The Screening And Identification Of Extracelluar Proteins Between Two Haemophilus Parasuis Strains Nagasaki And SW114

Haemophilus parasuis, a pleomorphic, gram-negative, nicotinamide adenine dinucleotide (NAD)-dependent rod in the family Pasteurellaceae, is a commensal organism of the upper respiratory tract of swines. With the recent changes in production methods, diseases caused by H. parasuis have received increasing attention worldwide because of the serious economic losses that these diseases cause in the pig industry. To date, a number of possible virulence-related factors have been identified in H. parasuis. However, the research on extracellular protein of H.parasuis of different virulent strains is limited. In this study, a comparative proteomics approach was used to distinguish between the 2D profiles of extracellular proteins in two H. parasuis strains, namely, SW114 and Nagasaki, in order to identify differently expressed extracelluar proteins in the two strains. Quantitative PCR was used to analyze the mRNA transcript levels of three randomly selected proteins of the two strains.1 Comparative analysis of the growth characteristics and virulence between the two H.parasuis strainsTurbidimetric and viable count methods were used to detect the growth curve of the two strains.The results showed the growth rate of SW114 was greater than Nagasaki obviously in logarithmic phase.The stable phase sustained nine hours in SW114 longer than six hours in Nagasaki/while the two strains were in the logarithmic phase when the OD600=0.6. Improved Karber method was used to detect the median lethal dose of the strains against female BALB/c mice,6-8-week-old and 18-22g.As results,The median lethal dose of the SW114 was 2.95×1012cfu, while the Nagasaki was 3.16×109cfu, so the virulence of Nagasaki was nearly 1000 times of SW114.2 Comparative proteome analysis of the extracellular proteins of Nagasaki and SW114The extracellular protein was extracted through Trichloroacetic acid(TCA).A comparative proteomics approach was used to distinguish between the two-dimensional electrophoresis profiles of extracellular proteins in Nagasaki and SW114. Protein spots were identified using matrix-assisted laser desorption/ionization tandem time-of-fiight mass spectrometry.The Nagasaki strain gel contained 32 protein spots, whereas (?)umber of protein spots in the SW114 strain gel was 25.22 spots exsisted in both Nagasaki and SW114. 10 spots were umique to Nagasaki.while another 3 spots were unique to SW114 A11 the 13 differentially expressed proteins were successfully identified, corresponding to 12 individual proteins. The ten proteins unique to Nagasaki were putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa). putative extracellular serine protease(autotransporter) (780aa). protective surface antigen D15,30S ribosomal protein S2. periplasmic serine protease do/hhoA-like protein, acid phosphatase. membrane protein, protein-disulfide isomerase, and iron ABC transporter substrate-binding protein. Meanwhile, the two proteins unique to SW114 were C4-dicarboxylate ABC transporter substrate-binding protein and peptide ABC transporter substrate-binding protein. Three virulence-related proteins,namely, putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa) and putative extracellular serine protease (autotransporter) (780aa) were identified in Nagasaki. C4-dicarboxylate ABC transporter substrate-binding protein and peptide ABC transporter substrate-binding protein were metabolic-related were found in SW114.3 Analyze the mRNA transcript levels of selected proteins of H.parasuisQuantitative PCR was used to analyze the mRNA transcript levels of three randomly-selected proteins. The afuA, AidA, and ompD15 genes encoding iron ABC transporter substrate-binding protein, putative adhesin AidA protein and protective surface antigen D15 respectively demonstrated significantly higher mRNA transcript levels (39.606.3.924. and 36.668. respectively) in Nagasaki than in SW114. These observations suggest the levels of differentially expressed proteins were directly proportional to their cellular mRNA levels. Putative adhesin AidA protein, putative extracellular serine protease (autotransporter) (771aa) and putative extracellular serine protease (autotransporter) (780aa) were virulence-related and C4-dicarboxylate ABC transporter substrate-binding protein and peptide ABC transporter substrate-binding protein were metabolic-related. These findings may-help understand the phenomenon that the growth rate of SW114 was greater than Nagasaki obviously in logarithmic phase and the virulence of Nagasaki was nearly 1000 times of SW114. Simultaneously, these results may help to understand the pathogenic mechanisms of H.parasuis and lay the foundation for prevention research.