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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566247
• 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，它要么无害地寄居在粘液层，要么在侵入结肠时引发疾病。在感知到寄生虫后，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和远距离分泌/释放的成分。对于阿米巴来说，通过与先天免疫细胞直接接触而选择性激活的传感器和细胞反应是未知的。与释放的 Eh 成分引起的反应相比，活 Eh 与宿主组织/细胞的直接接触引起的反应在幅度和质量上都不同。我们最近发现 Eh 通过接触依赖性事件激活 NLRP3 炎症小体，调节生物活性 IL-1ß 的释放，这与体内选择性释放 IL-1ß 以响应入侵寄生虫的情况相关。这表明 NLRP3 炎性体 1) 感知 Eh 与先天免疫细胞的直接结合，2) 是区分完整侵入性寄生虫的中心途径，仅在需要时/地点集中某些宿主防御。疾病动物模型研究表明，主要的 Eh 表面粘附素半乳糖凝集素对于激活抗 Eh 防御至关重要。 Gal-凝集素通过分别结合粘蛋白和未知宿主细胞受体上的 Gal/GalNAc 残基，介导 Eh 在定植期间和宿主细胞入侵期间与 MUC2 粘蛋白的附着。我们发现 NLRP3 激活需要通过表面相关半乳糖凝集素结合 Eh。天然可溶性半乳糖凝集素具有免疫刺激作用，可诱导强效 TNF-a，但不会激活 NLRP3 炎症小体。因此，作用于宿主细胞的特定寄生虫分子传递的信号传递有关寄生虫遭遇性质的关键信息，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，接触通过涉及半乳糖凝集素的粘附机制直接引发免疫激活，并假设含有半乳糖凝集素的接触依赖性信号突触参与信号级联，导致 NLRP3 炎性体激活。该研究的目的是 1，确定接触依赖性与接触非依赖性的先天免疫细胞反应，定义由表面相关半乳糖凝集素与天然可溶性半乳糖凝集素介导的反应，以及哪些需要激活 NLRP3 炎症小体，2 确定激活 NLRP3 炎症小体的接触依赖性半乳糖凝集素信号突触的分子机制。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/程度的先天免疫细胞特征。对于目标 2，我们已经确定 a5ß1 整合素是 Eh 诱导的 NLRP3 激活的表面受体，需要 EhCP5 的结合和激活。然而，尽管与 a5ß1 整合素结合，纯化的 EhCP5 仍会诱导微弱的激活，并且单独不会触发 NLRP3。完整的 a5ß1 整合素激活和 NLRP3 诱导同时需要表面结合的半乳糖凝集素。共聚焦显微镜显示 a5ß1 整合素、p-paxillin（主动整合素信号传导）和 NLRP3 快速定位到 Eh 接触位点，表明半乳糖凝集素调节 a5ß1 整合素募集。我们将测试固定在 Eh 表面的半乳糖凝集素是否将 a5ß1 整合素募集到含有半乳糖凝集素、EhCP5、a5ß1 整合素和 NLRP3 的活性信号复合物中。我们将确定 NLRP3 是否在 a5ß1 整合素激活后被招募，以及 a5ß1 整合素及其活性信号成分是否直接与 NLRP3 相互作用以调节其活性。