Identification Of F17-like Fimbrial Gene And Construction Of The Gene Mutant In Gallibacterium Anatis Chinese Isolates

The Gram-negative bacterium Gallibacterium anatis is a typical species in Gallibacterium genus,Pasteurellaceae family.G.anatis is commonly isolated from a wide range of birds,mainly including chickens,turkeys,ducks,geese.Increasing evidence indicate that besides constituting a part of the normal microflora of the upper respiratory tract and lower genital tract in chickens,G.anatis has been recognized as a major cause of salpingitis and peritonitis in egg-laying chickens,leading to decreased egg production and increased mortality.To date,a few virulence factors have been identified,but the G.anatis pathogenesis is not yet clear.A critical step in microbial colonization and invasion of mucosal tissues is adherence.As a mucosal microorganism,can adhere to plastic surfaces,form biofilms,and produce a glycoprotein-like hemagglutinin,but little is known about the importance of these factors during colonization and infection of the natural host.A variety of adhesins,including fimbriae,has been reported from other members of Pasteurellaceae.Fimbriae are long,hair-like,extracellular appendages on the surfaces of bacterial cells and are important virulence factors in many bacterial species.In the view of the above background,this study revealed the distribution and in vitro expression of the main fimbrial subtunit type Flf of F17-like fimbriae in G.anatis chinese isolates,and analyzed the immunogenicity and reactogenicity of major fimbrial subunit protein FlfA.In addition,to select the higher pathogenic bacterial isolates for the construction of flfA deletion mutant,G.anatis were assessed for their ability to adhere to and invade primary chicken oviduct epithelial cells(COCE).Finally,we tried to construct a flfA deletion mutant in G.anatis,and hoped to provide basic-materials for analyzing the importance of fimbriae in the G.anatis pathogenesis.The detailed contents of this project are as follows:1.The identification and analysis of flfA in G.anatis and the prokaryotic expression of FlfA proteinAccording to complete genome sequence of UMN179 in Gen Bank,a pair of primers was designed to clone the flfA gene.The result showed that flfA gene was widely existing in G.anatis Chinese isolates.The analysis of these sequences showed that the homology of flfA gene from 18 Chinese isolates was 71.1%-100%,and the homology with reference sequence was 70.2%-99.8%.The analysis of amino acid sequence of flfA demonstrated the FlfA protein had 6-8 dominant antigen epitopes,which mainly distributed in conserved amino acid sequences.According to the flfA gene of G.anatis Yu-PDS-RZ-1-SLG strain,which is highly virulent for mouse,a pair of primers was designed to clone the flfA gene.The PCR product was ligated into the prokaryotic expression vector p ET-28 a and introduced by transformation into E.coli BL21(DE3).The anti-FlfA polyclonal serum was obtained by immunizing rabbits with purified recombinant FlfA.Western blotting with anti-FlfA immune serum demonstrated recognition of a protein band corresponding to FlfA in 18 Chinese isolates.The results suggested that recombinant FlfA had good immunogenicity and reactogenicity and the potential to elicit cross-protective immunity against heterologous G.anatis strains.This study revealed the wide distribution of the main fimbrial subtunit type Flf and the potential of FlfA protein to be vaccine candidate,and provided effective detection method for the construction of flfA deletion mutant.2.Assessing for the ability of G.anatis to adhere to and invade primary chicken oviduct epithelial cells(COCE)In this study,the adhesive and invasive characteristics of G.anatis Yu-PDS-RZ-1-SLG strain were tested by immunohistochemistry,while the capacity to adhere to primary chicken oviduct epithelial cells(COCE)between 19 G.anatis strains was tested by bacterial CFU enumerations.The results of immunohistochemistry showed that Yu-PDS-RZ-1-SLG strain was not detected intracellularly at 0.5,1,1.5,2,3,4 hour after infection respectively,but the infection induced serious cell pathological change(CPE)and leaded to cell disruption.In addition,Yu-PDS-RZ-1-SLG strain was found adhering to the cell surface of COCE and the number of bacteria adhering to single cell was increasing during the time period of adherence assay.The results of bacterial CFU enumerations in adherence assay demonstrated great disparities of adhesive capacity between different strains.The results of immunohistochemistry further proved that Yu-PDS-RZ-1-SLG strain could not invade primary COCE.The disparities of adhesive capacity might suggest the different pathogenicity of strains.3.Screening of flfA deletion mutant in G.anatisAccording to complete genome sequence of UMN179 in Gen Bank,specific primers was designed to amplify the upstream and downstream homologous arms of the flfA gene.Using plasmid p BC-SK and suicide plasmid p RE112 as the backbone,we constructed the homologous recombinant plasimd p BC-flfA-Gmrand p REΔflfA.Then,the two recombinant plasmid were used to screen mutant strains by natural transformation and conjugation,respectively.And the linear recombinant DNA fragment and recombinant suicide plasmid were successfully introduced into the targeted strain.During natural transformation,we failed to obtain deleted mutant after screening about 3000 CFU.While during conjugation,the flfA gene single-exchange strain was easily obtained,but no flfA gene deletion mutant after screening about10000 CFU.The process of natural transformation was simple and controllable,however,the G.anatis strain with relatively high natural competence and suitable for genetic manipulation was very difficult to screen.This is the first study introducing DNA into G.anatis cells by conjugation,and we have made breakthrough in the process.To date,the screening is still going on.