Molecular mechanism for the spontaneous generation of pigmentless Porphyromonas gingivalis mutants

Abstract

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its protease activity has been implicated as an important virulence factor. Kgp is the major Lys-gingipain protease of P. gingivalis and appears to be involved not only in enzyme activity but also in hemagglutination and the pigmented phenotype due to heme accumulation and/or hemoglobin binding. However, little information concerning the molecular mechanism for the spontaneous generation of pigmentless P. gingivalis mutants is currently available. In this study, several spontaneous pigmentless mutants of P. gingivalis were isolated and characterized. The results revealed that a portion of the kgp gene had been deleted from the chromosomes of the pigmentless mutants. This deletion appears to result from recombination between the highly homologous DNA sequences encoding the adhesin domains of the tandemly arranged hagA and kgp genes on the chromosomes of P. gingivalis strains.

FIG. 1

Hemoglobin binding assay of P. gingivalis strains. The cell suspensions in PBS were incubated anaerobically with bovine hemoglobin at pH 7.4 and 37°C for 30 to 60 min and centrifuged. The absorbance of the suspensions at 415 nm was then measured. The decrease in absorbance was used to calculate hemoglobin binding. Shown are hemoglobin binding results with strains 381 (□), WK (◊), and WK-W (○) (A); with strains 381 (□), G102 (◊), and G102-W (○) (B); and with strains 381 (□), MT10 (◊), and MT10-W (○) (C). Bars represent the standard deviations of results of duplicate samples.

FIG. 2

Genomic maps of the proposed orientations of the hagA and kgp genes of P. gingivalis 381. The major restriction sites of hagA and kgp are indicated. Restriction sites: K, KpnI (nucleotides 3760, 5116, 6484, 7852, 10606, 12589, and 16087); B, BamHI (nucleotides 3309, 3672, 12484, and 15999); N, NcoI (nucleotides 4840, 6208, 7576, 8944, 17170, and 18643); S, SmaI (nucleotides 4583, 5951, 7319, 8687, 16913, 19245, and 19554); V, Van91I (nucleotides 1741 and 20629). Solid boxes on the map show the probes N (for the Kgp N-terminal protease domain), C (for the Kgp adhesin domain), hagAN (for the exact HagA N-terminal region), and kgpC (for the exact Kgp C-terminal region). Boxes with hatching labeled “hagA” and “kgp” represent the adhesin regions of the hagA and kgp genes, respectively. The stippled box represents the N-terminal domain and protease domain of Kgp. The boxes above the restriction enzyme map with hatching, vertical lines, and shading represent the proposed minimal deleted regions of WK-W, MT10-W, and G102-W, respectively.

FIG. 3

Southern blot analysis of genomic DNAs of P. gingivalis strains. (A to E) Southern blots of strain 381 (lanes 1), WK (lanes 2), WK-W (lanes 3), G102 (lanes 4), G102-W (lanes 5), MT10 (lanes 6), and MT10-W (lanes 7). The chromosomal DNAs of the P. gingivalis strains were digested with the following restriction enzymes and hybridized with the following probes: KpnI and probe N (A), KpnI and probe C (B), NcoI and probe N (C), NcoI and probe C (D), and Van91I and probe hagAN (E).