Involvement of an Skp-Like Protein, PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

Abstract

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

FIG 1

Localization of PGN_0300. P. gingivalis ATCC 33277 cells were subjected to fractionation, followed by SDS-PAGE and immunoblot analysis with anti-PGN_0300 antiserum. M, marker; LY, whole-cell lysate; CP/PP, cytoplasm plus periplasm; TM, total membrane; IM, inner membrane; OM, outer membrane; CBB, Coomassie brilliant blue staining.

FIG 2

Hemagglutination. P. gingivalis cells were grown in enriched BHI broth, washed with PBS, and resuspended in PBS at an optical density at 620 nm of 1.3. The suspension and a series of 2-fold dilutions were applied, from left to right, to the wells of a microtiter plate and mixed with sheep erythrocyte suspension.

FIG 3

Gene organization of the PGN_0300 (omp17) regions of the P. gingivalis ATCC 33277 chromosome. Black arrows indicate the direction of gene transcription. PGN_0299 is separated from omp17 by 57 bp, and omp17 is separated from PGN_0301 by 35 bp. PCR primers were designed to detect the presence of mRNA spanning PGN_0299, omp17, and PGN_0301 (small gray arrows). (B) RT-PCR products indicate that omp17 is cotranscribed with PGN_0299 (d) and PGN_0301 (e). Individual gene and operon products were amplified with unique primer sets. The primer sets used were PGN_0299PF and PGN_0299PR (a), PGN_0300PF and PGN_0300PR (b), PGN_0301PF and PGN_0301PR (c), PGN_0299PF and PGN_0300PR (d), and PGN_0300PF and PGN_0301PR (e). The genomic DNA from P. gingivalis ATCC 33277 was also employed in the experiments whose results are shown in panels d and e.

FIG 4

Rgp and Kgp activities in the omp17 mutant. P. gingivalis cells were grown anaerobically in enriched BHI medium at 37°C for 36 h. Kgp (A and C) and Rgp (B and D) activities of the cell lysates (A and B) and the culture supernatants, including vesicles (C and D), were measured. The statistical significance of the changes was evaluated using a two-sided t test. Error bars indicate standard deviations calculated from the enzyme activity. *, P < 0.05.

FIG 5

Immunoblot analysis of CTD-containing proteins in P. gingivalis cell lysate and culture supernatant. The whole-cell lysate (A) and culture supernatant (B) of P. gingivalis strains were analyzed by SDS-PAGE, followed by immunoblot analyses using anti-Kgp, anti-Rgp, and anti-HBP35 antibodies. M, marker; CBB, Coomassie brilliant blue staining.

FIG 6

Peptide map fingerprinting (PMF) analyses of proteins in the P. gingivalis culture supernatant. The vesicle-free supernatants of P. gingivalis strain ATCC 33277 and the Δomp17 strain were separated by SDS-PAGE, and the resulting gel was stained with Coomassie brilliant blue. Bands indicated by numbered arrows to the right are identified in Table 1.

FIG 7

Subcellular localization of CTD-containing proteins. P. gingivalis ATCC 33277 (A) and the Δomp17 strain (B) were subjected to fractionation, followed by SDS-PAGE and immunoblot analysis with anti-Kgp, anti-Rgp, and anti-HBP35 antibodies. M, marker; CP/PP, cytoplasm plus periplasm; TM, total membrane; IM, inner membrane; OM, outer membrane; CBB, Coomassie brilliant blue staining.

FIG 8

PorU protein is not localized to the membrane fractions of the Δomp17 strain. Whole-cell lysates (A) and the cytoplasm-plus-periplasm and membrane fractions (B) of P. gingivalis ATCC 33277 and the Δomp17 strain were subjected to SDS-PAGE and immunoblot analysis with anti-PorU. M, marker; CP/PP, cytoplasm plus periplasm; TM, total membrane; CBB, Coomassie brilliant blue staining.

FIG 9

Immunoblot analyses of P. gingivalis cell lysate using MAb 1B5 (anti-A-LPS antibody).

FIG 10

Survival rates of mice challenged with P. gingivalis strain W83 and the Δomp17 strain (Δomp17-83). BALB/c mice were subcutaneously inoculated with P. gingivalis cells injected into the dorsal surface (2 × 10¹¹ CFU), and then their survival was monitored daily for up to 14 days. The animal experiment, in which five mice were used for each bacterial strain, was performed three times. **, P < 0.05 versus the corresponding values for the wild-type littermates, as determined with the log-rank test.