Cloning And Expression Of S Gene From R.anatipestifer And Effect Of EcpS On Host Complement System

Riemerella anatipestifer(RA)is not only the pathogen of infectious serositis of duck,but also a great threat to waterfowls,such as ducks and geese.Nowadays,RA is almost founded in every duck farm and result in high morbidity and mortality in ducklings.It's difficult to control and prevent the disease due to its 21 serotypes,poor cross-protection and bacterial resistance to antimicrobial antigens;however,little is known regarding the molecular basis of its pathogenesis and the specific virulence factors involved.There are just a few virulence factors that have been determined,while most of virulence factors that have been reported still need more proof.The complement system is an important component of the innate immune system,which is a proteolytic cascade of plasma proteins.Following infection,pathogens can stimulate the activation of the complement system,leading to the formation of the membrane attack complex(MAC)to directly lyse invading bacteria.Moreover,C3 b can deposit on microbial surfaces and may lead to opsonisation by binding the C3 b receptors(CR)on cell membranes of macrophages.Bacterial pathogens exploit several strategies to evade detection by the complement system in order to increase the efficiency by which these pathogens can gain access and colonise the inner tissues where it may cause severe infections,such as inhibition of complement activity by secreting proteinases.Obviously,evasion of the complement system is critical for pathogens to cause invasive disease.To our knowledge,the secretion of proteases as an immune evasion strategy in RA remains poorly understood.In this study,we plan to construct four recombinant expression vectors of extracellular protease S(Ecp S)which was purified in our preliminary experiment,and aimed to determine how Ecp S interferes with complement activation and whether it could block complement-dependent neutrophil function.To further study the mechanism of Ecp S on the complement system,we dissected the individual complement pathways using pathway-specific complement ELISAs(psc-ELISA).Eventually,we also analyzed C3 b and C4 b deposition in order to study the level at which Ecp S affected these pathways and preliminarily screen the key proteins interacting with Ecp S in the complement system.In summary,we can use the study to further explore the characteristics of Ecp S and clarify the pathogenic mechanism of RA.The main results of this study are shown as follows:Four pairs of primers were designed according to the whole sequence of s gene of RA-AF strain.The four forward primers of the total introduced Bam H I sites,while the four reverse primers introduced Hind III sites.And there was no any possibility of overlap among amplified fragments,which length were 1062 bp for S1,1113 bp for S2,910 bp for S3 and 1021 bp for S4.The genomic DNA extracted from RA-AF strain(serotype 2)was used as template,and the target fragments amplified by polymerase chain reaction(PCR).Thereafter,the target fragments were cloned into the p MD19-T vector to generate the recombinant plasmids p MD-S,including p MD-S1,p MD-S2,p MD-S3 and p MD-S4,followed by transformation in E.coli DH5?competent cells.Subsequently,the positive clones selected by blue-white selection were verified by sequencing.Then,the target fragments was subcloned into the Bam H I and Hind III cloning sites of the E.coli expression vector p ET32 b to contruct the expression plasmid p ET32b-S,namely p ET32b-S1,p ET32b-S2,p ET32b-S3 and p ET32b-S4,which were transformed into E.coli BL21(DE3)competent cells.The transformants were screened by ampicillin and the inserted fragments were confirmed by PCR.Moreover,the target proteins were induced by auto-induction and detected by substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which showed that the recombinant proteins S1,S2,S3 and S4 were intracellular soluble and none of them could liquefy gelatin,all of which were used to prepare four oil emulsion vaccines by which we prepared antiserum to S protein in four healthy rabbits.Besides it,the Western-blot showed that all of groups were positive,which contains the groups of supernatants of RA-AF strain,incubated by previously four antisera.Moreover,these antisera could completely inhibit the activity of Ecp S in the gelatin liquefaction test.20% NDS was preincubated with different concentration of Ecp S at a moderate condition,and then RA was added and incubated under the same condition;colonies are then counted.In the phagocytosis killing assay,after preincubation of 5% NDS with different concentrations of Ecp S,both RA and duck neutrophils were added and incubated,and colonies are counted.The results showed that Ecp S potently blocked RA phagocytosis and killing by duck neutrophils(P<0.001).Subsequently,the haemolytic assays were performed using a modified version of the methods,and it could be found that Ecp S didn't block AP-mediated haemolysis;however,Ecp S did inhibit CP-mediated haemolysis(P<0.05).Additionally,the inhibition of Ecp S on duck complement system could be neutralized with rabbit anti-S1/S2 serum previously prepared.NDS was preincubated with binding buffer or different concentrations of Ecp S and then incubated with RA.C3 b deposited on the RA membrane was eluted by 3M KSCN after washing five times and was quantified using a duck C3 d ELISA kit.The protocol of C4 b deposition onto RA was similar to the C3 b deposition as described previously,except C4 b was detected using a duck C4 d ELISA kit after elution.According to the results,Ecp S partially inhibited the deposition of C3 b on RA at comparable concentrations as those observed for killing and phagocytosis of RA(p<0.05).Besides it,Ecp S also strongly inhibited C4 b deposition(p<0.001).We dissected the individual complement pathways using pathway-specific complement ELISAs.And until now,three different pathways of complement activation have been described: the classical pathway(CP),the alternative pathway(AP)and the lectin pathway(LP),which were specifically induced in the presence of serum using Ig M,LPS and mannan coated plates,respectively.The level of C3 b deposition was detected with specific Abs.And Ecp S specifically blocked C3 b deposition via CP and LP(p<0.001),whereas AP was not affected(p>0.05).Here we found Ecp S could potently inhibit the biological functions of host complement system.Furthermore,Ecp S inhibited the opsonisation of bacteria by C3 b.Ecp S specifically blocked C3 b and C4 b deposition via the classical and lectin pathways,whereas the alternative pathway was not affected.These results indicateed that secreting Ecp S was one of the mechanisms which RA had evolved to evade the host immune system.