Modeling Entamoeba histolytica host-parasite interactions

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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.