Date of Award

Fall 1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Robert E. Ratzlaff

Committee Member

Stephen Buescher

Committee Member

Daniel E. Sonenshine

Committee Member

Christopher Osgood

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, expresses a species specific, 39 kiloDalton protein of unknown function called P39. This protein is highly immunogenic and the presence of anti-P39 antibodies in patient sera is used as an indicator of B. burgdorferi infection. P39 is also a candidate for vaccine developement due to conserved expression of P39 among B. burgdorferi isolates. Since little is presently known about this important molecule, the present study was designed to characterize P39 and was carried out in six separate investigations. First, infectious and noninfectious isolates of B. burgdorferi were established in vitro, and the infectivity of each was assessed in a murine model. Second, the location of P39 within the spirochete was determined in infectious and noninfectious isolates by cell fractionation, and western blot analysis. A correlation was established between P39 localization and infectivity; infectious and noninfectious isolates localized P39 to both the outer envelope and inner membrane, but noninfectious isolates had increased levels of P39 in the inner membrane and cytosol as compared to infectious isolates. Third, flow cytometry was used to determine if P39 expression is uniform throughout a population of spirochetes. Within a cloned population, organisms expressed similar levels of P39 on the outer surface. In contrast, uncloned populations displayed variable surface P39 expression, and uncloned noninfectious B31, WCH1, and JD1 all expressed lower levels of P39 as compared to the respective infectious organisms. Fourth, detergent extraction studies were employed to determine the solubility of the P39 molecule. P39 was shown to be hydrophobic in nature and the solubility of the molecule was not altered in infectious or noninfectious isolates nor was it altered in different cell fractions. Fifth, P39 was immunoprecipitated from infectious and noninfectious isolates in an attempt to characterize molecules which associate with P39. P39 coprecipitated with four molecules in all isolates, and six additional molecules common only to noninfectious isolates. Finally, in an attempt to determine if P39 elicits immunopathological responses in an infected host, site directed DNA mutagenesis was used to inactivate the gene encoding P39. No viable P39 deletion mutants were obtained, thereby precluding any studies regarding immunopathology caused by P39. The results of this study demonstrate P39 is a hydrophobic, membrane associated protein, and is expressed by both infectious and noninfectious isolates. Furthermore, infectious and noninfectious isolates exhibited differences in P39 localization, and coprecipitation.

Comments

A Dissertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/gr4x-nc18

ISBN

9780591262254

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