Expression And Activity Of Sfm Fimbrial Operons From Escherichia Coli

Colibacillosis is a common bacterial disease in clinical,which has brought significant economic losses to the animal husbandry industry in China.Virulence adhesion and colonization is often considered to be the first and crucial step in the infection.Analyzing the genome sequence data of K88+Enterotoxigenic Escherichia coli(ETEC)strain C83902,sfm operon gene for encoding fimbriae was predicted and its function was identified by cloning,constructing mutant strain and phenotype analysis.By using BLAST to genomes of different E.coli strains on GenBank,the result showed that this operon majorly distributes in pathogenic E.coli,including ETEC,Avian pathogenic Escherichia coli(APEC)and Uropathogenic Escherichia coli(UPEC).It is helpful to further study the pathogenesis molecular mechanism of the interaction between E.coli and the host cells through the in-depth exploration of the pathogenicity of Sfm fimbriae,providing theoretical basis both for the prevention and control strategies of colibacillosis and the development of new vaccines.1.Cloning and expressing of Sfm fimbrial operon gene clusters from Escherichia coli and their bioactivityTo clone and express sfm operon gene clusters(sfmACDHF)from ETEC K88ac+C83902 strain in vitro,Sfm fimbrial operon gene clusters were amplified by long-PCR using the genomic DNA templates of E.coli C83902 strain and cloned into expressing vector pBR322.The recombinant plasmid with the inserts of sfm gene clusters was constructed and screened.The above recombinant plasmid DNA was transformed into non-fimbria E.coli SE5000 strain.The recombinant E.coli didn't agglutinate with chicken red blood cells,no fimbriae-like structure on the cell surface was observed by transmissible electromicroscope as well.Sfm fimbriae protein was extracted from the recombinant E.coli and a clear band of protein with size of approximately 35 kDa was visualized in Coomassie blue-stained gels after SDS-PAGE,and this protein band was recognized positively by anti-SfmH mouse polyclonal serum in Western blot assay,which successfully proved the expression of Sfm fimbriae.Adhesion assay showed that Sfm fimbriae didn't adhere to DF1 cells in vitro(P>0.05).Phagocytic assay showed that the ability of Sfm-expressing recombinant E.coli to be phagocytosed by HD-11 cells was reduced by 50%(P<0.01)compared to the control strains.This study is the first time to show the clone and express for Sfm fimbriae,providing theoretical basis both for exploration of new virulence factors and explanation of pathogenesis.2.Construction and function verification of sfmA gene mutant strain from APEC CE129In order to explore the function of Sfm fimbriae,the sfmA gene was knocked out by ?Red recombination system and the mutant strain was constructed,which was named as CE129?sfmA.Firstly,we generated the PCR products from strain CE129 by specific primers designed according to the known sfmA gene sequence.Secondly,the PCR products for the replacement of sfmA gene were constructed by employing template plasmid pKD3 and primers which contain a homologies 5' terminal to the target region and a homologies 3'terminal to chloromycin-resistance(cat)gene.With ? Red recombination system,the PCR products were introduced into wild type strain CE129 carrying a heat-sensitive plasmid pKD46 by electroporation,and sfmA gene was replaced by cat gene.The successful recombinant bacterial strains were selected by LB plates containing chloramphenicol.Then the chloromycin-resistance gene was eliminated by another heat-sensitive plasmid pCP20,which encoding the Flp recombinase.Using this system,sfmA gene in chromosome of strain CE129 was deleted.Finally,the isogenic gene deletion CE129?sfmA mutant was constructed and confirmed by PCR and DNA sequencing data.On the base of the mutant strain,we electroporated plasmid pBR322 which could express SfmA protein into second recombination mutant to construct the complementary strain CE129?sfmA/psfinA.Compared to the strain CE129,the results of serum bactericidal tests showed that the serum complement sterilization resistanceability of strain CE129?sfmA was lower by 20.1%(P<0.05)after incubation with fresh SPF chicken serum for 4 hours,the results of qualitative and quantitative biofilm tests showed that the biofilmforming ability of strain CE129?sfmA decreased by 13%(P<0.05),DF1 cell adhesion tests showed that the adhesion ability of strain CE129?sfmA decreased by 41%(P<0.01),HD-11 cell phagocytic assays showed that HD-11 cells had 30%(P<0.01)higher phagocytic ability for strain CE129?sfmA.Sequentially we used Real-Time PCR to detect the transcriptional changes of the major virulence factors in strain CE129?sfmA and strain CE129?sfmA/psfmA.The results showed that the transcriptional level of fimA(encoding the main structural subunit of type I fimbriae),papC(encoding usher protein of P fimbriae),fliC(encoding flagellin),ompA and iss(encoding outer membrane protein)decreased by 40%(P<0.01),43%(P<0.001),23%(P<0.01),21%(P<0.01)and 18%(P<0.05),respectively.As described above all,Sfm fimbriae plays an important role in bacterial adhesion,biofilm formation and the process of serum sterilization.Sfm fimbriae participates in the pathogenesis of APEC CE129 directly or indirectly.This may provide a new theoretical basis for the explanation of pathogenic mechanism of E.coli.