Modeling Entamoeba histolytica host-parasite interactions

溶组织内阿米巴（Eh）是一种寄生在黏液层上的寄生虫，当它侵入结肠时，要么无害，要么引发疾病。一旦感知到寄生虫，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直接接触寄生虫表面与远距离分泌/释放的成分。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对于阿米巴来说是未知的。活性Eh与宿主组织/细胞直接接触所引起的反应，与释放的Eh组分引起的反应相比，在强度和质量上都不同。我们最近发现Eh通过接触依赖事件激活NLRP3炎性体，以调节生物活性IL-1ß的释放，这与体内IL-1ß选择性释放以响应入侵寄生虫有关。这表明NLRP3炎性体1)感知Eh与先天免疫细胞的直接结合，2)是区分完整侵袭性寄生虫的核心途径，仅在需要时/在需要时聚焦某些宿主防御。动物疾病模型的研究表明，主要的Eh表面粘附素，即凝集素，对于激活抗Eh防御至关重要。Gal-凝集素分别通过结合Gal/GalNAc残基在粘蛋白和未知宿主细胞受体上，介导Eh在定植期间和侵袭期间与MUC2粘蛋白和宿主细胞的附着。我们发现NLRP3的激活需要Eh通过表面相关的凝集素结合。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，但不激活NLRP3炎性体。因此，特定寄生虫分子传递的信号作用于宿主细胞，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gal -凝集素的粘附机制直接激发免疫激活，并假设含有gal -凝集素的接触依赖信号突触参与信号级联，导致NLRP3炎性体激活。本研究的目的是：1，确定接触依赖性和接触非依赖性先天免疫细胞反应，定义由表面相关凝集素和天然可溶性凝集素介导的反应，以及哪些反应需要激活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，以定义先天免疫细胞特征，反映接触与非接触模式的质量/大小。对于目的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a5ß - 1整合素结合，纯化的EhCP5诱导弱激活，单独不触发NLRP3。充分激活a5ß - 1整合素和同时诱导NLRP3需要表面结合的gal凝集素。共聚焦显微镜显示a5ß - 1整合素、p-paxillin（活性整合素信号传导）和NLRP3快速定位到Eh接触部位，提示gal -凝集素调节a5ß - 1整合素的募集。我们将测试在Eh表面固定gal -凝集素是否将a5ß1整合素招募到含有gal -凝集素、EhCP5、a5ß1整合素和NLRP3的活性信号复合体中。我们将确定a5ß1整合素激活后NLRP3是否被招募，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。

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.