The Mechanism Of Action Of Phage Protein L5-03 Regulating Type Ⅵ Secretion System In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is one of the most opportunistic pathogen that can cause a variety of infections diseases.And it has strong resistance to a variety of antibiotics,so it is significantly to find new antibacterial targets and develop new antibacterial drugs.Bacteriophage can specifically infect and cleave bacteria.Phage Therapy is the use of bacteriophage and its products to treat and prevent various infections caused by bacteria,so it has broad application prospects in clinical treatment.However,the current understanding of the interaction mechanism between phage with its host and the function of phage genes is not detailed enough.Type VI secretion system(T6SS)is a new type of bacterial secretion system which participates in a variety of bacterial physiological functions.In P.aeruginosa,T6 SS is related to bacterial pathogenicity,biofilm formation,stress response and bacterial resistance.In this study,based on the regulation of T6 SS by P.aeruginosa PAO1 phage L5,the early gene L5-03 of phage L5 was screened by the lux reporter detection of each promoter of T6 SS to inhibit the inhibition of P.aeruginosa H1-T6 SS transcriptional expression of the core gene hcp1.The results of further study showed that L5-03 could inhibit the transcriptional expression of key genes hcp2 and hcp3 of T6 SS.In addition,western blot experiment also confirmed that L5-03 inhibited the expression of Hcp2 and Hcp3 protein.We also confirmed that L5-03 protein interacted with Hcp2 and Hcp3 proteins by bacterial two-hybrid.we further research found that overexpression of L5-03 significantly decreased pyocyanin production,motility ability and protease secretion of PAO1.Bacterial two-hybrid experiment proved that there was interaction between L5-03 protein with the core protein Pqs E of QS system and Lon protein which is a member of ATP dependent protease family.In short,the T6SS-lux reporter was used to screen the early gene L5-03 of phage L5 to inhibit the expression of T6 SS,and affected the QS system and virulence factors of P.aeruginosa.Finally,its mechanism was preliminarily elucidated.It was found that L5-03 protein could bind to some host proteins proteins Pqs E and Lon of Pseudomonas aeruginosa.which lay a foundation for further elucidating the mechanism of action between L5-03 and the host.And then provided a theoretical basis for the further research of the application of bacteriophage in clinical anti-infective therapy.