Functional Study Of Giardia Doudenalis Cathepsin B In The Interaction Between Giardia And Host Cells

Giardia doudenalis is a zoonotic protozoan parasite and one of the most common intestinal parasites.It is the main cause of waterborne diarrhea.The life cycle of Giardia includes two stages:cysts and trophozoites,in which the trophozoites colonize the duodenum and can attach to the surface of intestinal epithelial cells using adsorption discs.Giardia causes diarrhea,malabsorption,and weight loss through interactions with intestinal epithelial cells.Cysteine protease is an important virulence factor of protozoan parasites,which plays a crucial role in the pathogenesis of parasitic diseases and the life cycle of parasites.Giardia cysteine protease plays a critical role in the pathogenesis of parasitic diseases.Previous studies have demonstrated that Giardia cysteine protease are involved in gut barrier disruption and gut microbiota dysbiosis.Giardia cathepsin B,as the main component of Giardia cysteine protease,has high hydrolytic activity,can destroy the tight junctions and intestinal mucus barrier of host cells,and interact with host cell apoptosis and immune factors.Giardia cathepsin B is the main component of Giardia cysteine protease.Giardia cathepsin B has high hydrolytic activity,can disrupt the tight junctions of host cells and the intestinal mucus barrier,and is involved in host cell apoptosis and the degradation of immune factors.This study established various models of the interaction of Giardia trophozoites with goblet cells,intestinal epithelial cells and macrophages.The study used cathepsin B inhibitor and cathepsin B recombinant protein to explore the effect of cathepsin B on host cell mucin expression and secretion,apoptosis and cellular inflammatory response.The purpose of this study was to investigate the function of Giardia cathepsin B in the interaction between Giardia and host cells.In this study,the seven proteins cat B-1～7 of Giardia cathepsin B were bioinformatically analyzed,and the prokaryotic expression plasmid p Clod-cat B-1～7 was constructed,and the cat B-1～7 proteins were successfully expressed and purified.This study successfully established various cellular interaction models between Giardia trophozoites and intestinal epithelial cells,goblet cells and macrophages,and the study used the interaction model of Giardia and goblet cells to explore the effect of Giardia cathepsin B on the expression and secretion of mucin in host cells.We first detected the expression and secretion levels of Giardia-induced mucins by western blot.We used the Giardia cysteine protease inhibitor E-64 and the cathepsin B inhibitor CA-74 Me to evaluate the effect of Giardia cathepsin on goblet cell mucin by using q PCR,western blot and indirect immunofluorescence.The results showed that Giardia trophozoite stimulation induced the secretion of MUC2 protein from goblet cells,and the rapid secretion of goblet cell mucin caused by Giardia led to a rapid decrease in the level of intracellular MUC2 storage.In addition,q PCR results showed that Giardia stimulation could induce the up-regulation of goblet cell mucin m RNA level,which also confirmed that Giardia could enhance the mucin expression of host cells by regulating the m RNA level of cellular mucin.However,when Giardia cysteine protease or cathepsin B was inhibited,the ability of Giardia to induce upregulation of MUC2 m RNA levels in host cells was inhibited.This also confirms that Giardia cathepsin B is involved in the process.The results of western blot and immunofluorescence showed that after the inhibition of cysteine protease or cathepsin B,the reduction of intracellular mucin content induced by Giardia stimulation was inhibited to a certain extent,indicating that Giardia cathepsin B may also be involved in Giardiainduced goblet cell mucin secretion.In order to further confirm the regulation of cathepsin B on the expression and secretion of goblet cells,this study also used Giardia to directly stimulate goblet cells in vitro,and used q PCR and western blot to detect and analyze the m RNA and protein levels of MUC2,respectively.The results showed that cat B-1 was involved in Giardia-induced upregulation of goblet cell mucin expression,while cat B-6 also caused varying degrees of mucin expression down-regulation.This also indicates that the complex Giardia cathepsin B family,as an important virulence factor,plays a complex role in the regulation of mucin expression and secretion of host cells by Giardia.This also allows us to further confirm that Giardia cathepsin B plays an important role in the regulation of the host intestinal mucosal barrier by Giardia.In this study,the effect of Giardia cathepsin B on host cell apoptosis was evaluated using the interaction models of Giardia with intestinal epithelial cells and goblet cells.The study performed western blot analysis of the cleavage levels of PRAP and CASP3 in intestinal epithelial cells Caco-2 and goblet cells LS174 T caused by Giardia,and then inhibited using cystatin E-64 and cathepsin B.In this study,the effect of Giardia cathepsin B on apoptosis was evaluated using E-64 and CA-74 Me inhibitors using western blot method.The study used indirect immunofluorescence to evaluate the effect of Giardia cathepsin B on CASP3 cleavage levels in Caco-2 cells.Finally,the effect of cathepsin B on ROS in Caco-2 was analyzed and evaluated using ROS detection kit.The results showed that Giardia trophozoite stimulation can induce the cleavage of apoptotic effector proteins CASP3 and PARP in intestinal epithelial cells and goblet cells.This revealed that Giardia trophozoites can induce apoptosis in epithelial cells and goblet cells.While E-64 and CA-74 Me inhibited Giardia-induced CASP3 and PARP activation,Giardia cathepsin B plays a role in Giardiainduced apoptosis of intestinal epithelial cells and goblet cells.The detection results of ROS also showed that Giardia stimulation caused a large amount of ROS in Caco-2 of intestinal epithelial cells.However,the production of ROS caused by Giardia was suppressed to a certain extent after the inhibition of cathepsin B.This result also revealed that Giardia cathepsin B may regulate apoptosis by regulating ROS.The study of recombinant protein-stimulated Caco-2 cells found that cat B-1 and cat B-2 were involved in Giardia-induced apoptosis.In this study,we established an interaction model between Giardia and macrophages to explore the role of Giardia cathepsin B in the inflammatory response induced by Giardia.We detected the changes of m RNA levels of inflammatory factors IL-1β,TNFα,IL-6 and inflammasome NLRP3 induced by Giardia stimulation in RAW 264.7 and J774 A.1,respectively.To determine the role played by Giardia cathepsin B,we stimulated macrophages with recombinant proteins and detected the m RNA levels of IL-1β and TNFα.The results showed that direct stimulation of cat B-1 and cat B-6 recombinant proteins could induce the m RNA levels of IL-1β and TNFα in macrophages increase.Taken together,this study found for the first time that Giardia not only induces the rapid secretion of goblet cell MUC2,but also enhances mucin expression in host cells by upregulating the m RNA level of mucin MUC2.The Giardia cat B-1 and cat B-6 are involved in regulating goblet cell mucin expression.In addition,the study also found that Giardia cathepsin B plays an important role in Giardia-induced epithelial cell apoptosis and macrophage inflammatory response.cat B-1 and cat B-2 are involved in the regulation of intestinal epithelial cell apoptosis.cat B-1 and cat B-6 can regulate Giardia-induced macrophage inflammatory response by up-regulating the expression of IL-1β and TNF-α.This study not only provides a basis for future research on the pathogenic mechanism of Giardia and the immune regulation mechanism of host against Giardia infection,but also provides new ideas for the prevention and treatment of Giardia in clinical practice.