Entamoeba histolytica-induced IL-1ß secretion is dependent on caspase-4 and gasdermin D.

During invasion, Entamoeba histolytica (Eh) encounter macrophages and activate them to elicit tissue damaging pro-inflammatory responses. When Eh binds macrophages via the Gal-lectin, surface EhCP-A5 RGD sequence ligates α5ß1 integrin to activate caspase-1 in a complex known as the NLRP3 inflammasome. In this study, we investigated Eh requirements underlying macrophage caspase-4 and -1 activation and the role caspase-4 and gasdermin D (GSDMD) play in augmenting pro-inflammatory cytokine responses. Caspase-4 activation was similar to caspase-1 requiring live Eh attachment via the Gal-lectin and EhCP-A5. However, unlike caspase-1, caspase-4 activation was independent of ASC and NLRP3. Using CRISPR/Cas9 gene editing of caspase-4 and -1 and GSDMD, we determined that caspase-1 and bioactive IL-1ß release was highly dependent on caspase-4 activation and cleavage of GSDMD 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-1ß release. Activated caspase-4 and -1 cleaved GSDMD liberating the N-terminal p30 pore-forming fragment that caused the secretion of IL-1ß. These findings reveal a novel role for caspase-4 as a sensor molecule to amplify pro-inflammatory responses when macrophage encounters Eh.

The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1ß secretion.

Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5ß1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1ß release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1ß. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses.

Immune Evasion Mechanisms of Entamoeba histolytica: Progression to Disease.

Entamoeba histolytica (Eh) is a protozoan parasite that infects 10% of the world's population and results in 100,000 deaths/year from amebic dysentery and/or liver abscess. In most cases, this extracellular parasite colonizes the colon by high affinity binding to MUC2 mucin without disease symptoms, whereas in some cases, Eh triggers an aggressive inflammatory response upon invasion of the colonic mucosa. The specific host-parasite factors critical for disease pathogenesis are still not well characterized. From the parasite, the signature events that lead to disease progression are cysteine protease cleavage of the C-terminus of MUC2 that dissolves the mucus layer followed by Eh binding and cytotoxicity of the mucosal epithelium. The host mounts an ineffective excessive host pro-inflammatory response following contact with host cells that causes tissue damage and participates in disease pathogenesis as Eh escapes host immune clearance by mechanisms that are not completely understood. Ameba can modulate or destroy effector immune cells by inducing neutrophil apoptosis and suppressing respiratory burst or nitric oxide (NO) production from macrophages. Eh adherence to the host cells also induce multiple cytotoxic effects that can promote cell death through phagocytosis, apoptosis or by trogocytosis (ingestion of living cells) that might play critical roles in immune evasion. This review focuses on the immune evasion mechanisms that Eh uses to survive and induce disease manifestation in the host.

The future for vaccine development against Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amebiasis, one of the top three parasitic causes of mortality worldwide. In the majority of infected individuals, E. histolytica asymptomatically colonizes the large intestine, while in others, the parasite breaches the mucosal epithelial barrier to cause amebic colitis and can disseminate to soft organs to cause abscesses. Vaccinations using native and recombinant forms of the parasite Gal-lectin have been successful in protecting animals against intestinal amebiasis and amebic liver abscess. Protection against amebic liver abscesses has also been reported by targeting other E. histolytica components including the serine-rich protein and the 29-kDa-reductase antigen. To date, vaccines against the Gal-lectin hold the most promise but clinical trials will be required to validate its efficacy in humans. Here, we review the current strategies and future perspectives involved in the development of a vaccine against E. histolytica.