Chadee, Kris C.Quach, Jeanie2018-05-012018-05-012018-04-27Quach, J. (2018). Entamoeba histolytica-Induced Caspase-4 Activation Regulates IL-1β Secretion Through Caspase-1 (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/31866http://hdl.handle.net/1880/106580Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the top four parasitic causes of mortality worldwide. In 90% of infected individuals, Eh harmlessly colonizes the large intestine and results in a non-invasive and asymptomatic infection. In the remaining 10% of infected individuals, the parasite breaches the intestinal barrier causing amebic colitis and in rare cases, it can cause extra-intestinal lesions, mainly liver abscesses. During invasion, Eh encounter macrophages in the lamina propria and this intricate host-parasite interaction is critical in eliciting a tissue damaging raging pro-inflammatory response. When Eh binds macrophages via the Gal-lectin, surface EhCP-A5 ligates α5β1 integrin to activate caspase-1 in a complex known as the NLRP3 inflammasome. In this study, we investigated the parasite requirements underlying macrophage caspase-4 and -1 activation and the role caspase-4 play in augmenting pro-inflammatory cytokine responses. Surprisingly, caspase-4 activation was similar to caspase-1 requiring live Eh attachment via the Gal-lectin, EhCP-A5 and cellular stresses such as K+ efflux and ROS. However, unlike caspase-1, caspase-4 activation was independent of ASC and NLRP3. Using CRISPR/Cas9 gene editing of caspase-4 and caspase-1 in human macrophages, we determined that caspase-1 and bioactive IL-1β release was highly dependent on caspase-4 activation in response to Eh. Formaldehyde cross-linking to stabilize protein-protein interactions in transfected COS-7 cells stimulated with Eh revealed that caspase-4 specifically interacted with caspase-1 in a protein complex that enhanced the cleavage of caspase-1 CARD domains to augment IL-1β release. The mouse ortholog caspase-11, displayed similar requirements for its activation, however, it was not involved in regulating caspase-1 activation in the same way as caspase-4. These findings reveal a novel role for human caspase-4 as a critical sensor molecule to amplify downstream pro-inflammatory responses when macrophage encounters live Eh.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Entamoeba histolyticacaspase-4caspase-1IL-1betainflammasomeMicrobiologyParasitologyImmunologydoctoral thesis10.11575/PRISM/31866