Role Of Outer Membrane Protein Porins In Pathogenicity And Immunogenicity Of Avian Pathogenic Escherichia Coli

Escherichia coli usually colonizes the gastrointestinal tract of human,animals and birds.Although most types of E.coli are harmless,several pathovars have acquired specific virulence attributes,which confer an increased ability to adapt to new niches and allows then to cause a broad spectrum of disease.Pathogenic E.coli can be divided into two classes:intestinal and extraintestinal pathogenic E.coli(ExPEC)strains.ExPEC strains possess certain specific virulence traits that enable them to invade and colonize extraintestinal sites.Avian pathogenic E.coli(APEC),a subgroup of extraintestinal pathogenic E.coli(ExPEC),causes colibacillosis in birds resulting in economic loss because of treatment,condemnation of products,and death.The outer membrane(OM)of E.coli is an essential organelle.The OM allows E.coli to interact with its environment and has a critical function as a barrier to prevent the entry of toxic molecules into the cell.The OM is composed of phospholipids,lipoproteins,outer membrane ?-barrel proteins(OMPs)and lipopolysaccharide(LPS).This thesis studies four particular bacterial outer membrane proteins called OmpT,OmpF,OmpC and NmpC,focusing on their expression,purification,pathogencity and immunogenicity.1 Role of outer membrane protein T in pathogenicity of APECIn this study,An ompT gene was deleted from APEC mutant strain;(TW-XM)was constructed and characterized.The inactivation of ompT reduced significantly the adherence and invasion capabilities of APEC to mouse brain microvascular endothelial cell(BMEC)bEnd.3 cells at the rates of 43.8%and 28.8%respectively,compared with the wild strain TW-XM.Further studies showed that deletion of ompT gene reduced the bacterial virulence with 15.2-fold in duckl:ings and 9.7-fold in mouse models based on the measurement of the LD5o.Furthermore,experimental infection of animals revealed that,loss of ompT showed reduced APEC colonization and invasion capacity in brains,lungs and blood by 2-fold,1.96-fold,and 1.7-fold,respectively,compared with the wild-type strain TW-XM.These virulence-related phenotypes were partially recoverable by genetic complementation.The results of the quantitative real-time reverse transcription-PCR(qRT-PCR)indicated that the loss of ompT significantly decreased the expression levels of ompA,fimC and tsh in the mutant strain ?OmpT,when compared with TW-XM(p<0.01).2 Functional role of ompF porin in pathogenesis of APECHerein,we evaluated the potential of onpF outer membrane porin in the pathogenesis of avian pathogenic E.coli(APEC)strain TW-XM.The ompF was deleted to generate isogenic of mutant.The results showed that ?ompF reduced significantly the adherence by 41.3%and invasion capabilities of APEC to mouse brain micro vascular endothelial cell(BMEC)bEnd.3 cells in vitro by 51.9%compared with the wild strain TW-XM.In vivo experiment based on the measurement of the LD50 have also shown that,?ompF reduced the bacterial virulence by 9.8-fold in ducklings and 9-fold in mouse models.Animal infection experiments further revealed that,loss of ompF reduced TW-XM colonization and invasion capacity in brains,lungs and blood compared to wild-type strain TW-XM(P<0.01).These virulence-related phenotypes were partially recoverable by genetic complementation.The results of the quantitative real-time reverse transcription-PCR indicated that,the loss of ompF significantly decreased the expression levels of ompA,fimC genes in the mutant strains,compared to wild-type strain.3 Inactivation of OmpC contributes to decreased pathogenicity and biofilm formation in APECIn this study,we evaluated the potential of ompC outer membrane porin in the pathogenesis of avian pathogenic E.coli(APEC)strain TW-XM.The ompC was deleted to generate mutant(?ompC).We found that,?ompC reduced significantly the adherence by 46.1%and invasion capabilities of APEC to mouse brain microvascular endothelial cell(BMEC)bEnd.3 cells in vitro by 49.7%compared with the wild strain TW-XM.In vivo experiment based on the measurement of the LD50have also shown that,?ompC reduced the bacterial virulence by 12.3-fold in ducklings and 10.2-fold in mouse models.Animal infection experiments further revealed that,loss of ompC reduced colonization and invasion capacity in brains,lungs,blood and spleen compared to wild-type strain TW-XM(P<0.01).These virulence-related phenotypes were partially recoverable by genetic complementation.The biofilm formation of the onpC mutant was found to be attenuated compared to that of the wild type strain,whereas it was restored when the ompC gene was complemented.The results of the quantitative real-time reverse transcription-PCR(qRT-PCR)indicated that,the loss of ompC significantly decreased the expression levels off fimC and iBeA genesin the mutant strains,compared to wild-type strain TW-XM(P<0.01)4 Evaluation of nmpC gene in determining pathogenicity and Biofilm Formation in APECThis study,aimed to elucidate the potential of nmpC in pathogenesis and biofilm formation in APEC TW-XM.NmpC gene deleted from APEC mutant strain,?NNmpC,was constructed and characterized.We found that lack of nmpC significantly reduced the adherence and invasion abilities of APEC to bEnd.3 cells at the rates of 47%and 52%respectively,compared with the wild strain TW-XM.Further,we found that deletion of nmpC gene decreased pathogenicity in duck by 20-fold and 17-fold in the mouse based on the measurement of the LD50.Also reduced biofilm formation compared to the wild-type strain.Furthermore,inactivation of nmpC showed reduced colonization and invasion capacity in brains and lungs by 1.6-fold and 1.2-fold,respectively,compared with the wild-type strain TW-XM.Additionally,quantitative real-time reverse transcription-PCR indicated that loss of nmpC significantly decreased the expression levels of tsh in the mutant strain ?NmpC,when compared with parent.5 Recombinant proteins vaccine against infection of APECIn this study,three surface outer membrane porins,OmpT,OmpF and OmpC of APEC were cloned and expressed to investigate their immunogenicity and in vivo protection against APEC challenge in the mouse.Inmunization of mice via intraperitoneal injection with the purified three recombinant proteins and their combination stimulated strong immunoglobulin(IgG)antibody responses.After immunized with OmpT,OmpF,OmpC and their combination,challenged with 2.5 × 107 CFU(5 × LD50)of the highly virulent APEC strain TW-XM,the protection rate was 60%,40%,50%and 70%respectively.In addition,serum was tested for their inhibition ability in vitro against APEC isolates and we found that,a significant reduction(P<0.01)in adhesion of TW-XM.Further,we found that decrease significantly of bacterial loads in brain,blood and spleen in vaccinated compared to non-vaccinated mouse.These findings indicate that OmpT,OmpF and OmpC proteins are a promising vaccine target against APEC infections in poultry.In conclusion,the results of all our experiments demonstrated that inactivation of OmpT,OmpF,OmpC and NmpC decreased adhesion,invasion,colonization,proliferation capacities,which may suggested the involvement of APEC pathogenesis.Also our results indicated that OmpT,OmpF and OmpC are promising vaccine target against APEC infections in ducks.