Identification And Fuctional Characterization Of The Infection-and Stress-Associated Genes In Haemophilus Parasuis

Haemophilus parasuis is a common commensal bacterium of the upper respiratory tract of conventional pigs but,under appropriate conditions,can invade and cause severe systemic disease(Gl(a丨¨)sser's disease),characterized by fibrinous polyserositis,polyarthritis, meningitis and pneumonia.In the past decade,pig farms with high sanitary standards have suffered a significant increase in the incidence,morbidity and mortality associated with Gl(a丨¨)sser's disease,resulting in great economic losses worldwide.During the process of infection,bacteria have to adapt to all the changes of temperature,PH,osmotic pressure,oxygen,nutrition materias in vivo by the responses to the stress.So,broad definition of virulence factors include all factors related to in vivo and in vitro associated stress.Till now,the paucity of molecular biological studies on H.parasuis leads to a major obstacle to better understanding of H.parasuis virulence.Identification and functional characterization of the infection- and stress-associated genes will contribute to the understanding the mechanisms of by which H.parasuis survives in the host and the.study of vaccine and the target of medicine.In this study,selective capture of transcribed sequences(SCOTS) was used to identify H.parasuis genes up-regulated in necrotic porcine lung seven days post infection,under iron-limitation and heat stress. Immunogenicity of candidate pathogen protein was screened by animal assay.Yeast two hybrids methods were used to screen the cDNA library of porcine brain with protein intrested as the Bait protein.The results of this study are shown as follows:1.Identification and functional characterization of the infection- and stress-associated genes in H.parasuisH.parasuis genes preferentially expressed in the necrostic lungs of infected pigs. In this study,selective capture of transcribed sequences(SCOTS) was used to identify H. parasuis genes up-regulated in necrotic porcine lung seven days post infection.Thirty eight genes were identified that were up-regulated during infection of the lung tissue of pigs,compared to growth in culture medium.In two examples chosen gene expression was not confined to the lungs,there being variation between tissues.The data support biofilm formation being an important mode of growth for colonization and/or persistence. Results from the in vitro studies suggest that,as for other pathogens,iron- and oxygen restriction and heat stress are important environmental signals to regulate gene expression.H.parasuis genes in response to iron-limitation.Using SCOTS approach,a total of 36 up-regulated genes were identified under iron limitation compared to normal culture condition.The 36 genes could be divided into six functional groups:cell surface protein, DNA metabolism,energy metabolism,sugar metabolism,amino acid and protein synthesis,and transport and binding proteins.To validate the results of SCOTS,six genes were selected for transcript level analysis using qRT-PCR:recN and hrpA implicated in DNA replication and repair,ccmA and acrB related to respiration,and ptnC and gmhA involved in sugar metabolism.The resulted showed that all the 6 genes were up-regulated more than 33-fold under the iron-limitation compared with normal cultural condition.H.parasuis genes in response to heat stress.To investigate the transcriptional responses of H.parasuis to heat striess,the highly virulent strain 0165 of serotype 5 was grown 40℃.Using SCOTS approach,a total of 39 up-regulated genes were identified under iron limitation compared to normal culture condition.The 39 genes could be divided into six functional groups:DNA metabolism,energy metabolism,sugar metabolism,amino acid and protein synthesis,transport,regulator and binding proteins and unknown function.2.Immunogenicity of the infection-and stress-associated genes in H.parasuisBy analysising the function and signal peptide using bioinformatics,membrane location by FACS,interaction with St.Jude porcine lung cells(SJPLC) with indirect immunofluorescence by turns,five infection and stress- associated proteins conserved in other serotypes of H.parasuis,were chosen as the candidate of antigen protein.The results showed that the expressed protein PilA could provide protective efficacy.The expressed proteins could protect pigs from necrotic organs turning cellulose,which may be used to develop new effective vaccines of H.parasuis.3.Interactions between H.parasuis virulence factors and porcine brainYeast two-hybridization technology was used to study the interactions of three virulence factors,PilA,ArcB and SiaB,with porcine brain.Ubiquitin-conjugating enzyme E2A,adaptor-related protein complex 3(AP-3) and complement C1qA were identified to be interact with PilA,ArcB and SiaB respectively.These interactions were confirmed by pull-down assays.Further investigation of these interactions will contribute to understanding of pathogenesis mechanism of H.parasuis.