Characterization of the α- and β-mannosidases of Porphyromonas gingivalis

Abstract

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1 → 3 and α-1 → 2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1 → 6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.

Fig 1

Hydrolysis of α-mannobioses by sonicated cell extracts of P. gingivalis detected by TLC on Keiselgel F₂₅₄ HPTLC plates in n-butanol–ethanol–water (5:5:3, by volume). α-Mannosidase assays using α-1→2-, α-1→3-, and α-1→6-linked mannobioses were performed as described in Materials and Methods, and the assay mixtures contained 125 μg of disaccharide in 30 μl of 0.125 M sodium acetate buffer, pH 6.0, with 1.25 mM Ca²⁺. Ten-microliter aliquots were withdrawn and stored at 4°C, and this served as the zero time point. Ten microliters of sonicated cell extracts of P. gingivalis was added to the reaction mixture and incubated at 37°C for 20 h. Aliquots (10 μl) were withdrawn and centrifuged in an Eppendorf microcentrifuge. The supernatant was spotted onto Keiselgel F₂₅₄ HPTLC glass-coated plates, and TLC was performed in a tank equilibrated for 1 h in n-butanol–ethanol–water (50:50:30, by volume). The plates were dried in air, rechromatographed in the same solvent system, dried in air, sprayed with 3% sulfuric acid in methanol, air dried, and placed in an oven at 85°C to develop the spots. Lanes: 1, α-1→2 mannobiose; 2, mannose; 3, α-1→2 mannobiose plus P. gingivalis cell extract; 4, α-1→3 mannobiose plus P. gingivalis extract; 5, α-1→6 mannobiose plus P. gingivalis extract.

Fig 2

Hydrolysis of α-1→2 and α-1→3 mannobioses by P. gingivalis W50 and single-isogenic mutant strains analyzed by TLC on Keiselgel F₂₅₄ HPTLC plates in n-butanol–ethanol–water (5:5:3, by volume). The experiments were performed with P. gingivalis W50, ΔPG0032, ΔPG0902, ΔPG0973, ΔPG1711, and ΔPG1712 as described in the legend to Fig. 1. (A) α-1→2 mannobiose. (B) α-1→3 mannobiose. Lanes: 1, mannobiose standard; 2, mannose standard; 3, W50, time 0; 4, W50, 21 h; 5, ΔPG0032, 21 h; 6, ΔPG0902, 21 h; 7, ΔPG1711, 21 h; 8, ΔPG1712, 21 h; 9, ΔPG0973, 21 h.

Fig 3

Arg-gingipain and Lys-gingipain activities in whole cultures and culture supernatants of P. gingivalis W50 and mutant strains. (A) Arg-gingipain activities were measured as described in Materials and Methods. The cultures were adjusted to the same OD₆₀₀ with BHI broth prior to the assays. The results are expressed as activity (absorbance at 405 nm) units/ml of whole cultures or culture supernatants. (B) Lys-gingipain activities were measured as described in Materials and Methods. The cultures were adjusted to the same OD₆₀₀ with BHI broth prior to the assays. The data are expressed as activity (absorbance at 405 nm) units/ml of whole cultures or culture supernatants. Bars: 1, W50; 2, ΔPG0032; 3, ΔPG0902; 4, ΔPG0973; 5, ΔPG1711; 6, ΔPG1712. The filled bars represent cell-bound activity, and the gray bars represent activity in culture supernatants. The sum of the two activities represents the Arg-gingipain/Lys-gingipain activity in whole cultures.

Fig 4

SDS-PAGE of fluorescently labeled Arg- and Lys-gingipains in the culture supernatants of P. gingivalis W50, ΔPG0032, ΔPG0902, ΔPG0973, ΔPG1711, and ΔPG1712 strains. Culture supernatants (500 μl) from 6-day-old cultures of P. gingivalis W50 and mutant strains were treated with 750 μl of ice-cold acetone and left at −20°C for 1 h to effect complete precipitation of proteins. The suspension was centrifuged in an Eppendorf centrifuge at 13,300 rpm at 4°C for 20 min, the supernatant was discarded, and the protein pellet was air dried. Arg-gingipains and Lys-gingipains in the pellets were labeled with DNS-EGR-chloromethylketone as described previously (27). The samples were subjected to SDS-PAGE in 12.5% acrylamide gels, viewed under fluorescent light, and photographed immediately. The direction of migration is indicated by the arrow. The positions of mt-Rgps, Kgp, and Rgps are indicated.

Fig 5

SDS-urea-PAGE, followed by silver staining and Western blotting versus MAb 1B5 of LPSs isolated from P. gingivalis W50 and single-, double-, and triple-isogenic mannosidase mutant strains. LPSs from P. gingivalis W50 and single-isogenic mutant strains ΔPG0032, ΔPG0902, ΔPG0973, ΔPG1711, and ΔPG1712; double-isogenic mutant strain DMD; and triple-isogenic mutant strains 32/B2, 902/D2, and 973/E2 were isolated using the LPS isolation kit from Intron Biotechnology (South Korea). Aliquots containing 10 μg of LPS were subjected to SDS-urea-PAGE (28). (A) Silver staining was performed using the kit from Sigma Chemical Company, Poole, Dorset, United Kingdom. (B) Western blotting versus MAb 1B5 was performed as described previously (4). Δ32, ΔPG0032; Δ902, ΔPG0902; Δ973, ΔPG0973; Δ1711, ΔPG1711; Δ1712, ΔPG1712.