Secretion Detection And Functional Identification Of YPK?0952 Protein In Yersinia Pseudotuberculosis

In response to the changing environment,bacteria have evolved into a variety of secretion systems.The versatile effectors secreted by these secretory systems play essential roles in the exchange of nutrients,resisting environmental stress,and competition with eukaryotic and prokaryotic cells,that provides a favorable guarantee for the survival and growth of bacteria.T6 SS is a complex and secreted secretory system which was discovered in recent years.It is widely found in Gram-negative bacteria and is generally considered to be a weapon for bacteria-to-bacteria contact competition.Our previous studies showed that bacteria can also utilize T6 SS effector to participate in the uptake of metal ions,which played important roles in resistance to oxidative stress and bacterial pathogenicity.However,the understanding of T6 SS secretion system and its effector proteins is still very limited.The researches for T6 SS secretion system and its new biological functions will help improvethe understanding of bacterial secretion system,and reveal the interaction between bacteria and host,competition between bacteria and bacteria,and substance exchange between bacteria and the environment.Yersinia pseudotuberculosis is a zoonotic pathogen.Its genome contains four sets of complete T6 SS secretion system.In this study,Y.pseudotuberculosis T6SS3 was used as a research object.Through using the methods of secretion identification,enzyme activity detection,bacterial competition and GST pull-down,the biological functions of YPK?0952effector protein,which is highly homologous to Pyocins,was preliminarily explored.The main research contents and results are as follows:1.Through the alignment analysis,we found an unknown function gene ypk?0952 in the genome of Y.pseudotuberculosis.Its N-terminal coding product contains the conserved sequence PAAR domain of T6 SS effector proteins,and C-terminal has high homology with Pseudomonas aeruginosa Pyocin S3.2.The Y.pseudotuberculosis recombinant expression plasmid p ME6032-0952 was constructed.The secretion of YPK?0952 in wild type strain and T6 SS mutant was detected by western blot,which showed that YPK?0952 is an effector protein secreted by the type 6secretion system.YPK?0952 was also found to be a magnesium ion or zinc ion-dependent DNA hydrolase by DNA hydrolase activity assay.3.Y.pseudotuberculosis ?ypk?0952?ypk?0953 mutant strain was construted in this study.The analysis of the mutant strain and the wild type strain in intraspecific and interspecific competition showed that the deletion of ypk?0952 and ypk?0953 genes had significant effects on intraspecific competition of Y.pseudotuberculosis.4.Screening and validating the interaction proteins by GST pull-down and bacterial two-hybrid showed that YPK?0952 protein in Y.pseudotuberculosis could interact with YPK?0953 and membrane protein Omp A.In conclusion,this study found an effector protein YPK?0952 secreted by the Y.pseudotuberculosis T6 SS had DNA hydrolase activity,and played important role in bacterial intraspecific competition,which further improving the present knowledge for the function of T6 SS effectors.The study also provides a new idea for exploring the prevention and treatment of Yersinia infectious diseases.