Modeling Entamoeba histolytica host-parasite interactions

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Entamoeba histolytica (Eh) is a colonic protozoan parasite where 99% of infections are non-invasive. How the innate immune system determines the pathogenicity and level of risk posed by Eh are unanswered questions in this host-parasite interaction. When Eh invades, it is a dead end for the parasite as cysts production ceases and may kill the host. Eh invasion stimulates interleukin IL-1ß inflammatory responses. Even though Eh components can also induce inflammation through Toll-like receptor activation, it does not recapitulate the aggressive inflammatory IL-1ß production towards live Eh. This indicates that the host senses Eh and that other pathogen sensing pathways are activated to induce inflammation. We recently identified that NLRP3 inflammasome is the molecular sensor controlling the rapid IL-1ß response to Eh. Inflammasomes are cytosolic multimeric complexes that can be activated by pathogens. As inflammasome activation only occurs following direct contact with Eh, we hypothesize that inflammasome activation is a central mechanism used by the innate immune system to discriminate the threat posed by invading parasites. The specific aims are: 1) to determine the molecular mechanism of inflammasome activation by Eh and, 2) to quantify the function of inflammasomes in regulating the magnitude of the inflammatory response. To investigate this, we will assess inflammasome activation in macrophages (mo) using defined pharmacological inhibitors of the NLRP3 pathway and in mo that are genetically deficient for inflammasome components. Mechanistic studies will be done to elucidate a role for phagocytosis and phagosome maturation. Other studies will investigate if the danger signal ATP can trigger the inflammasome by blocking the P2X7 receptor and by enzymatic depletion of extracellular ATP. Inflammasome activation will be visualized in real-time using confocal microcopy with caspase-1 probes. Studies will be verified in WT and genetically deficient mice for inflammasome components or following inhibition of the inflammasome pathway. These studies will quantify a mechanism to define how Eh is able to successfully colonize without evoking unwanted host inflammatory responses.