Identification Of Fish Pathogen Pseudomonas Plecoglossicida Antibacterial T6SS Effector-Immunity Proteins And Preliminary Functional Analysis

Pseudomonas plecoglossicida is an important fish pathogen that can cause a large number of deaths in farmed fish such as yellow croaker.Previously,our group found that there were three distinct type Ⅵ secretion systems（named T6SS-1,-2 and-3）encoded in the genome of fish pathogenic P.plecoglossicida strain XSDHY-P;T6SS-2 mediated interbacterial competition,thereby promoting the colonization of P.plecoglossicida strains in the aquatic environment or host.However,the exact effectors that are responsible for T6SS-meidated interbacterial killing have not been elucidated in this strain.Previous reports have shown that antibacterial T6SS effectors are often encoded with their cognate immune genes that neutralize their toxicity.Moreover,genes encoding effector and immunity pairs（hereinafter referred as E-I）are often associated with Hcp,Vgr G,and PAAR-protein encoding genes.Using these known information,identified putative toxic T6SS effector in XSDHY-P strains through bioinformatics analysis and predicted their toxicity domains,and then proved that two E-I pairs play a role in the interbacterial competition mediated by T6SS by E.coli growth inhibition assays,plasmid degradation assays,and interbacterial competition assays.The key findings of my thesis are summarized as follows:1.P.Plecoglossicida XSDHY-P encodes five vgr G genes（three of which are encoded in the 3 main T6SS clusters and the other two are orphan vgr G genes）,of which vgr G-2 and two orphan vgr G（vgr G-4 and vgr G-5）genes are encoded downstream with 4 putative antibacterial effectors（Type six XSDHY-P effector;Txe）and their immune protein（Txi）.E-I pairs Txe1-Txi1 and Txe2-Txi2 are encoded downstream of Vgr G-2,Txe3-Txi3 is encoded downstream of vgr G-4,Txe4-Txi4 is found in vgr G-5 cluster.2.Bioinformatic analysis in this work show that above four putative effectors belongs to different nuclease families.Txe1,Txe2,and Txe4 contain a typical RHS conserved domain,and Txe1,Txe3,and Txe4 contain the PAAR motif.For the prediction of C-terminal domain（CTD）,Txe1 CTD did not have a homolog presented in the CDD database,but predicted structure by the Alphafold2 modeling showed that Txe1 CTD carries a catalytic structure similar to the His-Me finger of Caspase-Activated DNase（PDB ID:1v0d）.Txe2 belongs to the Tox-AHH nuclease（pfam14412,HNH/Endo ⅥI nuclease family）.Txe3 has the HNHc nuclease domain（pfam01844,HNH nuclease family）at its CTD.Txe4 has a Tox-SHH nuclease domain（pfam15652,HNH/Endo ⅥI nuclease family）.3.Txe1,Txe2 and Txe4 display striking toxicity to E.coli when expressed in the cytoplasm,while only Txe1 and Txe4 have a significant bacteriostatic role observed in the interbacterial competition assays.The coding sequences of txe1,txe2,txe3,txe4,txe1^CT（C-terminal domain of Txe1）,txe2^CT,txe4^CT,txe1-txi1（effector-immune coexpression）,txe2-txi2 and txe4-txi4 were cloned to arabinose-induced plasmid p BAD33.1.The recombinant plasmids were transformed into E.coli BL21（DE3）.Then growth inhibition assays and plasmid DNA degradation assays.The results show that Txe1,Txe2,Txe4,Txe1^CT,Txe2^CTand Txe4^CTinhibited the growth of E.coli and caused plasmid DNA degradation.Txi1,Txi2 and Txi4 are immunity proteins of Txe1,Txe2 and Txe4,respectively.The effector deletion strains were created in this work.Then interbacterial competition using XSDHY-P wild-type strain（WT）,effector-deletion mutants andΔT6SS-2 as predators,and E.coli XL10 as prey showed that only Txe1 and Txe4 mediated interbacterial killing.