Regulation Of Cell Cycle Progression And Parasite Development By TgAMPKβ In Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum apicomplexa.The parasite can infect almost all warm-blooded animals and humans,and has a world-wide distribution,posing great threats to livestock and public health.This parasite has a complex life cycle,which contains a sexual reproduction phase and an asexual reproduction phase.The asexual reproduction phase includes the rapid growing tachyzoites stage that causes acute infection and the slowly replicating bradyzoites stage that is responsible for chronic infection.Under the pressure of host immunity or drugs,tachyzoites are transformed to bradyzoites to resist external pressure.When the adverse pressure is improved,bradyzoites could be reactivated into tachyzoites.In addition,bradyzoites are key to connect asexual reproduction and sexual reproduction.At present,most therapeutic drugs for toxoplasmosis treatment only work for tachyzoites,and are very ineffective in killing bradyzoites.Therefore,studying the growth and development mechanisms of Toxoplasma gondii is important for developing new therapeutics.Previous studies have shown that bradyzoites differentiation is tightly coupled to cell cycle regulation.However,it is not clear how parasites respond to environmental signals to regulate cell cycle progression,growth and differentiation.Here,we focused on AMPK,a widespread protein complex in eukaryotes that is composed of α,β and γ.AMPK is involved in cell energy metabolism to maintain the energy homeostasis.In our study,the AMPK complex in Toxoplasma gondii was identified,and Tg AMPKβ was found to play an important role in the regulation of cell cycle and parasite development.(1)Identification of AMPK complex in Toxoplasma gondiiA highly homologous AMPKα subunit(Tg AMPKα)was found in the genome of Toxoplasma gondii by sequence alignment using the AMPK subunit sequences from yeasts and human.Using immunoprecipitation and LC/MS analysis,the potential Tg AMPKβ and Tg AMPKγ subunit was co-precipitated by Tg AMPKα.Then Co IP /LC-MS was performed using potential Tg AMPKβ and Tg AMPKγ as baits,to further confirm the components of the AMPK complex.The results showed that no matter which subunit was used as bait,the other two subunits were enriched with high abundance.Meanwhile,protein interaction experiments in vitro showed that Tg AMPKβ interacted with Tg AMPKα and AMPKγ.These results suggested that Tg AMPKα,Tg AMPKβ and Tg AMPKγ in Toxoplasma gondii constituted the only AMPK complex.(2)Tg AMPKβ plays a key role in the growth and development of type II strain ME49The CRISPR-Cas9 gene editing system was used to knock out the AMPKβ subunit in the type II strain ME49 to establish the ME49Δampkβ strain.The strain lacking Tg AMPKβ showed obvious growth defects and spontaneously differentiation into bradyzoites.We assessed the cell cycle of these parasites by determining the DNA count.The results showed that when AMPKβ was knocked out,the replication of DNA in the cell cycle process was slowed down and arrested at the G1 stage.These results showed that Tg AMPKβ was involved in the regulation of cell cycle and parasite growth and development of T.gondii under normal condition.Under alkaline stress,parasites without Tg AMPKβ differentiated into abnormal bradyzoites,and gradually died five days later.These results imply that Tg AMPKβ is essential for the maintenance of tachyzoites and the survival of parasite under alkaline condition.After detecting the cell division process of the mutant under the alkaline condition,the division mode of endodyogeny was impaired.In some parasites,endopolygeny that is seen in merozoites was observed.These results suggest that Tg AMPKβ is very important to maintain the reproduction mode of endodyogeny under alkaline culture condition.Virulence experiments in mice showed that infection with ME49 Δampkβ was self-limiting and the strain had significantly reduced virulence.(3)Tg AMPKβ is important for parasites’ response to environment stress by changing the phosphorylation of Tg AMPKαWhen the phosphorylation level of Tg AMPKα in different environments was detected,it was found that the phosphorylation level of Tg AMPKα in intracellular ME49 tachyzoites was very low under normal growth condition.However,alkaline induction could rapidly increase Tg AMPKα phosphorylation.Tg AMPKβ deletion made the phosphorylation of Tg AMPKα no longer responded to stress conditions.On the other hand,the phosphorylation of Tg AMPKα in the type I strain RH was very low under both normal and alkaline conditions.Tg AMPKβ deletion in the RH strain did not affect parasite growth and differentiation.This is consistent with the fact that RH strain do not differentiate into bradyzoites after alkaline stress.These results imply that Tg AMPKβregulates not only the cell cycle progression but also parasite growth and development,by affecting the phosphorylation level of Tg AMPKα.Interestingly,it doesn’t have much effect on parasites that have lost the ability to differentiate.(4)Tg AMPK regulates cell cycle and development by regulating the activity of target genes at transcriptional and protein modification levelsThe phosphoproteomic comparison between ME49 and ME49 Δampkβ found that:phosphorylation of a large number of transcription factors such as AP2 family 、 ZFP family and TFs was changed significantly after the deletion of Tg AMPKβ.Some of these factors have been confirmed to participate in cell cycle regulation such as Tg AP2IX-5.Tg AMPKβ may participate in the regulation of cell cycle,growth and development by regulating the phosphorylation level of transcription factors.By comparing the RNA-Seq data of ME49 with that of ME49Δampkβ,a large number of bradyzoites-specific genes(BAG1,EON1,ANK1,etc.)were higher expressed without alkaline stress in the ME49Δampkβ mutant.This explains the spontaneous bradyzoite differentiation of this strain.In this study we used reverse genetics,protein interaction proteomics,phosphoproteomics,transcriptomics and molecular biology methods to investigate the function of Tg AMPK complex in Toxoplasma gondii,by focusing on Tg AMPKβ.We found that Tg AMPK regulates the cell cycle and parasite growth of Toxoplasma gondii by regulating the transcription and post-translational modification of many target genes.The results are of great significance for understanding the regulatory mechanism of Toxoplasma gondii’s life cycle and developing new vaccines and other prevention and control methods.