Preliminary Study On The Function Of Tyrosine Hydroxylase Of Toxoplasma Gondii

Background:Toxoplasma gondii(T.gondii)is an obligate intracellular protozoan that infects most warm-blooded animals.It is one of the most widely distributed parasites in the world,both in terms of geographic location and host diversity.T.gondii replication:in various intermediate hosts,including humans is an asexual stage.When an intermediate host is infected,T.gondii rapidly spread throughout the host tissue.In all other hosts,Toxoplasma infection begins with a rapid growth stage called tachyzoites.Although most tachyzoites are cleared by the immune system,some tachyzoites are transformed into bradyzoites,forming relatively static cysts in the brain and muscle.In most normal immunized populations,Toxoplasma infection has a transient clinical symptom or even no clinical symptom,but for immunocompromised people,it will cause toxoplasmosis,or even death,and will cause miscarriage and abnormality for pregnant women.Serious damage such as fetal or stillbirth.T.gondii is also the primary source of congenital diseases,in most cases(about 80%),Toxoplasma infection causes a series of serious congenital defects,including the eyes'disease,developmental delay,and can be led to fetal death.Harmfulness due to acute infection with T.gondii,previous studies have focused more on the study of the tachyzoite stage,while the formation of relatively static cyst was thought to be ineffective in the host.However,studies in recent years have shown that rodents infected with T.gondii exhibit abnormal behavioral responses:they lose their instinctive aversion to the cat's odor,but to be gently attracted to the odor.This behavioral manipulation seems to be conducive for the transmission of parasites to the cat.The exact mechanism of this behavioral manipulation is unclear,but parasites in the brain that stimulate the host dopamine pathway have been considered a possible pathway.Some researchers have suggested that parasites alter DA levels in vitro or in vivo,even the cysts in brain can directly affect dopamine(DA)production and DA synaptic transmission in the host brain.In addition,dopamine receptor antagonist drugs used to treat schizophrenia can reduce the attractiveness of infected rodents,but the mechanism by which infection may alter dopamine is blurred.The genome of T.gondii contains two genes encoding the aromatic amino acid hydroxylase:TgAaaH1 and TgAaaH2.Tyrosine hydroxylase converts tyrosine to the dopamine precursor 3,4 dihydroxyphenylalanine(L-Dopa),which is the rate-limiting step for subsequent dopamine synthesis.Studies have also shown that the aromatic amino acid hydroxylase genes AAH1 and AAH2 in T.gondii contribute to the sexual reproduction of Toxoplasma from the intermediate host to the cat to explore the role in the development of T.gondii oocysts.Due to the AAH2 gene is expressed at low levels in the tachyzoite stage,but are up-regulated in bradyzoite stage,more researches were focussed on AAH2 gene function in the past.It has been reported that the AAH2 gene does not affect the function of Toxoplasma gondii in tachyzoite stage,and does not change the level of global or regional dopamine in the host brain,nor affect the DA-dependent neurobehavioral behavior of BALB/c mice chronically infected by toxoplasma.However,the function of the AAH2 gene is controversy.There are few studies on the function of the AAH1 gene which is expressed similar in the tachyzoite and bradyzotie stages.Whether AAH1 is likely to participate in the parasite to manipulate the host DA,which may affect the behavioral changes of the host?Then,we assume that AAH1 may also be involved in parasite manipulation of host dopamine,which may affect host behavioral changes.To explore this possibility,we constructed a defective strain of AAH1 gene in RH and PRU strain,and observed the parasites invasion,proliferation and growth of host cells in vitro.Infected animal models were constructed to directly detect the effects of AAH1 gene on the virulence of infected hosts and cyst formation in the brain tissues.Behavior of infected hosts was monitored to explore its function in the bradyzoite stage.Method:1.Real-time quantitative PCR was utilized to detect the copy number of the AAH1 gene in the genome of the type ? RH strain and the type ? PRU strain by constructing a single copy plasmid of pUC 19-SAG1-AAH1 as a basal control.2.AAH1 gene was knocked out by inserting pyrimethamine as the drug screen label in the seventh exon of the AAH1 gene using CRISPR/Cas9 technology to achieve gene disruption.The disruption of AAH1 gene was demonstrated at the genetic and transcription level.3.Crystal violet staining and immuno:fluorescence staining were used to observe the ability of T.gondii to invade HFF cells in vitro and proliferate and grow in cells.An acute mouse infection model established by intraperitoneal infection of tachyzoites was used to obeserve the oxicity function of T.gondii to the host to explore the function of AAH1 gene in the tachyzoite stage of T.gondii.4.Construction of a chronically mouse infection model by oral gavage of the cysts to observe the effect of AAH1 gene on the spatial distribution of cysts in the brain tissue of mice infected by T.gondii.And observe the behavior of mice chronically infected by T.gondii in the open field test and the Morris water maze to explore the function of the AAH1 gene in the bradyzoite stage of T.gondii.Results:1.AAH1 gene is a single copy in the RH and PRU strains.2.Successfully construction of RH?AAH1 and PRU?AAH1 strains.3.Establish mouse models of acute and chronic infection of T.gondii.4.The invasion,proliferation or growth in host cells,nor the virulence of mice acutely infected by T.gondii of AAH1 gene knockout strains were not affected by gene disruption.5.AAH1 gene knockout affects the global cyst burden and local cyst formation in the brain tissue of mice chronically infected by T.gondii.6.AAH1 gene knockout has no impact on the ability of mice chronically infected by T.gondii to explore in the new environment and the behavior of spatial learning in the Morris water maze,but may affect the host's behavior related to memory.Conclusion:The invasion,proliferation or growth in host cells,nor the virulence to the mice acutely infected by T.gondii were not affected by the disruption of AAH1 gene of T.gondii.AAH1 gene knockout does not affect the ability of mice chronically infected by T.gondii to explore in the new environment or behavior of spatial learning in the Morris water maze,but affect the formation of cysts in the brain tissue and the behavior related to memory in the Morris water maze.