Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Louisa B. Tabatabai

Second Advisor

Amy Andreotti

Abstract

Haemophilus parasuis is the causative agent of Glässer's disease, a respiratory illness in swine, which causes significant economic loss in the industry. To date, there is no commercially available cross-protective vaccine or diagnostic for this disease. Because of this, identification and characterization of putative virulence factors is crucial. In order to understand the mechanism of infection of H. parasuis, it is imperative to not only identify proteins traditionally associated with virulence but to also recognize differences that exist for those proteins at a molecular and/or expression level. Using a combination of bottom-up and isobaric-tagging for relative and absolute quantification (iTRAQ) proteomic strategies, N-terminal sequencing, molecular modeling and antigenic characterization techniques, putative virulence factors have been identified and characterized. In attempt to identify the P2 and P5 colonization factors of H. parasuis, tandem mass spectrometry was performed following 2-D SDS-PAGE immunoblots with a monoclonal antibody to OMP P5 of H. influenzae. While OMP P2 was identified using this method, OMP P5 was not. Additional proteins identified include: TRAP solute transporter, glucose-specific PTS system enzyme IIA, acetyl-CoA carboxylase biotin carboxylase subunit, purine nucleoside phosphorylase, chelated iron ABC transporter, probable sugar isomerase, Mn-dependent superoxide dismutase, putative iron ABC transporter, D-ribose transporter, alanine aminotransferase, phosphoribosylglycinamide formyltransferase, FbpA, and hypothetical proteins HPS 06844 and HPS 10240. Sequence and structural alignment of identified proteins with H. influenzae OMP P5 were used in order to elucidate common epitopes for antibody 4BF8 with no success. Molecular modeling and antigenic prediction analysis indicates that regardless of virulence, the OMP P5 protein comprises four surface exposed loops and that these loops have comparable antigenic propensity, with the presence of a linear B-cell epitope at the apex of both loops 3 and 4. Based on preliminary ELISA results using synthetic peptides and sera from animals infected with H. parasuis, a subunit vaccine strategy derived from the outer membrane loop 3 and loop 4 peptides of P5 was tested in an animal model, demonstrating that while the loop 3 peptide provided protection against heterologous challenge, the loop 4 peptide provided no protection. Molecular modeling analysis of OMP P2 indicates two forms of the protein that differ by approximately 3-5kDa. OMP P2 comprises eight surface exposed loops and the increase in molecular weight observed for OMP P2 in avirulent strains corresponds to extensions in the loop 3 and loop 5 regions. For OMP P2 of virulent serovars, loop 1, loop 4 and loop 8 have the highest antigenic propensity scores, with predicted continuous B-cell epitopes present on loops 1, 3, 4, 5, 6 and 8. In addition to the P2 and P5 colonization factors, non-traditional proteins may serve as colonization factors as epitopes of surface localized proteins may facilitate attachment. Following anti-P5 (4BF8) immunoblot and N-terminal sequencing, a 36kDa outer membrane protein of was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Studies in other bacterial pathogens indicated that surface localized GAPDH is involved with colonization and it is hypothesized that this protein may play the same role in H. parasuis. Given that few proteomic profiling studies have been performed on H. parasuis, the iTRAQ method was utilized to not only identify proteins, but to also quantify differential protein expression and evaluate how this differential expression may play a role in virulence of the organism. Results of the OMP P2 protein expression indicated a 1.5 to 2-fold increase in the presence of 7.5% CO2 when compared to protein expression in an aerobic environment. Additional differentially expressed proteins for the overall analysis include: MglB, hypothetical protein HPS_06844, Fe3+ ABC transporter, TRAP solute transporter, D-ribose transporter subunit RbsB, FbpA, ABC transporters, Cu-Zn SOD, iron binding proteins, chelated iron ABC transporter, hypothetical protein HPS_10240, acyl carrier protein, glucose-specific PTS enzyme IIA, heme-binding protein A, phosphoenolpyruvate-protein phosphotransferase, alanine aminotransferase, hypothetical protein HPS_07840, plp0007 from Legionella pneumophila, hypothetical protein GCWU000324_02210 from Kingella oralis, transaldolase B, UDP-sugar diphosphatase from M. haemolytica, tRNA pseudouridine synthetase C, gamma-glutamyl phosphate reductase and nitrate reductase. Overall, the research presented here not only provides one of the few proteomic profiles of H. parasuis, it presents a general methodology for the design of a subunit vaccine and corresponding diagnostic for extracellular pathogens.

Copyright Owner

Mandy Kay Zimmerli

Language

en

File Format

application/pdf

File Size

426 pages

Included in

Biochemistry Commons

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