Study Of The Function And Molecular Mechanism Of Type Ⅵ Secretion System AmpDh3 Effector In Pseudomonas Aeruginosa

Bacteria have developed multiple secretion systems to communicate with environment.Using the secretion systems,bacteria can secrete toxic proteins to the outer environment,or directly act on targets of neighboring or eukaryotic cells resulting in illness.Type Ⅵ secretion system(T6SS)is a multi-functional virulence mechanism widespread among Gram-negative bacteria.Pseudomonas aeruginosa(PA)is an opportunistic pathogen.The success of P.aeruginosa as a nosocomial pathogen is the result of its ubiquitous distribution and resistance to antibiotics.T6 SS plays a vital role in the infection of P.aeruginosa.P.aeruginosa encodes three different T6 SS clusters(H1-,H2-,H3-T6 SS,respectively).The sensor kinase Ret S regulates the Gac S/A-Rsm Y/Z-Rsm A signaling pathway through a cascade to inhibit T6 SS gene expression.Analyzed and compared of the proteins in the secretomes of the Δret S and Δret SΔclp V2 strains via liquid chromatography-tandem mass spectrometry(LC-MS/MS).The secretome analysis revealed that the secretion of 21 proteins is dependent on the H2-T6 SS,including the peptidoglycan hydrolase(AmpDh3).Relevant literature has already described the biological function and structure of the protein.Based on the foundation,the paper conducted a comprehensive study on the secretion mechanism,protein function and regulation mechanism of AmpDh3.Western-blot analysis confirmed that AmpDh3 is ecreted via H2-T6 SS,which is a substrate for the H2-T6 SS,and its secretion is dependent on the Vgr G2 b carrier protein.AmpDh3 is a peptidoglycan hydrolase of P.aeruginosa.We demonstrated AmpDh3 acts on the periplasmic space of cells and exerts hydrolase activity,and futher identified its key active site(H146A).In addition,we found and confirmed PA0808,a heretofore protein of unknown function,can act as an auto-immunity protein against AmpDh3 toxicity.PA0808 is periplasmic and that it mitigates the AmpDh3 antibacterial activity by cell location and toxicity assay.GST Pull-down assay showed that physical interaction between AmpDh3 and PA0808 exists.Previous studies show that T6 SS mediates inter-bacterial competition occurs between and within bacterial species.We performed bacterial competition assay.The findings suggest that P.aeruginosa can take a fitness advantageby exploiting AmpDh3 to compete with other bacteria in the complex environments.At last,the established phylogenetic trees revealed that the AmpDh3 and PA0808 orthologues is widely distributed in a variety of bacteria,indicating the wide range adaptation of bacterial competition weapon that promotes bacterial environmental and nutritional competition.In this study,we reveal that the P.aeruginosa H2-T6 SS delivers a peptidoglycan hydrolase,AmpDh3 that depends on Vgr G2 b carrier protein,to the periplasm of the recipient cells.Thereby AmpDh3 secure a fitness advantage in competition with other bacteria.Additionally,we demonstrate that PA0808,encoding a hypothetical protein of unknown function,serves in mitigating the deleterious auto-toxicity of AmpDh3,thereby protects themselves from poisoning.To gain further understanding the T6 SS function of P.aeruginosa and provide important pathway for the study of bacterial secretion system.