Inhibition of lysine decarboxylase in bacterial plaque

Objectives: In teeth-adherent biofilms (plaques), lysine decarboxylase, a bacterial enzyme, may injure the coronal, dentally attached cells of the epithelial attachment by starving them of lysine at the base of gingival sulci (Levine, et al., Microbial Path. 30, 179-192, 2001). The aim of this study was to identify lysine decarboxylase inhibitors for assessing this enzyme's activity in experimental gingivitis. Methods: Early stationary-phase cells of Eikenella corrodens (EC), or pooled whole-mouth plaque samples, were homogenized with a Potter-Elvehjem homogenizer in a 4-fold excess of 65 mM NaCl and the supernatant fluid after centrifugation sterilized by Millipore-filtration. A goat was immunized by multiple subcutaneous injection of 0.25 mg EC protein in Freunds adjuvant, repeated after 2 and 4 weeks to induce IgG antibodies. Enzyme activity was assayed at 37^oC in 0.15 ml sodium phosphate buffered saline pH 7 containing 1 mM pyridoxal phosphate, 50% (v/v) pre-immune or immune serum, or 5 – 20 mM difluoromethyllysine (DFML), an irreversible inhibitor. Proteins were precipitated with trichloroacetic acid (10% v/v) on ice. Cadaverine was measured using trinitrobenzenesulfonic acid (Levine et al., 2001). Results: On incubation with EC or plaque extracts (75 ng or 58 μg protein respectively), cadaverine production increased asymptotically as the lysine concentration increased from 0 to 25 mM. Maximal catalysis was 80-120 nmol cadaverine/min/ml/mg protein for EC and 0.03% of that for plaque. DFML (20 mM) and immune serum antibodies inhibited enzyme activity in both plaque and EC extracts, but DFML became inactive on storage. On Western immunoblots of EC extracts, immune but not pre-immune serum antibodies detected a prominent 80 kDa protein previously identified as lysine decarboxylase. Much smaller amounts were in plaque extracts. Conclusion: Immunizing Beagle dogs with EC protein should inhibit biofilm lysine carboxylase activity, permitting the effect on experimental gingivitis to be determined. Supported by NIH-NIDCR 5 R21 DE014583-02.