Research On Mechanisms Of Adaptor Protein PA3293 Translocate H2-T6SS Effector PA3290 In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a movement type nonfermentation conditions of gram-negative pathogenic bacteria,which can cause severe acute and chronic infection.The bacteria secrete a variety of proteins into the extracellular environment to mediates the interactions between its and the surrounding environment.Bacteria have evolved at least seven kinds of specialized productions system to complete the task.? secretion system(T6SS)is one of this kinds of secretion system,which is a means of contact dependent protein secretion and responsible for transfer toxin to the target bacteria and eukaryotic cells.Studies have shown that Pseudomonas aeruginosa PA3290 is an effector,it has the activity of phospholipase A2 and can hydrolysis of adjacent cell membrane phospholipids by enter the periplasmic space of the adjacent cells by T6 SS.Therefore,Pseudomonas aeruginosa is used as the research material to study the mechanism of Pseudomonas aeruginosa adaptor protein PA3293 in the translocation of H2-T6 SS effector PA3290(Tle1).The results are as follows:1.The competition index between the donor and the recipient bacteria were significantly decreased when the clpV2 knocked out of H2-T6 SS as the same of effector protein PA3290 mutant,while the other two sets of T6 SS trends and wild-type donor bacteria did not show,so Tle1 was H2-T6SS-dependent antibacterial effector protein.2.PA3291,PA3292 and PA3291-3292 were complementary to the recipient strain ?3290-3292 Ptac(pME6032),but the tendency were significantly reduced when the two were simultaneously complementary,so Tle1 has two immune proteins.3.Bioinformatics analysis showed that Pseudomonas aeruginosa PAO1 which exist with chimeric daptor protein PA3293 is homologous with Vibrio cholerae N16961 which the embedded joint protein tap-1.4.The competitor index of Tle1,Tap-1 and VgrG4 a were significantly lower than those of wild-type,and the competition index between the corresponding donor and the receptor was significantly higher than that of wild-type to return the wild-type trend,indicates that Tle1-mediated bactericidal action depends on the adapter proteins Tap-1 and VgrG4 a,through the experiments of bacterial two-hybrid and ?-galactosidase activity,it was found that Tap-1 interacts with Tle1 and VgrG4 a directly.5.The interaction between Tap-1 and Tle1 or VgrG4 a was identified by truncation of the protein,bacterial two-hybrid and ?-galactosidase activity.The results showed that the interaction sequence of Tap-1 and Tle1 is located at the the 3'-terminal 180-210 bp of PA3293 encode of amino acid;the interaction sequence of Tle1 and Tap-1 is located at the 600-1464 bp of PA3290 encode of amino acid;the interaction sequence of Tap-1 and VgrG4 a is located at the 3'-terminal 180-210 bp of PA3293 encode of amino acid;the interaction between VgrG4 a and Tap-1 is located at the 3'-terminal 12-18 bp of PA3294 encode of amino acid.In order to validate the effect protein PA3290 in Pseudomonas aeruginosa rely on VgrG and Tap-1 as the same of effector protein TseL of Vibrio cholerae N16961 in the process of translocation.The function of PA3290 was verified by competitive experiments,and the immunological proteins corresponding to the effector were identified on the basis of their functional studies.In addition,the international of effector,adapter protein and VgrG were identified by bacterial two-hybrid.And is of great significance for the secretion mechanism on the study of effector,adapter protein and VgrG.