Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Kimberly Jefferson

Abstract

Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is of major clinical importance due to its ability to significantly affect pregnancy outcome and enhance the transmission and acquisition of HIV. BV is characterized by a dramatic shift in the vaginal microflora; in most BV cases, the predominant bacterial species is Gardnerella vaginalis. It has been demonstrated that G. vaginalis forms an adherent biofilm on the vaginal epithelium of women with BV. Furthemore, evidence suggests that the high rate of recurrence associated with BV is related to incomplete eradication of the biofilm. The overall goal of this study was to characterize G. vaginalis virulence properties, including biofilm formation, in order to better understand the pathogenesis of BV and to improve available treatment methods. In an effort to tease apart the uncertain etiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. Only G. vaginalis demonstrated all three virulence properties, including robust biofilm formation. It has been shown that the biofilm phenotype allows its constituent bacteria to be resistant to many negative environmental stimuli. Therefore, we studied the susceptibilities of biofilm vs. planktonic cultures to H2O2 and lactic acid. Biofilms tolerated higher concentrations of both chemicals; however, when the biofilm was proteolytically disrupted, sensitivity to the chemicals returned to planktonic levels. Since our data suggested a critical role for a protein in biofilm formation, a partial genome sequence of G. vaginalis was searched for sequence homology to known biofilm adhesins using the tBLASTn program. This revealed an open-reading frame encoding a hypothetical protein with significant homology to the staphylococcal Bap protein. Antibody towards a portion of the identified gene product was produced in rabbits by inoculation of a recombinant peptide to an antigenic region of the protein. Antibody inhibition assays against biofilm formation, adherence, initial adherence and aggregation were conducted. Relative expression levels of the biofilm-associated protein were analyzed under different conditions by western blot analysis. Finally, the protein was expressed in heterologous hosts and analyzed for an increase in biofilm formation.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2010

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