The Effect And Mechanism Of Innate Immunity Mediated By TLRs In The Process Of Giardia Pathogenicity

Giardia duodenalis,also known as Giardia lamblia(Giardia),is a flagellated,low-grade,single-celled eukaryote that evolved status is in the middle of the transition from prokaryotes to eukaryotes,which has important research value in evolutionary biology and has become a hot research object.Giardia is an important parasitic and zoonosine protozoan,and Giardiasis caused by it is called "traveler's diarrhea".It is clinically characterized by abdominal pain,diarrhea,vomiting,and stunted growth of infants and young children.Giardia can also cause cholecystitis and liver damage,severe cases the death of infants and immunocompromised patients.The United States has included Giardia on the black list of bioterrorism.According to reports,the total infection rate of Giardia is 2.52% in China.Giardia detection is one of the mandatory index in the world's urban health standards for supply and drinking water.At present,metronidazole is commonly used in the treatment of Giardiasis,and metronidazole has significant side effects on pregnant women and lactating women.To date there is no ideal way to control Giardia.The reason for this is the lack of detailed understanding of the cellular and molecular biology features of Giardia,and no key targets for drugs or vaccines have yet been found.Toll-like receptors(TLRs)are important pattern recognition receptors involved in innate immunity.They can recognize the conserved structural components of pathogenic microorganisms and activate the body's innate immune response.TLRs can recognize a variety of pathogenic parasites such as Toxoplasma gondii,Cryptosporidium,Plasmodium,and Trypanosoma,and play an important role in anti-infection.So far,few studies have been conducted on the activation of host TLRs by Giardia,and the roles and mechanisms of TLRs in the pathogenesis of Giardiasis have not yet been elucidated.In this study,we researched the role and mechanism of TLR2 receptor-mediated innate immune response in the pathogenesis of Giardia,the role and mechanism of TLR3 receptor-mediated innate immune response in the pathogenesis of Giardia,and the effect as well as molecular basis of Giardia virus on Giardia morphology,growth and development,in order to clarify the role and mechanism of TLRs in Giardia treatment process.The role and mechanism of innate immune response mediated by TLR2 receptor in the pathogenesis of Giardia.We detected the activation of TLR2 in host macrophages by Giardia through fluorescence quantitative method,the results showed that Giardia could activate TLR2;we detected the activation of the host macrophage signaling pathway and the secretion of cytokines by Western blot and ELISA,respectively,the results showed that Giardia can rely on TLR2 to activate P38,ERK and AKT signaling pathways,and significantly inhibit the secretion of TNF-?,IL-12 p40,IL-6 through TLR2-AKT signaling pathway;we studied the effect of TLR2 in the process of Giardia infection through animal experiments,the results showed that Giardia can inhibit the secretion of cytokines such as IL-12 and p40 by activating the TLR2-AKT signaling pathway after infection with Giardia,resulting in an increase in trophozoites and intestinal lesions in mice,and leading to more serious Giardiasis.Giardia virus is a kind of double-stranded RNA virus parasited in Giardia trophozoites,the shape is spherical icosahedral symmetry and its diameter is about 33 nm,it is widely distributed in the nucleus and cytoplasm.Studies have found that the proliferation rate of Giardia significantly decreased after infection by Giardia virus within 96 h.At present,there are still few studies on the effect of Giardia virus on the growth and development of Giardia.Certain protozoal viruses(ds RNA viruses),such as Leishmania virus,Trichomonas vaginalis virus,etc.,affect the virulence of the protozoa carrying them by activating the host's TLR3.At present,researchers have not found any correlation between Giardia virus and Giardia virulence,and whether Giardia virus aggravates Giardiasis has not yet been elucidated.Therefore,in this article,we will conduct a detailed study of the molecular mechanism of Giardia virus affecting the growth,development and the pathogenicity of Giardia.The role and mechanism of innate immune responses mediated by TLR3 receptor in the pathogenesis of Giardia.First,we used Giardia virus to infect a virus-free strain Giardia to construct a new VIG strain.The activation of TLR3 in host macrophages by different Giardia strains was detected by fluorescence quantitative method,the results showed that TLR3 could recognize the virus-carrying strain and VIG strain but could not identify the virus-free strain.Further analysis showed that TLR3 could recognize the ds RNA in first two of them.We detected the activation of the host macrophage signaling pathway and the secretion of cytokines by Giardiasis from different strains by ELISA,the results showed that the secretion of cytokines caused by activating the TLR3 signaling pathway in the virus-bearing strain and the VIG strain was significantly more than virus-free strains,further analysis showed that the Giardia virus ds RNA also activates TLR3 signaling pathway to cause secretion of cytokines;animal experiments were used to study the role of TLR3 in different insects Giardia infection process,the results showed that the strains of carrying virus and VIG strains were able to increase the secretion of TNF-?,IL-6,and IL-12 p40 by activating the TLR3 signaling pathway after infecting the host compared with the virus-free strain Giardia,resulting in a reduction of trophozoites in the intestine of mice,Gut lesions are reduced,resulting in a lighter Giardiasis.Effect of Giardia virus on the morphology,growth and development of Giardia and its molecular basis.Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used to observe the morphology and ultrastructure of trophozoite after viral infection,the results showed that virus-infected non-viral trophozoites had tail nodules and nodules contained glycogen and virus-like particles.By statistical analysis of the proliferation of trophozoites and induction of cyst formation rate in vitro of different strains,the results showed that virus carrying strains and VIG strains trophozoites proliferated faster than non-virus strains,and the rate of induction cyst was lower.The RNA-seq method was used to perform transcriptome sequencing analysis of the VIG strain and the virus-free strain Giardia,the results showed that Giardia virus infection can affect the proliferation and cyst of Giardia by controlling the Giardia trophozoite sugar metabolism signaling pathway.In summary,in this study we found that Giardia can activate TLR2-AKT signaling pathway in the host,inhibit the secretion of pro-inflammatory cytokines,resulting in more serious Giardiasis in wild-type mice.Activation of TLR3 signaling pathway by virus-carrying strains and VIG strains increased the secretion of pro-inflammatory cytokines and caused milder Giardiasis in wild-type mice.Further studies showed that the molecule recognized by TLR3 is the Giardia virus ds RNA,and Giardia virus can affect the growth and development of Giardia through sugar metabolism pathways.The above results showed that Giardia evades the immune attack by activating the host TLR2;while the host TLR3 exerts its effect against Giardia infection,thus revealing a new mechanism of Giardia disease.