| Toxoplasma gondii is a serious zoonotic parasite among apicomplexans.The secretory proteins produced by special organelles such as rhoptries（ROPs）,dense granules（GRAs）and micronemes（MICs）play an important role in the invasion,replication and immune evasion of T.gondii.The folding,degradation and various post-translational modifications of secreted proteins and membrane-bound proteins of eukaryotes all require the participation of ATP as an energy source or cofactor in the ER.However,as a nucleotide macromolecule,ATP cannot directly enter the ER through the biofilm system,so a specialized transporter is required to transfer it into the endoplasmic reticulum,and at the same time,the ADP generated by decomposition is transferred out of the ER.As a parasitic protozoa with professional secretion ability,T.gondii theoretically requires a large amount of ATP in the ER to participate in the processing and folding of secreted proteins.However,it is still unclear how the cytoplasmic ATP enter into the ER and what role it plays in the growth of T.gondii.In this study,an ATP/ADP transporter located in the ER of T.gondii was identified by various methods,and its function was studied.（1）Screening of ATP/ADP transporters in the ER of T.gondiiDue to the lack of research on the ER ATP/ADP transporter,there is no accurate information for gene screening in T.gondii.Therefore,the BLAST algorithms were used for searching the ATP/ADP transporter with the ER-ANT1 and AXER in ToxoDB.Blastp analysis revealed only three sequences encoding putative solute carriers family protein were high homologous to ER-ANT1,but no genes with homology to AXER were found,experiments show that they are located in the mitochondria instead of the ER.Meanwhile,we screened sixty-nine sequences by setting the CRISPR phenotype value and transmembrane domain in the ToxoDB database.Among the sixty-nine selected genes,we focused on TGGT1＿254480,which was annotated as UDP-galactose transporter family protein in the ToxoDB database,and predicted to have ten transmembrane helices.Besides,TGGT1＿254480 contains a C-terminal dilysine motif（-KKQC）that serves as an ER retention signal,C-terminally dilysine motif shown in other proteins to limit exit from the ER.Localization experiments found that TGGT1＿254480 was located in the ER of T.gondii and named TgANT.（2）Studies on the transport capacity and substrate specificity of TgANTTo test whether TgANT might act as an ER ATP/ADP transporter,we cloned the c DNA of TgANT into the plasmid pet16b and expressed it in E.coli（BL21,DE3）.IFA showed that the TgANT was expressed and inserted into the bacterial membrane.Uptake studies were carried out with radioactively labeled[α-32P]ATP and showed ATP was imported into intact bacterial cells harboring TgANT.By contrast,the uninduced E.coli was not able to absorb ATP or ADP at significant rates.Competition experiments with different potential substrates showed that the heterologously expressed TgANT in E.coli were highly specific for ATP and ADP.The ability of E.coli to uptake radioactive ATP was inhibited in the presence of non-radioactive ATP or ADP.Back exchange experiment showed that TgANT is an ATP/ADP antiporter（3）Effects of TgANT on growth of Toxoplasma gondii in vitroSince TgANT has a low phenotype score（-4.8）,it is very important for T.gondii and cannot be deleted directly from the parasite.Therefore,an inducible knockdown system（iTgANT）was used to deplete TgANT expression.The result showed that iTgANT strains treated with ATc did not form any visible plaques compared with the iTgANT strains without ATc treatment.Moreover,the intracellular replication speed of iTgANT strains was also significantly reduced and reduced ability to invade after treatment with ATc.Taken together,these data indicate that TgANT is essential for Toxoplasma’s normal growth.（4）The cause of severe growth defects in Toxoplasma gondii caused by TgANT depletionIn order to prove that the severe growth defect of T.gondii is caused by the interruption of ATP supply in the ER,we found two conserved domains of TgANT and the two conserved domains were mutated using amino acids with similar hydrophilicity and hydrophobicity.The results showed that mutant TgANT no longer had the ability to absorb ATP,and the mutated TgANT could not complement the growth defect of iTgANT strain,but heterologous mammalian ATP/ADP transporter AXER can rescue the corresponding phenotypic defects.At the same time,the deletion of TgANT leads to the interruption of ATP supply in the ER of T.gondii,resulting in ER stress in T.gondii,and this irreversible ER stress eventually greatly increases the level of apoptosis.This study focused on the ER ATP/ADP transporter of T.gondii,demonstrating that the ER of T.gondii requires specialized transporters to transport cytosolic ATP into the ER,and the supply of ATP in the ER is essential for parasite growth and shows a completely different importance from mammalian and plant cells.Our study provides new insights and theoretical basis for further understanding of the molecular mechanism of growth regulation of T.gondii and the development of new prevention and control technologies. |
