Study On The Function Of α-amylase And Starch Branching Enzyme In Amylopectin Metabolism Of Toxoplasma Gondii

Toxoplasma gondii（T.gondii）,a vital intracellular parasite,can infect proliferate and survive in almost all mammalian cells.Toxoplasmosis has become a global,serious public health problem due to the extensiveness of the host.Tachyzoites infected with immunocompetent hosts can transform to bradyzoites,which cause chronic infection and exist in brain and striated muscle tissues.Nevertheless,bradyzoites can be reactivated to tachyzoites under an advantageous environment,which may lead to disseminated infections.There are great differences in the energy metabolism in the different stages of infections.The most typical difference is the abundant accumulation of amylopectin granules in bradyzoites,which is almost absent in tachyzoites.Some researchers have proposed that amylopectin may be the source of energy for bradyzoites,while others believe that amylopectin can provide energy for the conversion of bradyzoites to tachyzoites.However,this hypothesis has not been well proven.Until now,the physiological functions of amylopectin have not been clearly elucidated.Therefore,our study focused on the amylopectin metabolism pathway in T.gondii,taking the α-amylase（α-AMY）in the amylopectin digestion pathway and the starch branching enzyme（SBE）in the synthesis pathway as the research objects.Biochemistry,genetic and metabolic flux analysis were used to study the biological functions of α-AMY and SBE to reveal the exact physiological significance of amylopectin.Specific work done is as follows:（1）First,the bioinformatics analysis was applied to analyze the amino acid sequence and evolutionary relationship of α-AMY（TGME49₂49960）in T.gondii,and it was found to be closely related to α-amylase in plants.Then,the enzyme activity of recombinant protein α-AMY proved that it could catalyze amylopectin to produce glucose.ME49Δα-amy strain was successfully obtained by direct knockout method,and α-AMY deletion had no effect on the growth of tachyzoites in vitro,but it could significantly reduce the virulence and the brain cysts formation.In addition,knocking out α-AMY only caused a slight accumulation of amylopectin in tachyzoites,but obvious accumulation of starch granules was observed in bradyzoites.The above results indicated that α-AMY indeed contributed to amylopectin digestion,but it had little effect on tachyzoites growth in vitro,and mainly played a role in bradyzoites.（2）We speculated that the ME49Δα-amy strain might have the potential to become a candidate vaccine against T.gondii infection.Therefore,our study established a mouse model of Toxoplasma infection to explore whether the ME49Δα-amy strain can stimulate the hosts to produce immune protection against acute and chronic infection.The results showed that the survival rate of mice immunized with ME49Δα-amy reinfection with different strains of tachyzoites or tissue cysts was close to 100%.In addition,immunization with ME49Δα-amy can effectively eliminate the proliferation of parasites in mice and prevent the formation of cysts.The significant increase in Th1 type and Th2 type cytokines and Toxoplasma-specific Ig G levels confirmed that cellular and humoral immunity had a common effect on T.gondii infection.High expression levels of Th1-type cytokines from spleen cells after 145 days of immunization indicated that Th1-type cellular immune response played a leading role in immune response.（3）SBE in amylopectin synthesis pathway was studied,and amino acid sequence alignment and evolutionary tree were utilized to analyze the starch branching enzyme（TGME49₃16520）,revealing that relatively conserved Tg SBE had a close evolutionary relationship with SBE in plants.The research on SBE direct knockout strain revealed that SBE deletion led to amylopectin synthesis defects,but had no significant effect on the growth,virulence and brain cyst formation of tachyzoites under normal culture conditions in vitro,indicating that SBE indeed contributed to the starch synthesis pathway.Deletion of SBE reduced the influx of exogenous glucose and the replication ability of tachyzoites in a single carbon source,which indicated that amylopectin could also be used as a carbon source to provide energy under nutritionally deficient conditions.（4）Δα-amyΔsbe strain with amylopectin synthesis defects was constructed,which had no significant change in tachyzoites growth,and the virulence was still reduced,but the brain cyst formation was restored.This further revealed that amylopectin did not contribute much to the growth of tachyzoites,but played a vital role in the growth and development of bradyzoites.Cysts with defective amylopectin metabolism were abnormal morphology and avirulent to mice,which indicated that amylopectin was essential for the maintenance of chronic infections,and suggested that amylopectin,as a form of energy storage,might play an important role in the reactivation of bradyzoites to tachyzoites.In conclusion,this study further improved amylopectin metabolic pathway in T.gondii through studying the biological functions of α-AMY in amylopectin digestion pathway and SBE in the synthesis pathway,clarified the role of amylopectin in tachyzoites and bradyzoites,and demonstrated that amylopectin,as an important carbon source,was critical to parasites growth under an unfavorable environment and the reactivation of bradyzoites to tachyzoites.The findings obtained from our study provides a new avenue for the development of Toxoplasma vaccines and anti-chronic toxoplasmosis drugs.