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Dissertation
Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Jun 1999
DOI:
https://doi.org/10.17918/00009502
Abstract
Neisseria gonorrhoeae Opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. Although gonococci possess alternative surface-exposed molecules involved in adhesion and invasion, the expression of Opa proteins appears to be necessary for gonococcal disease. Since little is known about the interaction of gonococci with host cell "constituents" that mediate post-adhesion and post-invasion processes, including intracellular replication and survival, we wanted to identify novel roles for Opa in the infectious process. Specifically, we wanted to use the yeast two-hybrid system as a relatively rapid screen to identify human epithelial cell proteins that specifically interact with gonococcal Opa proteins. Prior to the initiation of these studies, the yeast two-hybrid system had not been employed to screen a human cell cDNA library for the binding partners of a prokaryotic outer membrane protein. Therefore, a major aim of this thesis was to explore the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from N. gonorrhoeae strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified 5 different OIPs, designated OIP1-OIP5, three of which are homologous to characterized human proteins-thyroid hormone receptor interacting protein (TRIP6), pyruvate kinase isoenzyme M2 (PK), and preferentially expressed antigen of melanoma (PRAME). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro binding assays and in vivo co-localization studies, independent of the yeast two-hybrid system. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, we also demonstrated that intracellular pyruvate is essential for gonococcal growth and survival. These results illustrate a novel mechanism in bacterial pathogenesis, i.e., the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results further demonstrate that the yeast two-hybrid system is a valuable tool for rapidly identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.
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Details
- Title
- Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins
- Creators
- John Michael Williams
- Contributors
- Richard F. Rest (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- xv, 175 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Medicine (1993-1996, 1998-2002); Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888845404721