrin and its active signaling components interact directly with NLRP3 to regulate its activity.

溶组织内阿米巴（Eh）是一种寄生虫，它可以无害地寄居在粘液层，也可以在侵入结肠时引发疾病。 在感知寄生虫时，先天免疫细胞必须确定对宿主的危险程度并启动适当的反应。 区分寄生虫威胁程度的一种方法是区分与寄生虫表面的直接接触与在一定距离处分泌/释放的组分。阿米巴与先天免疫细胞直接接触，选择性激活的感受器和细胞反应是未知的。活Eh与宿主组织/细胞的直接接触引起的反应是不同的幅度和质量相比，释放Eh组件引起的反应。我们最近发现Eh通过接触依赖性事件激活NLRP 3炎性小体，以调节生物活性IL-1 β的释放，这与体内IL-1 β响应入侵寄生虫而选择性释放有关。这表明NLRP 3炎性体1）感知Eh与先天免疫细胞的直接结合，以及2）是区分完整的侵入性寄生虫的中心途径，以仅在需要时/在需要时集中某些宿主防御。在疾病动物模型中的研究已经证明，主要的Eh表面粘附素，半乳糖凝集素，是激活抗Eh防御的关键。半乳糖凝集素介导Eh附着在粘蛋白和未知的宿主细胞受体上的Gal/GalNAc残基，分别在殖民化和宿主细胞入侵期间的粘蛋白和MUC 2粘蛋白。我们确定NLRP 3激活需要Eh通过表面相关的半乳糖凝集素结合。天然的可溶性半乳糖凝集素是免疫刺激性的，并诱导强大的TNF-α，但它不激活NLRP 3炎性体。因此，由作用于宿主细胞的特定寄生虫分子传递的信号传递了关于寄生虫遭遇的性质的关键信息，这些信息被区分并转化为与寄生虫威胁类型相匹配的反应。我们的数据表明，接触直接引发免疫激活通过一个涉及半乳糖凝集素的粘附机制，并假设一个接触依赖性信号突触含有半乳糖凝集素从事信号级联导致NLRP 3炎性小体激活。本研究的目的是：1、确定接触依赖性与接触非依赖性的先天免疫细胞应答，定义由表面相关的Gal-凝集素与天然可溶性Gal-凝集素介导的那些应答，以及哪些应答需要NLRP 3炎性体的激活，2、确定激活NLRP 3炎性体的接触依赖性Gal-凝集素信号传导突触的分子机制。对于目标1，我们将使用多重细胞因子阵列来评估多达60种感染相关细胞因子的分泌，以定义反映接触与非接触模式的质量/幅度的先天免疫细胞特征。对于目标2，我们已经鉴定了α 5 β 1整联蛋白是Eh诱导的NLRP 3活化的表面受体，其需要EhCP 5的结合和活化。然而，尽管与α 5 β 1整联蛋白结合，纯化的EhCP 5诱导弱活化，单独不触发NLRP 3。完全的α 5 β 1整联蛋白活化和NLRP 3诱导同时需要表面结合的Gal-凝集素。共聚焦显微镜显示α 5 β 1整联蛋白、β-桩蛋白（活性整联蛋白信号传导）和NLRP 3快速定位于Eh接触位点，表明Gal-凝集素调节α 5 β 1整联蛋白募集。我们将测试Eh表面上的Gal-凝集素是否将α 5 β 1整联蛋白募集到含有Gal-凝集素、EhCP 5、α 5 β 1整联蛋白和NLRP 3的活性信号传导复合物中。我们将确定NLRP 3是否在α 5 β 1整联蛋白活化后被募集，以及α 5 β 1整联蛋白及其活性信号组分是否直接与NLRP 3相互作用以调节其活性。