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. 2010 Jul;156(Pt 7):2180-2193.
doi: 10.1099/mic.0.038331-0. Epub 2010 Apr 8.

Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Devon O Osbourne  1 , Wilson Aruni  1 , Francis Roy  1 , Christopher Perry  2 , Lawrence Sandberg  2 , Arun Muthiah  1 , Hansel M Fletcher  1
Affiliations

Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Devon O Osbourne et al. Microbiology (Reading). 2010 Jul.

Abstract

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

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Figures

Fig. 1.
Fig. 1.
(a) Model of the VimA protein (confidence score 0.54). The secondary structures are highlighted in red (α-helices) and yellow (β-strands). (b) Predicted binding site residues (green): Leu : 55 Gly : 56 Ser : 57, Phe : 71 Arg : 72 Ala : 73, Val : 77 His : 78 Met : 84, Tyr : 95 Ser : 96 Lys : 97, Tyr : 58 Ser : 59, Arg : 75 Arg : 76, His : 88 Phe : 91, and Gln : 108 Trp : 297. The N- and C-terminal residues are marked by blue and red spheres, respectively.
Fig. 2.
Fig. 2.
TEM micrographs of W83 (a) and FLL92 (b) showing capsule (CP) with vesicles (MV). AFM micrographs of W83 (c) and FLL92 (d). Immunogold localization of P. gingivalis FimA in W83 (e) and FLL92 (f), visualized by TEM after incubation with anti-FimA serum conjugated to 10 nm gold particles (GP).
Fig. 3.
Fig. 3.
Immunoblot analysis using anti-FimA antibody with outer membrane (OM) and total protein (TP) fractions of P. gingivalis ATCC 33277, W83 and FLL92. A 43 and 41 kDa protein corresponding with the expected size of FimA was observed in 33277 and in the vimA mutant, respectively (arrows).
Fig. 4.
Fig. 4.
Outer-membrane preparations from P. gingivalis W83 and FLL92 were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked in PBS then incubated with lectin-peroxidase overnight. Peroxidase activity was detected using the DAB peroxidase substrate kit. Differential lectin binding was observed using ABA, ConA, DBA, ECA, LPA, SBA and SGA.
Fig. 5.
Fig. 5.
Autoradiography of total protein (TP) and extracellular fractions (EX) of W83 and FLL92 grown overnight in the presence of 3H-labelled palmitic acid. (a, b) Brilliant blue-stained SDS-PAGE gel (a) and 3H-labelled palmitic acid nitrocellulose membrane (b) with W83 and FLL92 TP and EX fractions. (c, d) Densitometric analysis of 3H lipid-labelled proteins of W83 (grey) and FLL92 (black) total protein (c) and extracellular (d) fractions. A 54 kDa protein (B1) was three times as abundant in W83 compared with FLL92 and the 27 kDa protein (B3) was twice as abundant in FLL92 compared with W83 (see arrows).
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References

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