The Role Of Autophagy-related Protein 6 In Invasion Of Toxoplasma Gondii

Toxoplasmosis is a common zoonotic disease with high infection rate and cause severe consequences to immune compromised human.The clinical drugs used for treatment of toxoplasmosis are poorly effective and have many side effects.There is an urgent need to find new drugs against T.gondii.Depletion of autophagy-related gene 3,4 and 8(TgATG3,TgATG4 and TgATG8)significantly affects the growth of T.gondii.Therefore,autophagy-related genes are one of the most promising drug targets against T.gondii.ATG6 is a core molecule of complex Ⅰ(including Vps34,Vps15,Vps30/ATG6,ATG38 and ATG14).In previous studies,the accumulation of GFP-TgATG8 puncta in starvation-or monensin-treated T.gondii is inhibited by the PtdIns 3-kinase inhibitors of wortmannin and 3-methyladenine.These data might suggest that the complex I is necessary for the regulation of TgATG8.But whether TgATG6 can be a drug target against T.gondii has not yet been confirmed.To investigate the function of the T.gondii orthologue of ATG6(TgATG6),relationships of TgATG6 and other ATG6 orthologues was analyzed through phylogenetic analysis.The subcellular localization of TgATG6 was detected in a parasite strain overexpressing recombinant TgATG6 fused with mCherry.By using CRISPR/Cas9 method,we generated TgATG6-depleted parasites.The effect on the development of parasites caused by TgATG6 deficiency was analysed by flow cytometry.The results suggested that TgATG6 had close relationship to plant ATG6.TgATG6 signal was found in the cytosol of intracellular parasites and accumulated into spots in starvation-treated parasites.Depletion of TgATG6 had no effect on replication of T.gondii but caused a defect in invasion ability of the parasites.We conclude that TgATG6 could be a assistant drug target aginst T.gondii.Furthermore,TgATG8 protein was successfully expressed and used to immunize mice.Polyclonal antibodies against TgATG8 were prepared.Thus a method to detect the conjugation and the subcellular localization of TgATG8 was established.In summary,the function of TgATG6 was investigated and its potential for being a drug target was evaluated through gene knockout method.Our research provided the basis for screening drugs against T.gondii.