Construction And Characterization Of PPECC33₀0229 Deleted Mutant Of Extraintestinal Pathogenic Escherichia Coli.isolated From Swine

During the past decade,Type VI secretion system（T6SS）has always been a hot topic for domestic and foreign scholars,since it was discovered in 2006.However,the research on T6 SS of Ex PEC is at a preliminary stage now.T6 SS is a dynamic apparatus that translocates proteins from effector cells to target cells.It is conserved in 25% Gram-negative bacteria,including Pseudomonas aeruginosa,Vibrio cholera and Escherichia coli.T6 SS plays a crucial role in bacterial pathogenicity,by delivering effector proteins into both eukaryotic cells and prokaryotic cells.PCN033,isolated from the brain of swine,is one of the porcine Ex PEC.The T6 SS gene cluster was found in the genome of PCN033 by whole genome sequencing and further sequence alignments.We further found a functionally unknown gene PPECC33₀0229（hereinafter referred to as 0229）exists in the cluster of T6 SS.Therefore,we explored the function of 0229 to reveal the potential role in the process of bacterial competition and pathogenicity using PCN033 as parental strain.We constructed the mutant strain and complementation strain,then series of biological characteristics were compared with the parental strain.1.Construction and identification of gene deletion mutant Δ0229 and complementationstrain Δ0229-0229.The method of homologous recombination mediated by suicide plasmid p RE112 was adopted.The upstream and downstream homologous arms of 0229 were amplified with PCN033 as template by PCR,arms were conneted to the shuttle plasmid p Bluescript II SK（+）,and then connected to the suiside plasmid p RE112 by restriction enzyme.The successfully recombinant plasmid was transferred into x7213,it was used as donor strain and then conjugated with the recipient strain PCN033.Chloramphenicol-resistant and sucrosesensitive transconjugants were screened out after two single exchange processes.Ultimately,the mutant Δ0229 was confirmed by PCR using outside and internal gene identification primers.Gene 0229 was amplified by PCR,and then amplified product was digested and ligated into shuttle plasmid p HSG396.The recombinant plasmid was transferred to Δ0229 deletion strain,chloramphenicol resistant plate was used to screen positive clones,We confirmed the successful construction of the complementary strain Δ0229-0229 through PCR identification.2.Study on the correlation of the bacteria competitive ability.The growth competition assay model was performed,PCN033 and its derivative were used as predators,the E coli.K12 strain W3110 was used as prey.The results showed that the W3110 survival rate of Δ0229 group was significantly down-regulated comparing to parental strain PCN033.Indicating that gene 0229 may play a role in the competition between bacteria.3.Study on the correlation of the pathogenicity.We conducted experiment to compare the intracellular survival rate of PCN033 and Δ0229 after phagocytosed used RAW264.7.The survival rate of Δ0229 is higher than that of the PCN033 at 2 h and 4 h,there is no significant difference,but it is significant at 6h.On the other hand,in the HBMEC adhesion and invasion assay,Δ0229 was significantly upregulated comparing with PCN033,and this phenotypeis basically restored when gene 0229 was returned.These results showed that 0229 may be associated with meningitis caused by PCN033 adhesion and invasion to HBMEC.Four weeks Kunming mice were used as animal models,to compare the tissue colonization ability of Δ0229 with PCN033.The brain,spleen,lung and kidney tissues of mice were obtained,and the numbers of bacteria in tissues were counted.The data shows that the numbers of bacteria in Δ0229 is higher than that in PCN033.Indicating that the Δ0229 may has stronger tissue colonization capacity.The blood sterilization test was carried out by using the blood from native animal swine.After interacting with blood at 1 h,2 h and 3 h,the survival rate of Δ0229 is higher at all the three time points compared with PCN033,however there is no significant difference.4.Detect the transcriptional level of T6 SS related genes by Quantitative Real-time PCR.The transcriptional level of T6 SS genes in PCN033 and Δ0229 was detected by Quantitative Real-time PCR.The results showed that the transcriptional level of some genes in Δ0229 are up-regulated.Therefore,we assume that the phenotypic differences may be the result of the differential expression of T6 SS.5.The prokaryotic expression of 0229 encoding protein aec28.The genomic of PCN033 was used as a template to amplify gene 0229,and then the amplified product was digested and ligated into expression vector p ET28 a,Transferred the recombinant plasmid into E.coli / BL21（DE3）.We confirmed the protein aec28 was successfully expressed by SDS-PAGE and Western blot,while the protein concentrated expressed in the inclusion body.