Research On The Function And Regulation Of Type â…¥ Secretion System (T6SS) In Vibrio Ansuiliarum

Vibrio anguillarum is one of the most common bacterial pathogens in aquaticdiseases that can infect fish, shellfish and other aquatic animals. V. anguillarum cancause vibriosis, a lethal hemorrhagic septicemia, which leads to extensive losses inboth freshwater and marine aquaculture. So far, the pathogenic mechanism of V.anguilllrum has not been fully elucidated. As one of the many causative agents,bacterial secretion systems can translocate toxins and enzymes synthesized inbacterial cells to host tissues and cells and cause pathogenesis, of which, secretionsystems are one key factor. Research on secretion systems will help to reveal thenormal life activities of V. anguillrum and provide valuable theory and basis for thecontrol of vibriosis.Type VI secretion system （T6SS）, is a new bacterial secretion system found in2006, and its mechanism has not yet been clarified. In previous studies of ourlaboratory, most of the sequences of the T6SS gene cluster were screened from theFosmid genomic library of V. anguillarum M3. In this study, the T6SS upstreampromoter region in V. anguillarum was cloned using Genome walking based on theprevious work. And T6SS operon structure was studied by RT-PCR using inter-geneprimers with cDNAs in different growth stages as templates. Then, seven genemutants of T6SS were constructed using in-frame deletion, of which the phenotypicassays and transcription detection were made for identification of T6SS functions.Interactions between transmembrane proteins were established by analysis of in vitroexpression and co-immunoprecipitation, which can provide experimental evidencefor the T6SS assembly model. Detection of T6SS secreted proteins under differentconditions were performed for a preliminary understanding of T6SS secretion characteristics in V. anguillarum. In-frame deletion mutants of σ⁵⁴and σ³⁸factorswere constructed, then phenotypic tests and transcriptional detection were made inorder to identify their functions. Regulation relationships among T6SS, σ⁵⁴and σ³⁸were analyzed at the transcriptional level by qRT-PCR technology, and regulatorynetworks were constructed next. The main results obtained in this study are depictedas follows:1. A sequence sized2584bp in the upstream of T6SS was cloned, in which a weakpromoter was found by the bioinformatics analysis. Meanwhile, through the wholegrowth process of V. anguillarum, including the logarithmic growth phase andstationary phase, T6SS gene cluster from gene vaa107to vaa121was controlled bythe same operon and promoter.2. Gene in-frame deletion mutants were constructed in vaa017, vaa109, vaa110,vaa115, vaa116, vaa117and vaa120in V. anguillarum. Phenotypic assays showedthat T6SS had significant positive regulations on the growth of V.anguillarum in ironlimited conditions, motility and biofilm production, however, no significant impacton the growth under normal culture conditions was detected. Then the qRT-PCRdetection displayed that expression of extracellular metalloprotease EmpA and PrtVwas positively regulated by T6SS.3. Bioinformatics analysis indicated that the T6SS protein VAA115and VAA120were transmembrane proteins. Results of co-immunoprecipitation showed that therewere interaction relationships between the lipoprotein VAA113and VAA115,VAA113and VAA120, which indicated that the three structural proteins maycollaborate to play an important role in T6SS transport channel assembly.4. Detection of T6SS secretion protein VAA017using Western blot showed thatno existence of VAA017was detected under different culture conditions; however,VAA017was detected in other V. anguillarum strains such as L73. This showed thatthere is a great difference in secretion of T6SS effector proteins among V.anguillarum strains.5. Construction of a V. anguillarum σ factor RpoN (σ⁵⁴) and RpoS (σ³⁸) genein-frame deletion mutants and corresponding complementary strains was completed, and the phenotypic assays showed that RpoN and RpoS had a significant positiveregulation in growth under iron limited conditions, motility, biofilm formation,exopolysaccharides synthesis and secretion, EmpA expression and secretion,gelatinase activity, virulence and in vivo survival ability. Additionally, RpoN alsohad a strict positive control in the formation of bacterial flagellum. qRT-PCRdetection was then performed, and the results were consistent with the phenotypicassays.6. Using qRT-PCR detection combined with the phenotypic analysis, we initiallyconstructed a regulatory network among T6SS, RpoN and RpoS in V. anguillarum.With this work, we found that RpoN and T6SS had a mutual positive regulationwhile T6SS was positively regulated possibly through VanT by RpoS. At the sametime, we found that RpoN had a negative regulation on RpoS, while RpoS positivelyregulated RpoN mainly.Summed with the above results, this study has focused on the functions, assemblystructure, secretion characteristics and regulatory networks of T6SS in V.anguillarum. And this work will lay an important theoretical foundation for thefuture research on the T6SS mechanism.