Dentilisin activity affects the organization of the outer sheath of Treponema denticola

Abstract

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178-5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with a prtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.

FIG. 1

Construction of a prtP mutant with the ermF-ermAM cassette.

FIG. 2

Southern blot analysis of T. denticola ATCC 35405 and prtP mutant K1. Chromosomal DNAs from T. denticola ATCC 35405 (lanes 1 and 3) and prtP mutant K1 (lanes 2 and 4) were digested with HindIII and hybridized with a digoxigenin-labeled KpnI-PstI fragment from the prtP gene (lanes 1 and 2) or the KpnI-BamHI fragment from pVA2198 (lanes 3 and 4). Numbers at right are kilobase pairs.

FIG. 3

Gelatin zymography of sonicates of T. denticola ATCC 35405 (lane 1) and K1 (lane 2).

FIG. 4

SDS-PAGE analysis of sonicates of T. denticola ATCC 35405 and K1. Samples from lanes 1 to 4 were treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without boiling); lanes 3 and 7, T. denticola ATCC 35405 (with boiling); lanes 4 and 8, T. denticola K1 (with boiling). After electrophoresis, the gel was stained with Coomassie brilliant blue R-250. Arrowheads indicate the high-molecular-mass oligomeric protein and the Msp band.

FIG. 5

Immunoblot analysis of T. denticola ATCC 35405 (lane 1; boiled) and K1 (lane 2; boiled) with antidentilisin serum.

FIG. 6

Immunoblot analysis of T. denticola ATCC 35405 and K1 with anti-T. denticola ATCC 35405 whole-cell serum. Samples from lanes 1 to 4 were treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without boiling); lanes 3 and 7, T. denticola ATCC 35405 (with boiling); lanes 4 and 8, T. denticola K1 (with boiling). Arrowheads indicate the high-molecular-mass oligomeric protein and the Msp band.

FIG. 7

Immunoblot analysis of T. denticola ATCC 35405 and K1 with anti-T. denticola ATCC 35404 Msp serum. Samples from lanes 1 to 4 were treated with PMSF. Lanes 1 and 5, T. denticola ATCC 35405 (without boiling); lanes 2 and 6, T. denticola K1 (without boiling); lanes 3 and 7, T. denticola ATCC 35405 (with boiling); lanes 4 and 8, T. denticola K1 (with boiling). Arrowheads indicate the high-molecular-mass oligomeric protein and the Msp band.

FIG. 8

Mean areas of lesions at infection sites after challenge with T. denticola ATCC 35405 and prtP mutant K1. Mice were injected with live T. denticola ATCC 35405 (open bars) or mutant K1 (hatched bars), and lesion areas were determined at the indicated times following infection.