Function Of Alkaline Protease In Pseudomonas Aeruginosa

Background:Pseudomonas aeruginosa(P.aeruginosa)is a kind of gram negative bacteria,which is an opportunistic pathogen andcauses various kinds of infectious diseases,including burn wounds,middle ear,cornea,urinary tract and respiratory tract,especially in patients with lung cystic fibrosis.P.aeruginosa produce a large number of virulence factors,such as elastase,lipase,phospholipase,exotoxin A,iron acquisition protein,alkaline protease and rhamnolipid,which play the important role in the preventive and treatment of P.aeruginosa's infection.AprA is a zinc finger protease which contains 479 amino acids.The molecular weight of AprA is50,433 Da.It has been reported that AprA is invoeld in immune envasion by cleavaging complement molecules such as C1 q,C2 and cytokines such as IFN-g and TNF-?.However,the function of AprA in the pathogenecity of P.aeruginosais far from clear.P.aeruginosa also expresses AprA's inhibitory factor(Apr I),which is secreted in the periplasmic space.Apr I contains 131 amino acids,the molecular weight of Apr I is14,410 Da.B subtype of Flagellin is a substrate of AprA,whichcontains 488 amino acids.The molecular weight of B subtype of Flagellin is 49,242 Da.NETs(neutrophil extracellular traps)is an extracellular structure released by neutrophils.NETs is mainly composed of DNA,Histone,Neutrophil elastase(Neutrophil Elastase NE),Myeloperoxidase(MPOS),and Cathepsin G.NETs is found in many vertebrates including fish,poultry,mouse and human.It has been reported that NETs is important for clearance of pathogen including G+and G-bacteria,fungi and parasites.Phage display technology refers to infusion protein of Fab or sc Fv and gene IIIon the surface of filamentous phage,a bacterial viruses that can infect and replicate within Escherichia coli,and DNA encoding the heavy chain(VH)and light chain(VL)are batchcloned into viral genome to creat fusion protein with phage coat proteins p Ill or p VIII.After several rounds of screening,the specific antibody can be selected and enriched.The advantage of phage display is that it can couple phenotype and genotype to selecte antibodies with high specificity.Complement system is an innate immune defense system consistingof a number of proteins,which includes classical complement pathway,alternative complement pathway and lectin pathway.Complement system is essential for clearance of pathogens and other immunoregulatory effect.Purpose of research:We have investigated the function of AprA in the pathogenecity of P.aeruginos.The recombinant AprA,Apr I and Flagellin protein were expressed and purified.The potential role of AprA was investigated.Research Methods:(1): Expression,purification and preliminary function study of AprA,Apr I and Flagellin from P.aeruginos.Expression of AprA,Apr I and Flagellin protein: The genes encoding AprA,Apr I and Flagellin were constructed into expression vectors respectively,and AprA protein was obtained by denaturing and renaturation.Apr I and Flagellin proteins were purified by immobilized metal-ion affinity chromatography(IMAC)according to the manufature.(2): Preparation and preliminary function study of specific antibody against AprA.The complement-mediated erythrocyte cleavage assay(CH50)showed that AprA significantly inhibited the activation of classical complement pathway in a dose-dependentmanner.In order to study the biological function of AprA,we prepared the polyclonal antibody against AprA(anti-AprA)by immunizing white rabbits with recombinant AprA protein.Simultaneously,a human monoclonal antibody YG4 was selected by three rounds with phage antibody library and recombiant expressed by mammalian expression system.anti-AprA and YG4 were found to bind AprA with high specificity by an enzyme-linked immunosorbent assay(ELISA),and the EC50 value of YG4 binding to AprA was 74.5ng/ml.(3): Inhibition of NETs formation by AprA degrading the Cit-H3The formation of NETs was induced via stimulation of human neutrophils with PMA in vitro.It was found that structure of NETs was disrupted with addition of AprA.Our further investigation showed that AprA could specifically degrade Cit-H3,a key component of NETs.Conclusions:?.Our further investigatons showed that AprA was capable of cleaving Flagellin in a time-dependent manner,which was blocked by Apr I.?.we found that anti-AprA and YG4 were able to enhance the inhibitory effect of AprA on complement pathway activation respectively,suggesting that the antibodies simulated by AprA had the antibody-dependent enhanced effects.?.AprA could mediate the immune escape of P.aeruginosa by inhibiting the formation of NETs via degrading of Cit-H3.