| Various mechanisms can be use to efficiently transport proteins outside of the cellular in bacteria.We called these secreted proteins as effectors which play an important roles in the interactions of bacterial cells with abiotic environment,other bacteria and the host organisms.At present six major secretion systemshave been reported in Gram-negative Bacteria.Type ? secretion systems?T6SS?was discovered in 2006,as a novel secretion system in Pseudomonas aeruginosa and Vibrio cholerae where it was associate with pathogenicity.The T6 SS like a syringe that delivers effectors directly inside a target cell which can target either eukaryotic or prokaryotic cells.There are two ways that T6 SS identify and secretion effectors: either fused to structural core components?called specialised effectors?or by noncovalent interaction with the core components?named cargo effectors?,In both cases,effectors are correlation with components of the expelled Hcp-Vgr G-PAAR composite structure.Rhs protein as the bacteria T6 SS secretion protein which play an important role in contact-dependent growth inhibition?CDI?systems.Rhs is a kind of large molecular weight effector which is composed of conserved N-terminal,core domain and variable C-terminal.C-terminal is the critical area of virulence.The C-terminal usually code zymoproteins and perforinproteins,such as metallopeptidase,DNase,RNase,deaminase which can target cell wall,cell membrane and cell nucleus,respectively.Rhs I gene usually exists in downstream of the Rhs gene?Rhs Immunity Protein?.Rhs I can neutralize toxicity of Rhs,so as to avoid the effect of proteins secreted by bacteria itself.Through bioinformatics analysis,we found the four classic Rhs protein in extraintestinal pathogenic Escherichia coli PCN033,and respectively named Rhs1,Rhs2,Rhs3 and Rhs4.The Rhs2 is in the T6 SS gene cluster where downstream of the vgr G2,but Rhs1,Rhs3 and Rhs4 are out of the T6 SS gene cluster of which Rhs1 is in downstream of the vgr G4.Meanwhile we found that all of four Rhs contained with PAAR structure.Then we found Rhs1 is similar to perforin 4oebA by protein database analysis,but Rhs2,Rhs3 and Rhs4 have no result.Therefore we conduct research as follow: 1.Construction of gene deletion mutants ?Rhs1CT,?Rhs2CT,?Rhs3CT,?Rhs4CT.Amplified upstream and downstream homologous arm of Rhs1 CT,Rhs2CT,Rhs3 CT and Rhs4 CT according to parent bacteria PCN033 and then connected the upstream and downstream homologous arm to the plasmid p RE112.So far we have successfully constructed recombinant plasmid: p RE112-?Rhs1CT,p RE112-?Rhs2CT,p RE112-?Rhs3CT,p RE112-?Rhs4CT.Then translated recombinant plasmid into Escherichia Coli X7213.Next we used X7213 as donor bacterium,parent strain bacteria as receptors,joint transferred combinant plasmid to parent bacteria genome,acquired single exchange strains.Then through no salt LB and sucrose LB medium alternately passaged single exchange strains,we can screen out double exchange strains which is sensitive to Cm and tolerate to sucrose.Finally,we detected the double exchange strains with internal primers and exogenous primers.If Rhs CT gene primers couldn't expand the stripe and exogenous primers expand a small band compared to the wild strain,we acquire the Rhs CT gene deletion strains.2.Prokaryotic expression and toxicity studies of Rhs CT.First amplified the orf of Rhs1 CT,Rhs2CT,Rhs3 CT,Rhs4CT and respectively connected to the plasmid p ET-28 a,translated the recombinant plasmid into Escherichia BL21.Then explored condition of prokaryotic expression,however only Rhs4 CT were expression in inclusion body,Rhs1 CT,Rhs2CT,Rhs3 CT can't be expression under any condition.Therefore we revived the expression vector of BL21 strain and the empty vector of BL21 strain and regulation their OD600 to a consistent.Next we sucked up diluted bacterium solution into K50 medium and K50+IPTG medium,induced for one night.Finally we observed that BL21 strain which contained Rhs1 CT,Rhs2CT and Rhs3 CT gene grows slower than BL21 strain contained empty vector.So we draw a conclusion that the proteins Rhs1 CT,Rhs2CT and Rhs3 CT have toxic effect on bacterial growth.3.Identification of immunity protein.Inorder to avoid damage form effectors which secreted by itself,bacteria often expression a series of immunity proteins which can neutralize toxicity of effectors.We predicted immunity proteins Rhs1 I,Rhs2I and Rhs3 I by ORF finder.And then fused Rhs CT which removed the termination codon and Rhs1 I together into plasmid p ET-28 a.Next we conducted induction experiment as above.Finally we successfully identificated Rhs2 I and Rhs3 I.Unfortunately,due to the effector protein and immune protein interaction mechanism is not clear and there is no basis when predicting immune protein,we haven't found the Rhs1 I.4.Rhs CT gene deletion strains and parental strains pathogenicity studies.We want to explore the function of Rhs CT in pathogenic processes by living level and cellular level.First of all,we intravenous injected gene-deleted strains and parental strains at the same dose of 107 amount into mice.After compared the microbe amount in blood,brain,lung and spleen.We found that compared with parental strains group,Rhs1CT-deleted group's microbe amount seriously decreased in blood,brain and lung.But Rhs2CT-deleted group,Rhs3CT-deleted group and Rhs4CT-deleted group maintained constant.Based on this result,we focused on Rhs1CT-deleted strain in follow-up experiments.We injected Rhs1CT-deleted strain and wild strains at the same dose of 2×106 into mice abdominal cavity and recorded death rate.The results show that,compared with wild strains,the ability of Rhs1CT-deleted strain cause mice death decreased obviously.Under the circumstance of 37? and 5%CO2 in vitro,we compared the ability of bacteria survive in phagocytes and the ability of invasion or adhesion to the epithelial cell.Compared with wild strains,Rhs1CT-deleted strain ability of survival in RAW264.7 decreased obviously.After deleted Rhs1 CT gene,adhesion and invasion ability also significantly decreased. |
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