Functional Identification Of Acetyltransferase Of Pseudomonas Aeruginosa T6SS Effector TseP1 And Analysis Of Its Biological Effects

Pseudomonas aeruginosa is a gram-negative bacterium and is a widely infected conditional pathogen,which can be transmitted through water,soil,utensils and other ways.In addition,studies have shown that the type Ⅵ secretion system(T6SS)in P.aeruginosa plays an important role in the adaptation to the environment and pathogenesis.Typically,the biological function performed by T6 SS is mainly determined by the function of its own effectors.At present,it is known that T6 SS has a variety of functions,including mediating metal ion uptake,inhibiting bacterial growth,and most of the effectors that determine these functions have been identified,and its mechanism has been well analyzed.However,some recent studies have shown that T6 SS also plays an important role in mediating bacteria-plant interactions,but so far the effectors that mediate this kind of T6 SS research.Based on the blank of this kind of T6 SS,we identified a T6 SS effector PA0115 that can act on plants with P.aeruginosa PAO1 as the starting stain,and named Tse P1(Gene ID: Q9I717).In this study,T6 SS effector Tse P1 in P.aeruginosa PAO1 was used as the research object,explored the Tse P1 on the colonization of PAO1 in plants,analyzed the acetyltransferase activity of Tse P1,identified the target protein of Tse P1 in plants,and analyzed the biological effects of Tse P1.The main results of the study are as follows:(1)We combine PAO1 with PAO1,tse P1 mutant strains(Δtse P1)and genetic complementary strains that mixed 1:1 competitive infection with mung beans,it was found that tse P1 mutations did not affect the ability of PAO1 to competitively colonize in mung beans.However,when mung beans were infected alone by PAO1(p ME6032),Δtse P1(p ME6032)and Δtse P1(p ME6032-tse P1),the number of Δtse P1 colonization in mung beans was significantly lower than that of wild type,indicating that Tse P1 has a promoting effect on the colonization of PAO1 in mung beans.(2)We analyzed the protein structure of Tse P1 through bioinformatics and found that the Tse P1 is an acetyltransferase with multiple acetyltransferase domains.(3)We the expression and purification of Tse P1 protein,and determined the acetyltransferase activity.It was found that the optimal substrate for Tse P1 was L-Arginine,the optimal temperature was 55℃,and the optimal p H was 9.The thermal stability of Tse P1 was poor,and the p H stability range of Tse P1 was small.In addition,Tse P1 contained 15 conserved amino acid residues,of which 9 amino acids had an effect on the acetyltransferase activity of Tse P1,and R65,R80,R88,Y125 were the key active sites of Tse P1.(4)In order to explore the interaction mechanism between Tse P1 and plant hosts.Through yeast double hybridization technology,we used the previously established double-hybrid c DNA library of the Dog’s Head Jujube yeast as a screening object to screen the target proteins of Tse P1 in Dog’s Head Jujube.And the 9 strains of possible target proteins were screened.Based on the above results,we know that Tse P1 is an acetyltransferase,and promotes the colonization of P.aeruginosa PAO1 in plants.Based on this phenomenon,we speculate that Tse P1 may promote the infection of plant cells by acetylating the target protein in plants,inhibiting the plant immune response.