The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

Abstract

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α₅β₁ integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α₅β₁ integrin and NLRP3 into the intercellular junction, where α₅β₁ integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α₅β₁ integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X₇ receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α₅β₁ integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α₅β₁ integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α₅β₁ integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection. Amebiasis caused by the enteric protozoan parasite Entamoeba histolytica is among the three top causes of death from parasitic infections worldwide, as a result of amebic colitis (dysentery) and liver or brain abscess. When Eh invades the intestinal barrier and contacts host tissue there is a profound inflammatory response, which is thought to drive the disease. One of the central outstanding questions has been how the immune response is escalated at sites of invasion. Adherence of the parasite to host cells has long been appreciated in the pathogenesis of amebiasis, but was never considered as a “cue” that host cells use to detect Eh and initiate host defense. Here we introduce the idea, and demonstrate, that an intercellular junction forms between Eh and host cells upon contact that engages the NLRP3 inflammasome. The NLRP3 inflammasome belongs to a group of “danger” sensors that are uniquely designed to rapidly activate highly inflammatory host defenses. In this work, we identified a surface receptor on macrophages that normally functions in adhesion and polarization recognizes a protein on the outer surface of Eh. Intriguingly, Eh also secretes this protein. However, the full activation of the surface receptor leading to inflammasome activation only occurs when the Eh protein is immobilized on the parasite surface. Thus, we uncovered a molecular mechanism though which host cells distinguish direct contact, and therefore recognize parasites that are immediately present in the tissue, to mobilize a highly inflammatory response. We believe this concept is central to understanding the biology of amebiasis.