Characterization Of Type ? Secretion System In Enterohemorrhagic Escherichia Coli:Identification,Function And Regulation

Enterohemorrhagic Escherichia coli(EHEC)is one type of major contagious and foodborne pathogens.Once infected,the host may exhibit severe symptoms,such as abdominal pain,diarrhea,hemorrhagic colitis and hemolytic uremic syndrome or even death.On the other hand,EHEC can intimately adhere to the enterocyte membrane and efface the microvilli,causing the so called A/E(Attaching and Effacing)lesion mediated by type III secretion system.Gram-negative bacteria possess multiple secretion systems,which can form transmembrane channels in bacterial cell surface to translocate specific substrates across the cell envelope to outside environment or directly into host cells for a variety of purposes including toxicity to hosts.Hcp(Hemolysin coregulated protein)is a secreted protein without an N-terminal signal peptide in Gram-negative bacteria.The newly discovered secretion system,type ? secretion system(T6SS),is specifically responsible for the translocation of Hcp.Deletion of T6 SS or hcp can lead to loss of virulence in pathogens such as Vibrio cholerae.Pseudomonas aeruginosa can always be the predominant species with antimicrobial ability mediated by T6 SS.However,the role of T6 SS in bacterial pathogenesis remains unclear.In order to investigate the role of T6 SS in the pathogenesis of EHEC,we identified a typical 36.5 kb T6 SS gene cluster,which has 32 ORFs named from z0243 to z0275,including 13 conserved T6 SS elements.In addition,we identified z0706 and z0707 as the homolog of hcp and vgrG,respectively.We used in vitro and in vivo assays including macrophage and mouse model to characterize T6 SS and determine its role in the pathogenicity of EHEC.Firstly,we constructed the deletion mutant of T6 SS by Lambda Red homologous recombination.The growth rate and motility of ?T6SS are comparable to these of the wild type(WT).Then,we collected the secreted proteins of the two strains and determined their components by LC-MS/MS.After validated by Western blotting and ?-lactamase fusion assay,we found that Z1921 was a novel substrate of T6 SS in EHEC.Subsequently,Z1921 was identified as a Mn-calatase by physiological and biochemical analysis,so we named Z1921 KatN according to its homologs in Salmonella.KatN can degrade hydrogen peroxide and might contribute to bacterial resistance to oxidative stress.In logarithmic phase,RpoS and OxyR can both positively regulate the transcription of katN,maintaining the cytoplasmic abundance of KatN,regardless of whether hydrogen peroxide is present or not.Secondly,the expression of T6 SS gene cluster could not be elevated by changing the sets of culture conditions to different temperature,pH,medium,and salinity,or in the different growth phases.H-NS,a histone-like nucleoid structuring protein,can mediate the silencing of laterally acquired genes in bacteria,including the genes located in pathogenicity islands.We compared the transcriptomes of WT and ?hns by RNA sequencing.When hns was deleted,954 genes showed significant changes,78% of them were up regulated,including the genes located in the T6 SS gene cluster.This result was confirmed by quantitative RT-PCR and Western blotting.Finally,we challenged 5-week-old female BALB/c mice with equal amount of WT or ?T6SS.We found that the mice infected with WT showed a lower survival rate compared to those infected with ?T6SS,which suggested that T6 SS contributed to EHEC virulence.As a consequence of the absence of hns,deletion of either T6 SS gene cluster or katN did not affect bacterial virulence in mice model,suggesting H-NS contributed to the bacterial virulence by regulating T6 SS.When EHEC was phagocytized by murine macrophage RAW264.7,its expressions of T6 SS gene cluster and katN were obviously elevated.However,when hns was deleted,the expresssions of T6 SS gene cluster and katN were increased at a higher level.EHEC could not survive in the macrophage when T6 SS gene cluster or katN were deleted in ?hns background.This suggested that T6 SS contributed to EHEC survival in macrophage.In summary,T6 SS contributed to the virulence of EHEC in mice,particularly in the early stage of infection.When phagocytized by macrophage,EHEC can sense the oxidative stress by RpoS and then up regulate the expression of katN.KatN could be subsequently secreted by T6 SS to eliminate the reactive oxygen species in host cell to improve EHEC survival in macrophage.The findings of this project not only deepen our understanding of EHEC pathogenicity,but also provide the subsequent investigation under the same purposes with a new horizon.Of course,all the work can provide new insights into treatment of EHEC infection related diseases,drug development and so on.