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Title: IDENTIFICATION OF IRON-REGULATED PROTEINS OF HAEMOPHILUS PARASUIS BY COMPARATIVE 2-D ELECTROPHORESIS, WESTERN BLOTTING, AND MALDI-TOF

Author
item Zehr, Emilie
item Tabatabai, Louisa

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 5/18/2003
Publication Date: 5/18/2003
Citation: ZEHR, E.S., TABATABAI, L.B. IDENTIFICATION OF IRON-REGULATED PROTEINS OF HAEMOPHILUS PARASUIS BY COMPARATIVE 2-D ELECTROPHORESIS, WESTERN BLOTTING, AND MALDI-TOF. AMERICAN SOCIETY FOR MICROBIOLOGY. 2003. Abstract #Z-024.

Interpretive Summary:

Technical Abstract: Background: Haemophilus parasuis (HP) is the causative agent of Glässer's disease and is considered a remerging pathogen of swine in high-health status herds. Historically, classification of HP serotypes has been through pathogenicity studies and the standard culture/biochemical, agar-gel diffusion, and the rapid identification system (RapIDNH) methods. Information on the capsular polysaccharide, outer membrane proteins, fimbriae, toxins and DNA of HP is not available. To date, only a 16S rRNA diagnostic study of HP serotypes has been done. In a previous study, we showed differences among nonvirulent, virulent, and highly virulent strains by random amplification of polymorphic DNA (RAPD) analysis and protein profiles of known HP serotypes and selected field isolates. In this study, we have identified some of the virulence factors associated with HP. Methods: Cell lysates from HP grown in iron-restricted (Frey's broth containing 2,2' dipyridyl) and in iron-replete broth were treated with DNase and RNase, then precipitated with trichloroacetic acid. Dissolved precipitates were subjected to 2-D electrophoresis, and compared for up-regulation or down-regulation of proteins. Nonimmunoreactive and immunoreactive proteins were detected by Western blotting using convalescent porcine antiserum as a probe. Selected proteins were picked from gels, digested with trypsin, and analyzed by MALDI-TOF for identification using Prospector's MS-Fit software. Results: Putative homologues have been identified. Iron up-regulated and immunoreactive proteins include adhesion and penetration protein, OMP P2, OMP P1, and hemoglobin binding protein HgbA. Iron down-regulated and immunoreactive proteins include EF-Tu, Fur, urease accessory protein, and tail-specific protease. Nonimmunoreactive proteins include heme-hemopexin utilization proteins A (down-regulated) and B (up-regulated). Conclusion: We have combined 2-D electrophoresis, Western blotting, and mass spectrometry to identify nonimmunoreactive and immunoreactive proteins of HP. The proteins will be further analyzed to study their role in HP infection.