The Discovery Of The Virulence Factor HpHtrA And Its Proteolytic And Chaperone Activities In Haemophilus Parasuis

Haemophilus parasuis,an opportunistic organism in the upper respiratory tract of swine,is the pathogen of Glasser's disease,resulting in serious economic loss in the pig farms worldwide.But virulence levels vary greatly among serotypes and potential virulence factors and pathogenic mechanisms are still largely unknown.Therefore,the screening and identification of virulence factors in H.parasuis play crucial roles in efforts to prevent and control this disease.Our studies focused on comparison of virulence factors between virulent and avirulent isolates.We employed two-dimensional gel electrophoresis and mass spectrometry to analyze and identify membrane proteins known or thought to be major virulence gene products.Mutations of newly identified putative virulence factors were generated via recombinant techniques,and the biochemical characteristics of the products of these were investigated.Ultimately,our findings allowed determination of function for many of the virulence factors uncovered in this study.A brief summary of our conclusions is presented below.1.The identification of key differences in membrane proteomes between virulent andavirulent strains of H.parasuisTo identify the differences in membrane proteomes of the virulent and avirulent strains,and to discover the important virulence factors of H.parasuis,two-dimensional gel electrophoresis and mass spectrometry were employed to analyze the expression levels of membrane proteins in virulent strain SC-1 and avirulent strain SC 105.The results showed that 189±15 protein spots were detected in the SDS-PAGE gels of SC-1,and 191±16 were detected in those ofSC105.Further analysis showed that 9 protein spots were specific to SC 105,while 10 spots were found to be SC-1 specific.All differentially expressed protein spots were subsequently identified using MALDI-TOF MS and MALDI-TOF MS/MS.Differences in protein expression were further confirmed by qRT-PCR.Based on the experiments and bioinformatic analysis,the periplasm serine proteinase(HpHtrA)was identified to be a differentially expressed protein between the virulent and avirulent strains,suggesting that it is a putative virulence factor of H.parasuis.2.The construction of an HphtrA mutant of H.parasuisHtrA protein has been reported to be an important virulence factor in many pathogens.In our previous research,HpHtrA protein was shown to be overexpressed in the virulent strain,which might be involved in the pathogenesis in the host.To detect the relationship of HpHtrA to virulence,the HphtrA mutant was constructed by using the modified gene deletion method.First,the flanking sequences of HphtrA and kanamycin resistance gene were used to construct the suicide plasmid pMDHK.And then one natural competent strain SC 1401 was detected from eight isolates using the natural transformation method with the suicide plasmid.After that,two suicide plasmids pMDHKS and pMDH were constructed.With the natural competent strain and the two suicide plasmids,the target gene was deleted unmarked through two-step homologous recombination.This research improved the existing natural transformation method,which makes it possible to construct unmarked multiple-gene deletion mutants by two-step natural transformation.In this study,one HphtrA mutant was constructed,which is of great help for the study on virulence correlation and functions of HpHtrA.3.The role of HpHtrA in the virulence in H.parasuisTo detect the virulence correlation of HpHtrA,the wild type and ?htrA strains were both used.When cultured at 37?,the wild type and ?htrA strains showed no difference in growth.When cultured in different conditions,the ?htrA strain was more sensitive to high temperature than the parental strain,but no obvious difference was observed between the two strains to the oxidative and PH stresses.In the autoagglutination experiment,the autoagglutination activity of the ?htrA strain was significantly higher than that of parental strain.In the serum bactericidal experiment,the survival rate of the AhtrA strain was 2.8%,but the survival rate of the parental strain was 75.5%,which showed that the ?htrA strain is more sensitive to the serum bactericidal activity than the parental strain.The AhtrA strain also displayed a higher level of biofilm formation than the parental strain.In the virulence experiment,when injected with the same dose of bacteria(1.4×109CFU/mouse),the mice injected with the parental strain all died,while only half of the mice injected with the?htrA strain died,which showed a decrease in virulence of the ?htrA strain when compared to that of the parental strain.All the results above showed that HpHtrA is required for the virulence of H.parasuis,which is an important virulence factor.4.The proteolytic and chaperone activities of HpHtrA in H.parasuisTo further identify the functions of this protein,rHpHtrA and its mutant proteins were expressed and used to perform the chaperone-like activity and proteolysis assays.Bioinformatic analysis showed that HpHtrA is composed with an N-terminal signal sequence,a chymotrypsin-like proteolytic domain and two C-terminal characteristic PDZ domains.Purified rHpHtrA protein and active HpHtrA in H.parasuis showed to be of different molecular weight.rHpHtrA exhibited both chaperone and protease activities.The protease activity was temperature-dependent and reached its maximum at 40 ?.rHpHtrA also showed self-degradation activity.The chaperone and protease activities of rHpHtrAH113R and rHpHtrAD143V both decreased significantly,but rHpHtrAS219A showed no change when compared to that of rHpHtrA.The results showed that His113 and Asp 143 of HpHtrA are the active sites,but Ser219 is not.HpHtrA was not involved in the secretion of extracellular proteins at 37?.The protein showed chaperone activity to membrane proteins of H.parasuis,and is involved in the degradation of denatured NikE,DppA and OmpA.This research enhances our understanding of the functions of HpHtrA,which may contribute to the discovery of drugs targeting to HpHtrA.