| Toxoplasma gondii is an obligate intracellular parasite,which infects a wide range of hosts,seriously endangers human health and affects animal food production.Many proteins in Toxoplasma gondii need to undergo post-translational modifications to perform their functions.Post translational modifications mediated by glycosyltransferase family play an important role in the glycosylation of Toxoplasma gondii.In the past,researches on glycosylation of Toxoplasma gondii proteins mainly focused on glycosyltransferase family,but little was known about how glycosylated donors were produced during glycosylation.UDP-N-acetylglucosamine(UDP-Glc Nac)is catalysed by UDP-N-acetylglucosamine pyrophosphorylase.UDP-N-acetylglucosamine is the first glycosylation donor in Nglycosylation and Skp1 glycosylation of Toxoplasma gondii.It is also one of the important monosaccharide donors in other glycosylation modification processes.It plays an important role in the formation of glycosylation modification of Toxoplasma gondii protein,However,the enzymes responsible for the catalytic production of UDP-Glc NAc in Toxoplasma gondii have not been characterized.In this study,we found that there was a glyconucleotide synthesis pathway in Toxoplasma gondii by bioinformatics analysis,and then found the potential UDP-Glc NAc pyrophosphorylase(Tg ME49＿264780)in Toxoplasma gondii by amino acid sequence alignment and phenotypic value screening 。 According to its annotation in the database,it was named as UTPG.The biological functions of UTPG gene in the growth and development of Toxoplasma gondii were studied by genetic methods,including the following three aspects:(1)Effect of UTPG gene on the growth and development of Toxoplasma gondii in vivoThe UTPG gene was conditionally knocked out by CRISPR / cas9 gene editing technology and TIR1 auxin induced conditional degradation protein system.The conditional knockout strain and reinfection strain were constructed.A series of phenotypic experiments were carried out on the obtained knockout strain.It was found that the deletion of UTPG gene affected the normal growth and development of Toxoplasma gondii in vivo,deletion of UTPG gene is lethal to Toxoplasma gondii.(2)Effect of amino acid sites in UTPG gene on its biological functionBy mutating the key amino acid sites in the amino acid sequence of the protein expressed by UTPG gene,it was found that G330 A could not rescue the phenotype defect caused by UTPG gene deletion,and it would also cause the death of Toxoplasma gondii,while R334 A and K341 A could rescue the growth phenotype defect caused by UTPG gene deletion,These results indicate that G330 is very important for UTPG gene to play its normal biological function,while R334 and K341 are not very important.(3)UTPG gene is a potential UDP-Glc Nac pyrophosphorylase in Toxoplasma gondiiThrough the UDP-Glc NAc pyrophosphorylase(UAP1)gene in the heterologous yeast,it was found that the UAP1 gene of heterologous yeast could support the normal development of Toxoplasma gondii without UTPG gene,and could completely restore the previous phenotypic defects,indicating that UTPG gene is a potential UDP-Glc NAc pyrophosphorylase in Toxoplasma gondii.In conclusion,the phenotype of the constructed monoclonal strains was studied by conditional knockout of UTPG gene and construction of point mutation and heterologous strains.It was found that UTPG gene is very important for the normal growth and development of Toxoplasma gondii,and G330 site is the key amino acid site for UTPG gene to play its biological function,At the same time,UTPG gene has potential catalytic activity of UDP-Glc NAc pyrophosphorylase,and the UDP-Glc NAc produced by UTPG is essential in the glycosylation of Toxoplasma gondii.These results indicate the importance of the UDP-Glc NAc pyrophosphorylase,which is involved in the formation of UDPGlc NAc,in the growth and development of Toxoplasma gondii,and lay a foundation for further understanding the glycosylation modification of Toxoplasma gondii,and provide a new idea for the development of new drugs according to the related pathway of glycosylation modification. |
