Nontypable Haemophilus influenzae (NTHi) is a Gram-negative bacterium which causes otitis media (ear infections), pneumonia, bronchitis, and sinusitis. For many years, the P6 protein has been a leading candidate for a protein vaccine against NTHi, because it is conserved among NTHi strains, is believed to be surface exposed, and contains binding sites for specific antibodies. However, recent structural analysis suggests that P6 may be inserted into the outer membrane in two orientations. Based on the interaction with its homologues in Escherichia coli (E. coli), we proposed that P5, a transmembrane protein in NTHi, plays a role in P6's apparent dual membrane orientation by either helping it to flip to the surface or by deceptively binding to P6 antibodies. Although the structure of the transmembrane region of P5 has been solved, the structure of the C-terminal end was not known until very recently. We attempted to use nuclear magnetic resonance (NMR) spectroscopy to solve the structure of the C-terminal end of NTHi P5, but the protein piece we engineered was most likely denatured or aggregated in solution. Also, flow cytometry experiments on the knockout of a P5 homologue, OmpA, in E. coli showed improved surface antibody binding to the P6 homologue, Pal: a contradictory result to our proposed hypotheses. Therefore, we discontinued our work with P5. A second goal of this work was to determine the epitope for the P6 antibody 4G4 through site directed mutagenesis, antibody binding assays, and NMR spectroscopy. Experimental results using the P6 T42E mutant showed no difference in antibody binding to 4G4 antibodies, suggesting that either the binding is not dependent on that single amino acid or that a different mutation is required to alter antibody binding.

Library of Congress Subject Headings

Bacterial vaccines--Design; Proteins--Structure

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Department, Program, or Center

School of Chemistry and Materials Science (COS)


Michael, Lea


Note: imported from RIT’s Digital Media Library running on DSpace to RIT Scholar Works in December 2013. Physical copy available from RIT's Wallace Library at QR189.5.B33 M36 2012


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