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Dissertation
Regulation of sialyltransferase and lipooligosaccharide expression: effects on serum resistance and interactions of Neisseria gonorrhoeae with human cells
Doctor of Philosophy (Ph.D.), Allegheny University of the Health Sciences
May 1997
DOI:
https://doi.org/10.17918/00007398
Abstract
Neisseria gonorrhoeae (gonococci (GC)) are Gram negative bacteria that cause sexually-transmitted infections and reside extracellularly and intracellularly. Extracellularly, GC are found at mucosal surfaces along with serum components such as complement, antibodies, pyruvate, lactate, glucose, and cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA). It is in this milieu that GC are resistant to normal human serum (NHS); serum resistance is lost when most GC strains are grown in vitro, unless they are co-cultured with CMP-NANA. NANA is transferred primarily to a gonococcal 4.5-kDa lipooligosaccharide (LOS) structure by a GC sialyltransferase (Stase). We investigated LOS and Stase expression and serum resistance in strain F62 grown in different carbon sources, attempting to mimic environmental conditions that GC encounter in vivo. Pyruvate-grown GC, compared with glucose-grown GC: (i) expressed significantly more Stase activity; (ii) expressed significantly more of the sialylateable 4.5-kDa LOS species; (iii) incorporated up to threefold more radiolabelled CMP-NANA onto the 4.5-kDa LOS; and (iv) were significantly more serum resistant at limiting CMP-NANA concentrations. These results suggest that expression of Stase activity and LOS are coregulated by environmental factors that exist in vivo. In different niches, gonococcal LOS sialylation, Stase, serum resistance, and interactions with host cells may be highly regulated. Since GC also reside intracellularly within human epithelial cells and neutrophils, we also questioned whether Stase itself modulates the interactions of GC with these cells. To that end, we isolated 5 mutants deficient in Stase activity (Stase null). Stase null mutants: (i) could not sialylate their LOS when grown with CMP-NANA; (ii) remained serum sensitive when grown with CMP-NANA; (iii) were rescuable to Stase by transformation with chromosomal DNA from WT F62; (iv) adhered to and invaded the human cervical epithelial cell line ME-180 at levels similar to those of WT F62; and (v) stimulated the oxidative burst in, adhered to, and were phagocytically killed by human neutrophils at levels similar to those of WT F62. These results indicate that expression of Stase activity is not required for interaction of GC with human cells.
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Details
- Title
- Regulation of sialyltransferase and lipooligosaccharide expression
- Creators
- David James McGee
- Contributors
- Richard F. Rest (Advisor) - Drexel University, Allegheny University of the Health Sciences (1996-1998)
- Awarding Institution
- Allegheny University of the Health Sciences
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Allegheny University of the Health Sciences; Philadelphia, Pennsylvania
- Number of pages
- xxiv, 203 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology [Historical]; Allegheny University of the Health Sciences (1996-1998); School of Medicine (1996-1998)
- Other Identifier
- 991021888912804721