Effect of deletion of the rgpA gene on selected virulence of Porphyromonas gingivalis

Abstract

Background/purpose: The most potent virulence factors of the periodontal pathogen Porphyromonas gingivalis are gingipains, three cysteine proteases (RgpA, RgpB, and Kgp) that bind and cleave a wide range of host proteins. Considerable proof indicates that RgpA contributes to the entire virulence of the organism and increases the risk of periodontal disease by disrupting the host immune defense and destroying the host tissue. However, the functional significance of this proteinase is incompletely understood. It is important to analyze the effect of arginine-specific gingipain A gene (rgpA) on selected virulence and physiological properties of P. gingivalis.

Materials and methods: Electroporation and homologous recombination were used to construct an rgpA mutant of P. gingivalis ATCC33277. The mutant was verified by polymerase chain reaction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell structures of the mutant were examined by transmission electron microscopy and homotypic biofilm formation was examined by confocal laser scanning microscopy.

Results: Gene analysis revealed that the rgpA gene was deleted and replaced by a drug resistance gene marker. The defect of the gene resulted in a complete loss of RgpA proteinase, a reduction of out membrane vesicles and hemagglutination, and an increase in homotypic biofilm formation.

Conclusion: Our data indicate that an rgpA gene deficient strain of P. gingivalis is successfully isolated. RgpA may have a variety of physiological and pathological roles in P. gingivalis.

Keywords: Porphyromonas gingivalis; biofilm formation; mutant; outer membrane vesicles; rgpA.

Figure 1

A sketch gene map of Porphyromonas gingivalis ATCC33277 (WT) and the rgpA mutant strain (rgpA-). In the genome of mutant strain, rgpA was replaced by ermF-ermAM cassette (erm). PGN_1969 is the upstream gene and PGN_1971 the downstream gene adjacent to rgpA.

Figure 2

Construction and verification of rgpA mutant strain. DNA products were analyzed by 1% agarose gel electrophoresis. (A) Upstream (lanes 1 and 2, ∼1.1 kb) and downstream (lanes 3 and 4, ∼1 kb) of rgpA, ermF-ermAM cassette (lanes 3 and 4, ∼2.2 kb) were respectively amplified by polymerase chain reaction. (B) Overlap extension products of the above three DNA fragments, named up_erm_down (lanes 1 and 2, ∼4.3 kb). (C) Restriction enzyme reaction was performed to identify the recombinant plasmid pPHU281_up_erm_down. pPHU281_up_erm_down was linearized by restriction enzyme SacI (lane 2, 11.2 kb), and cleaved into two fragments (lane 1, 2 bands, 6.9 kb and 4.3 kb) by EcoRI. (D) The genomic DNA of putative strain was amplified with specific primer pairs RgpA F and RgpA R. Mutant strains (lanes 1–4, no DNA band) were rgpA-deficient genotypes. (E) Genomic DNA of the putative strain was amplified with specific primer pairs upF' and ermR. It had been confirmed that ermF-ermAM cassette was inserted into the mutant strain (lane 1–4, ∼3 kb) genome at the rgpA locus. In (D) and (E), Porphyromonas gingivalis ATCC33277 (lane 5) and plasmid pPHU281_up_erm_down (lane 6) were used as negative control and positive control, respectively. M = DNA marker, bp.

Figure 3

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of gingipain extracts of Porphyromonas gingivalis ATCC33277 (WT) and the rgpA mutant (rgpA-) with Coomassie brilliant blue staining. M = molecular weight marker, kDa.

Figure 4

Hemagglutination and pigmentation of the rgpA mutant strain. (A) Hemagglutination activity. Porphyromonas gingivalis cells were grown in enriched trypticase soy broth, washed with phosphate-buffered saline, and suspended in phosphate-buffered saline at an optical density at 660 nm of 0.4. The suspension and its dilutions in a two-fold series were applied to the wells of a microtiter plate from left to right and mixed with sheep erythrocyte suspension (2.5%). (B) Colonial pigmentation. The mutant cells formed glossy roundish black colonies on blood agar plate after incubation anaerobically for 7 days. WT = wild type; rgpA- = rgpA mutant strain.

Figure 5

Transmission electron micrographs of Porphyromonas gingivalis ATCC33277 (WT) and rgpA mutant (rgpA-). Bubbles on the cell surfaces indicate the presence of outer membrane vesicles (red arrows).

Figure 6

Homotypic biofilm formation of Porphyromonas gingivalis ATCC33277 (WT) and rgpA mutant (rgpA-). Bacterial cells were incubated with a glass slip in trypticase soy broth for 12 hours. Homotypic biofilms developed on the glass slip were stained with FITC (green) and observed with a confocal laser scanning microscope. Representative images are shown.