Construction Of The Bag1 Knockout Strain Of Toxoplasma Gondii And Functional Analysis Of The Bag1 Gene

Toxoplasma gondii(T.gondii)is a special intracellular parasitic protozoon,which can infect almost all warm-blooded animals and human beings,results in a worldwide distributed zoonotic disease alled toxoplasmosis and has become one of the most important infectious diseases in the world.T.gondii is extremely harmful to the immunocompromised population,which can cause encephalitis,retinochoroiditis,miscarriage,teratogens and even dead birth in pregnant women in the primary infection.T.gondii has a complex life cycle including sexual and asexual stages.The sexual life cycle only occurs in family feline’s intestine epithelia;therefore,cats are the definitive hosts for T gondii.While in human,asexual reproduction is the only way for Toxoplasma gondii to reproduce.Unlike in humans or other animals,T.gondii can reproduce sexually in family feline’s intestine epithelia.The asexual reproduction stage of T.gondii mainly includes the tachyzoite stage with rapid proliferation,and the dormant bradyzoite stage.The mutual transformation between the tachyzoites and bradyzoites is the biological basis for T gondii’s opportunistic pathogenesis.In vitro transformation of tachyzoite to bradyzoite can be triggered by stress,alkaline culture and so on,which may cause severe disease even death for the patients with immunodeficiency.Toxoplasma gondii can undergo phenotypic transition from tachyzoites to tachyzoites.The mutual transformation of tachyzoites and bradyzoites is the biological basis for T.gondii’s opportunistic pathogenesis.Under the conditions of immune pressure human or animals,or stress,alkaline induction and other conditions in vitro,T.gondii can undergo the transformation from tachyzoites to bradyzoites,which causes transition of acute infection to chronic infection,and T.gondii bradyzoites can live in the tissue cysts for life-long period;When the host immunity is damaged,T.gondii bradyzoites can be activated into tachyzoites,causing severe toxoplasmosis and even become life-threatening.T.gondii in the body has the unique ability to migrate to distant tissues and organs,including the immune privileged areas,such as lens,central nervous system,testis,placenta,and fetus.After the formation of cysts,it is difficult to be cleared by the immune system due to the blocking and protective effect of the cyst wall.The bradyzoite antigen 1(bag1)of T.gondii is one of the main specific antigens of the bradyzoites.Bag1 is expressed at the early stage of the transformation of tachyzoites to bradyzoites.It has been reported that bag1 plays a very important role in cyst formation,and the specific immunoreactivity of recombinant bag1 also shows that bag1 has the potential to be develop into a vaccine.This research constructed the bagl knockout strain of T.gondii based on ME49-Δku80 strain,and explored the role of bag1 gene in the transformation of tachyzoites to bradyzoites.This study successfully constructed ME49-Δku80-Δbag1 strain by using CRISPR/Cas9 technology.It was found that the transformation of tachyzoites to bradyzoites and bradyzoites to tachyzoites induced in vitro was blocked when bag1 gene was knocked out.No significant difference was found in the virulence between ME49-Δku80-Δbagl and ME49-Δku80 in the survival analysis for the mice after infection.The cyst formation was significantly blocked for ME49-Δku80-Δbagl,compared to ME49-Δku80,which further proved that the bag1 gene deletion significantly blocked the transformation of tachyzoites to bradyzoites.It laid a foundation for the mechanism study of the mutual transformation between bradyzoites and tachyzoites of T gondii.