Identification And Function Study Of The Potential T6SS Effector Protein TseM In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen,and the VI secretion system（T6SS）is one of its important pathogenic factors.There are three T6SS gene clusters independently in the P.aeruginosa genome,which are H1-T6SS,H2-T6SS,and H3-T6SS.Current research shows that H1-T6SS mediates interactions between bacteria,while H2-T6SS and H3-T6SS not only participate in the interaction with bacteria,but also participate in the interaction with eukaryotic host cells.At present,some effector proteins secreted by H1-T6SS have been identified,and little is known about the effector proteins secreted by H2-T6SS and H3-T6SS.The identification of effector proteins is of great significance for elucidating the function of T6SS.The research object of this paper is PA14 strain which is more toxic than PAO1 in Pseudomonas aeruginosa.The regulation of Pseudomonas aeruginosa PA14 H3-T6SS was studied and the potential effector protein TseM was identified and studied.The specific findings are as follows:1.Bioinformatics analysis of H3-T6SS of Pseudomonas aeruginosa PA14 revealed that there are two genes PA14＿33970（named tseM）and PA14＿33980 in the H3-T6SS gene cluster of Pseudomonas aeruginosa PA14,which are absent in the H3-T6SS gene cluster of Pseudomonas aeruginosa PAO1.KEGG database showed that the conserved protein encoded by tseM consists of two Peptidase＿M91 motifs which were defined as effector proteins.Further alignment analysis found that TseM has 33%similarity with the pathogenic E.coli T3SS effector protein Nle D,suggest that TseM may be a substrate effector protein of Pseudomonas aeruginosa PA14.2.By constructing the H3-T6SS promoter β-galactosidase activity reporter strain and the regulatory factor mutant strain ΔoxyR,ΔompR,ΔrpoS,to construct a chromosomal fusion reporter strain,it was found that the expression of H3-T6SS was negatively regulated by the global oxygen regulatory protein OxyR and osmoregulatory protein OmpR,and positively regulated by theσ-factor Rpo S.EMSA experiments further showed that OxyR and OmpR can directly bind to the H3-T6SS promoter,indicating that their regulation of H3-T6SS is directly,while Rpo S has an indirect positive regulatory effect on H3-T6SS.3.By constructing the mutant strainΔtseM and its genetic complementary strain,the survival rate under H₂O₂ stress conditions was tested.The study showed that compared with the wild type,the survival rate of ΔtseM mutant strain under 0.5 mM H₂O₂ stress conditions was significantly reduced,and the survival rate of the complementary strain can be restored to the wild type level,indicating that tseM participates in the stress resistance.4.PAR method revealed that TseM protein can bind to Zn²⁺,indicating that TseM is a Zn²⁺binding protein.The results of intracellular ion experiments indicate that TseM may not be involved in antioxidant stress through transporting Zn²⁺.5.In vitro purification of GST-TseM protein,doing GST-Pulldown experiment with T6SS component VgrG3,it was found that TseM protein can interact with VgrG3,suggesting that TseM may be a H3-T6SS dependent secretion of substrate protein.6.The biofilm formation and motility experiments showed that compared with wild type,ΔtseM significantly reduced the biofilm formation capacity and swimming and swarming ability in Pseudomonas aeruginosa PA14.