Altered gingipain maturation in vimA- and vimE-defective isogenic mutants of Porphyromonas gingivalis

Abstract

We have previously shown that gingipain activity in Porphyromonas gingivalis is modulated by the unique vimA and vimE genes. To determine if these genes had a similar phenotypic effect on protease maturation and activation, isogenic mutants defective in those genes were further characterized. Western blot analyses with antigingipain antibodies showed RgpA-, RgpB-, and Kgp-immunoreactive bands in membrane fractions as well as the culture supernatant of both P. gingivalis W83 and FLL93, the vimE-defective mutant. In contrast, the membrane of P. gingivalis FLL92, the vimA-defective mutant, demonstrated immunoreactivity only with RgpB antibodies. With mass spectrometry or Western blots, full-length RgpA and RgpB were identified from extracellular fractions. In similar extracellular fractions from P. gingivalis FLL92 and FLL93, purified RgpB activated only arginine-specific activity. In addition, the lipopolysaccharide profiles of the vimA and vimE mutants were truncated in comparison to that of W83. While glycosylated proteins were detected in the membrane and extracellular fractions from the vimA- and vimE-defective mutants, a monoclonal antibody (1B5) that reacts with specific sugar moieties of the P. gingivalis cell surface polysaccharide and membrane-associated Rgp gingipain showed no immunoreactivity with these fractions. Taken together, these results indicate a possible defect in sugar biogenesis in both the vimA- and vimE-defective mutants. These modulating genes play a role in the secretion, processing, and/or anchorage of gingipains on the cell surface.

FIG. 1.

SDS-PAGE of acetone-precipitated P. gingivalis extracellular proteins. Acetone-precipitated proteins from culture supernatants of P. gingivalis cells grown to exponential phase (A) or stationary phase (B) were separated by SDS-PAGE and stained with Simply Blue Safe stain. All lane contained 20 μg of proteins. Lane 1, P. gingivalis W83. Lane 2, P. gingivalis FLL92. Lane 3, P. gingivalis FLL93.

FIG. 2.

Extracellular protease profile of P. gingivalis FLL92 and FLL93 from the exponential and stationary growth phases. Western blot analysis of extracellular proteins from P. gingivalis grown to the exponential and stationary growth phases separated by SDS-PAGE and immunoreacted with gingipain-specific antibodies. Lane 1, P. gingivalis W83. Lane 2, P. gingivalis FLL92. Lane 3, P. gingivalis FLL93. Panels A and D, rabbit anti-RgpB. Panels B and E, chicken anti-RgpA. Panels C and F, chicken anti-Kgp.

FIG. 3.

Analysis of membrane proteins of P. gingivalis. Membrane preparations of P. gingivalis grown to the stationary phase were analyzed by SDS-PAGE and stained with Simply Blue Safe stain and subjected to immunoblot analysis with antigingipain antibodies. Panel A, stained proteins. Panel B, rabbit anti-RgpB. Panel C, chicken anti-RgpA. Panel D, chicken anti-Kgp. All lanes contained approximately 20 μg of protein. Lane 1, P. gingivalis W83. Lane 2, P. gingivalis FLL92. Lane 3, P. gingivalis FLL93.

FIG. 4.

Most active gingipains are released into the vesicle supernatant of P. gingivalis FLL92. Membrane vesicle preparations of P. gingivalis W83 and FLL92 isolated from a 2-day-old culture were dialyzed, and fractions were then analyzed for Rgp (BApNA) or Kgp (ALNA) activity. Solid bars represent the activity of membrane vesicles after dialysis. and striped bars represent protease activity found in the dialysis supernatants. Panel A, P. gingivalis FLL92. Panel B, P. gingivalis W83.

FIG. 5.

RgpA and Kgp analysis of membrane vesicles. Membrane vesicles isolated from P. gingivalis W83 and isogenic mutants FLL92 and FLL93 were separated by SDS-PAGE and then subjected to immunoblot analysis with anti-RgpA (A) and anti-Kgp (B) antibodies. All lanes contained 20 μg of protein. Lane 1, W83. Lane 2, FLL92. Lane 3, FLL93.

FIG. 6.

Mass spectrometry analysis of TonB receptor-linked protein RagA from FLL93 membrane vesicles. Membrane vesicles isolated from culture supernatants of P. gingivalis W83 and FLL93 were separated by SDS-PAGE and stained with Simply Blue Safe stain. A band greater than 200 kDa from FLL93 membrane vesicles was excised and analyzed by mass spectrometry. Panel A, stained protein gel. Panel B, mass spectrometry data.

FIG. 7.

Lipopolysaccharide and polysaccharide profiles from P. gingivalis W83, FLL92, and FLL93. Lipopolysaccharides and polysaccharides isolated from P. gingivalis strain W83 and isogenic mutants FLL92 and FLL93 from the stationary phase were separated by SDS-PAGE and silver stained. Panel A, lipopolysaccharides. Panel B, polysaccharides.

FIG. 8.

Membrane and extracellular proteins are glycosylated in P. gingivalis FLL92 and FLL93. Membranes or extracellular preparations of P. gingivalis strains W83, FLL92, and FLL93 from the stationary phase were separated by electrophoresis, and oxidized carbohydrates from the glycoproteins were stained. Lane 1, P. gingivalis FLL92. Lane 2, P. gingivalis FLL93. Lane 3, P. gingivalis W83. Panel A, glycoprotein staining. Panel B, negative control (no oxidation step). Panel C, total protein staining.

FIG. 9.

Immunoblot analysis of membrane with monoclonal antibody 1B5. Membrane preparations from P. gingivalis W83 and isogenic mutants FLL92 and FLL93 were separated by SDS-PAGE and analyzed for certain carbohydrate modifications of membrane-associated (mt) Rgps and/or lipopolysaccharide modifications of membrane proteins with monoclonal antibody 1B5. All lanes contained 20 μg of protein.

FIG. 10.

Activation of inactive Rgps by purified RgpB. We incubated 750 μg of proteins from 37.5% extracellular acetone fractions containing the inactive gingipains of P. gingivalis FLL92 (A and C) and FLL93 (B and D) with 2.5 units of RgpB for 1 h at 37°C. Panels A and B, Rgp activity. Panels C and D, Kgp activity.