Role of VimF in Gingipain Maturation in Porphyromonas gingivalis

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.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Basic Sciences

Year

2013

Author dc:creator

• Muthiah, Arun S.

Contributors dc:contributor

• Fletcher, Hansel M.
• Boskovic, Danilo
• Johnson, Mark
• Perry, Christopher
• Soto-Wegner, Ubaldo

• Medical Genetics
• Medical Microbiology
• Gingivitis; Cardiovascular Diseases; Periodontitis; Periodontal Diseases; Respiratory Tract Infections; Arthritis, Rheumatoid; Porphyromonas Gingivalis
• Gingivitis
• Periodontitis
• Periodontal disease
• Cardiovascular disease
• Respiratory infections
• rheumatoid arthritis
• Gingipain maturation
• Virulence modulating protein F
• Porphyromonas gingivalis

Statement dc: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.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/139

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-1138