Elucidation of amoebic factors evoking for host cell responses and intracellular signaling pathway induced by Entamoeba histolytica

Abstract

Entamoeba histolytica is an enteric tissue-invasion protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. Amoebic major virulence factors include Gal/GalNAc lectin, amoebapore, amoebic cysteine proteases (CP). When amoebic trophozoites adhere to the intestinal epithelium and mucin, tissue pathogenesis induced by amoebae can be initiated. During tissue invasion, E. histolytica also interacts with extracellular matrix (ECM) proteins such as fibronectin, collagen, and laminin. Therefore, adhesion to host tissue components is important for successful expression of amoebic virulence. However, the mechanism of amoebic adhesion to tissue components is not fully understood and information regarding the host’s and parasite’s factors involved in adhesion leading to parasitic pathogenesis is limited.In chpter I, Entamoeba adhesion using various Entamoeba strains with mutations in major amoeba virulence factors was investigated. Amoebic adhesion to ECM protein occurred rapidly and increased in a dose-dependent manner. The adherent capacity of the amoebic Gal/GalNAc lectin-silenced strain L5 and the non-pathogenic Rahman strain was dramatically reduced compared to the pathogenic wild-type E. histolytica. Interestingly, the Rahamn strain exhibited decreased CP activity compared to the wild-type amoeba. Next, to address whether CP is responsible for amoebic adhesion to ECM protein, Entamoeba were pretreated with a CP inhibitor, CP modifier, or specific CP antibodies. Pretreated Entamoeba with these molecules resulted in a marked decrease in Entamoeba adhesion to ECM protein. In addition, inhibitor of cysteine protease (ICP1-/-) strain with the hyper-CP secretion showed a significant increase in amoebic adherence to host components compared to control strain. These results strongly suggest that both amoebic CP activity and Gal/GalNAc lectin are important factors affecting Entamoeba adhesion to host tissues, causing tissue inflammation induced by E. histolytica. In chapter II, mast cell responses induced by E. histolytica-derived secretory products (EhSP) and host cell death induced by various Entamoeba strains was investigated to elucidate amoebic factors that evoke the host cell response. Tissue-residing mast cells are reported to be a major player in the mucosal inflammatory response to infections with various pathogens. However, the amoebic factor(s) responsible for mast cell activation and host cell death are not fully understood. Mast cell degranulation via the non-exocytotic mode and a significant increase in IL-8 production were observed in EhSP-stimulated HMC-1 cells. Entamoeba secreted, heat-resistant (56 ºC for 30 min) protein components were responsible for EhSP-induced IL-8 production that occurred via a PAR2-independent mechanism. Because IL-8 production in immune cells is related to intracellular reactive oxygen species (ROS), EhSP-stimulated ROS production in HMC-1 cells was examined using various pharmacological inhibitors. The results showed that ROS generated by NADPH oxidase and COX-1 was enhanced in EhSP-stimulated HMC-1 cells. Investigation of amoebic factors responsible for IL-8 production was performed using EhSPs derived from various Entamoeba mutant strains. As a result, HMC-1 cells stimulated with EhSP from non-pathogenic Rahman and cysteine protease-deficient ICP1+/+ strains showed a reduction in IL-8 production as compared to the pathogenic strain. Similarity, LDH release in HepG2 cells triggered by the ICP1+/+ strain was significantly reduced compared to pathogenic Entamoeba and control transfectants. Especially, the non-pathogenic Rahman strain abolished DNA fragmentation as well as LDH release in HepG2 cells compared to the pathogenic E. histolytica strain. Taken together, these results indicate that CPs participate in host IL-8 production and cell death induced by E. histolytica. In chapter III, amoebic factors responsible for E. histolytica-induced intracellular signaling molecules activation in host cells were investigated using various Entamoeba strains. Although there are many reports about amoebic virulence factors, the amoebic factor(s) associated with host cell responses and the intracellular signaling molecules responsible for amoeba-induced host cell death are not fully understood. In order to identify the amoebic factors evoking host cell responses and the intracellular signal pathways induced by E. histolytica, the activation of various intracellular signal molecules induced by amoebae in HepG2 cells using various Entamoeba strains modified with respect to major amoebic virulence factors was investigated. At first, wild-type E. histolytica strongly induced the cleavage of caspases, calpain and ROS generation in HepG2 cells. Cleavage of caspase-3, but not of calpain, was inhibited in the non-pathogenic Rahman strain. Co-incubation with E. histolytica induced a decrease in tyrosine phosphorylation and in O-GlcNAc levels within 2 min in HepG2 cells. DeGlcNAcylation in HepG2 cells induced by Entamoeba occurred in a contact-dependent manner. In addition, when Entamoeba-induced deGlcNAcylation in HepG2 cells was inhibited by pretreatment with PUGNAc, Entamoeba-induced host cell death was also inhibited, suggesting an import role of GlcNAcylation in host cells. Moreover, both the Rahman and ICP1+/+ strains remarkably inhibited Entamoeba-induced dephosphorylation and deGlcNAcylation compared to the wild-type Entamoeba and the control transformant, respectively, demonstrating the critical role of CP in intracellular signal molecule activation in HepG2 cell death induced by E. histolytica. Finally, the specific proteases involved in the degradation of cytoskeletal proteins during Jurkat T cell death induced by E. histolytica were investigated. Amoebic trophozoites induced marked degradation of paxillin, Cas, vimentin, vinculin, and talin, as well as α- or β-spectrin, in Jurkat T cells. The cleavage effects of E. histolytica were strongly retarded by pretreatment with a calpain inhibitor, but not with a pan-caspase inhibitor. In addition, calpain knockdown with siRNA in Jurkat T cells effectively inhibited E. histolytica-induced PARP, paxillin, -spectrin, -spectrin and talin degradation in Jurkat T cells, as compared to scrambled siRNA, suggesting that calpain plays a crucial role in the cleavage of cytoskeletal-associated proteins during cell death induced by E. histolytica. These results suggest that amoebic adhesion to target cells induces hepatocyte cell death via the activation of cell death-associated molecules and the disturbance of cellular function-regulating intracellular signal molecules. Furthermore, amoebic CP as well as Gal/GalNAc lectin is closely associated with host cell death processes induced by E. histolytica. Taken all together, these results suggest that amoebic CP as well as Gal/GalNAc lecin are closely associated with amoebic adhesion to host cell, induction of host cell responses and various intracellular signal molecules activation of host cell. These studies will be useful in understanding the relationship between amoebic factors and host signaling mechanisms underlying host cell death in amoeba-invaded lesions during human amoebiasis.