Abstract
Previously, we have reported that gingipain activity in Porphyromonas gingivalis, the major causative agent in adult periodontitis, is post-translationally regulated by the unique Vim proteins including VimF, a putative glycosyltransferase. To further characterize VimF, an isogenic mutant defective in this gene in a different P. gingivalis genetic background was evaluated. In addition, the recombinant VimF protein was used to further confirm its glycosyltransferase function. The vimF-defective mutant (FLL476) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimF-defective mutant (FLL95) in the P. gingivalis W83 genetic background. While hemagglutination was not detected and autoaggregation was reduced, biofilm formation was increased in FLL476. HeLa cells incubated with P. gingivalis FLL95 and FLL476 showed a 45% decrease in their invasive capacity. Antibodies raised against the recombinant VimF protein in E. coli immunoreacted only with the deglycosylated native VimF protein from P. gingivalis. In vitro glycosyltransferase activity for rVimF was observed using UDP-galactose and N-acetylglucosamine as donor and acceptor substrates, respectively. In the presence of rVimF and UDP-galactose, a 60 kDa protein from the extracellular fraction of FLL95 which was identified by mass spectrometry as Rgp gingipain, immunoreacted with the glycan specific mAb 1B5 antibody. Taken together, these results suggest the VimF glycoprotein is a galactosyltransferase that may be specific for gingipain glycosylation. Moreover, galatose is vital for the growing glycan chain.
Highlights
Porphyromonas gingivalis, a Gram-negative, anaerobic bacterium, is a major etiological agent implicated in adult periodontal disease and is associated with other systemic diseases, including cardiovascular disease [1,2,3,4,5]
In this report we have used both genetic and biochemical approaches to further confirm the role of specific bacterial host factors in gingipain biogenesis
Since the initial report of the S-layer glycoprotein of archaea Halobacterium halobium, some of the well-known examples of bacterial glycoproteins belong to genera Campylobacter, Mycobacterium, Neisseriae, Pseudomonas, Chlamydiae, Escherichia and Porphyromonas [7,19]
Summary
Porphyromonas gingivalis, a Gram-negative, anaerobic bacterium, is a major etiological agent implicated in adult periodontal disease and is associated with other systemic diseases, including cardiovascular disease [1,2,3,4,5] This data, taken together, implies a significant impact of this organism on the overall health of humans. The maturation pathway of the gingipains including its secretion facilitated by a novel POR secretion system (PorSS) is linked to carbohydrate biosynthesis This pathway is regulated by several proteins including the PorR, PorT, Sov, Rfa, VimA, VimE, VimF and other components of PorSS [Reviewed in [9,10,11]]. The role of VimF in this process is still unclear
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