2.2. Detection of dentipain in the outer sheath fraction

The outer sheath fraction was isolated from the wild-type strain and the two mutant strains using Triton X-114 as previously described (Brusca & Radolf, 1994). Briefly, the cells were harvested, washed twice with phosphate-buffered saline (PBS), and then resuspended in Tris-buffered saline (TBS), comprising 100 mM Tris-HCl (pH 7.4) and 150 mM NaCl, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2% Triton X-114. After incubation at 4 °C for 3 h, the insoluble materials were removed using centrifugation (5530 × g, 4 °C, 10 min). The supernatant was incubated at 37 °C for 3 min before separation into detergent and aqueous phases using centrifugation (5530 × g, 25 °C, 10 min). The detergent phase was washed four times via phase separation with TBS, as described above. The proteins in the detergent phase were then precipitated with cold acetone at −20 °C. The precipitate was washed twice with hexane:isopropanol (3:2), and the pellet was dissolved in 50 mM Tris-HCl (pH 7.4) containing 1 mM PMSF via sonication (100 W for 5 min). The protein profile and immunoreactivity with anti-dentilisin and anti-Msp antibodies of the outer sheath fraction are shown in Supplemental Fig. 1.

In separate experiments, sonicates of T. denticola strains were obtained to verify the location of dentipain. Briefly, T. denticola strains were harvested, washed with PBS twice, and suspended in PBS containing 1 mM PMSF. The obtained cell suspension was disrupted using sonication at 200 W for 10 min with Bioruptor (Sonicbio Co., Ltd.). The supernatant was collected using centrifugation at 20,000 × g for 10 min. The resulting outer sheath fractions and sonicates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10%–20% Multi gel II gradient gel (Cosmobio, Tokyo, Japan). The proteins were transferred on to an Immobilon polyvinylidene difluoride membrane (Millipore) using the Trans-Blot Turbo system (Bio-Rad Laboratories, Hercules, CA, USA), and dentipain was detected using a rabbit antibody against recombinant dentipain (Ishihara, et al., 2010) diluted 1:5000, as previously described (Ishihara, et al., 1998). Dentipain bands were detected using peroxidase-conjugated goat anti-rabbit antibody (Citiva, Tokyo, Japan), the Image Quant LAS 4000 system (Citiva), and ECL Prime Western Blotting Detection Reagent (Citiva).