Abstract

Gingipain activity in Porphyromonas gingivalis, the major etiological agent in adult periodontitis, is post-translationally regulated by unique Vim proteins including VimF, a putative glycosyltransferase. To ascertain the VimF mediated phenotype we first inactivated the vimF gene in P. gingivalis ATCC 33277 (FLL476), a less virulent fimbriated strain. We observed that the vimF-defective mutant (FLL476) showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 background. While hemagglutination was not detected and autoaggregationn was reduced, biofilm formation was increased in FLL476. Also, invasive capacity decreased for this mutant. Furthermore, fimbrial structures were missing in FLL476, suggesting the role of VimF in fimbrial processing. We were able to restore the wildtype phenotype by complementing the defect. Secondly, we cloned, expressed and purified the vimF gene and demonstrated its ability to glycosylate gingipains. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. Further, in the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified my mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb IB5 antibody. Finally, the polyclonal antibody raised against rVimF that did not react well with native VimF from P. gingilvalis, reacted with a 47 kDa protein when the fractions were first deglycosylated before probing with the antibody. Galactose is vital for growing glycan chain leading to maturation/activation of gingipains. Taken together, these results suggest that VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation.

LLU Discipline

Microbiology and Molecular Genetics

Department

Basic Sciences

School

School of Medicine

First Advisor

Fletcher, Hansel M.

Second Advisor

Boskovic, Danilo

Third Advisor

Johnson, Mark

Fourth Advisor

Perry, Christopher

Fifth Advisor

Soto-Wegner, Ubaldo

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

January 2013

Date (Title Page)

9-1-2013

Language

English

Library of Congress/MESH Subject Headings

Gingivitis; Cardiovascular Diseases; Periodontitis; Periodontal Diseases; Respiratory Tract Infections; Arthritis, Rheumatoid; Porphyromonas Gingivalis

Subject - Local

Gingivitis, Periodontitis, Periodontal disease, Cardiovascular disease, Respiratory infections, rheumatoid arthritis, Gingipain maturation, Virulence modulating protein F, Porphyromonas gingivalis

Type

Dissertation

Page Count

154 p.

Digital Format

Application/PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses & Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

Share

COinS