ZnCl₂ Inhibits Fusobacterium nucleatum H₂S production by repressing L-Cysteine Desulfhydrase

Background: Fusobacterium nucleatum is a periodontal pathogen commonly associated with human halitosis (oral malodor) by the production of volatile sulfur compounds (VSCs). The enzymes, L-cysteine desulfhydrase (CDHase) catalyzes L-cysteine to produce hydrogen sulfide (H₂S), and L-methionine-α-deamino-γ-mercaptomethane-lyase (METase) catalyzes L-methionine to produce methyl mercaptan (CH₃SH). Zinc-containing compounds have been incorporated into oral hygiene products to reduce halitosis due to their reported sulfur-affinity and antimicrobial activity. We hypothesize that ZnCl₂ regulates the expression of CDHase or METase to suppress the production of VSCs. Objective: To investigate the mechanism of ZnCl₂-induced VSC reduction through CDHase and METase in F. nucleatum. Methods: F. nucleatum were grown anaerobically in the presence of ZnCl₂ (2-100 µg/ml) and in vitro growth, VSCs production and metabolic activity were determined. Rabbit polyclonal antibodies against CDHase and METase of F. nucleatum were generated using specific peptides sequences. Cytosolic proteins of F. nucleatum grown in the presence of sub-MIC levels of ZnCl₂ (0 to 42.6 µg/ml) were extracted, separated by SDS-PAGE, followed by detection of CDHase and METase using the Western Blot assay. Results: The MIC of ZnCl₂ for F. nucleatum was 85 µg/ml. When grown in 42.6 µg/ml of ZnCl₂, in vitro production of H₂S and CH₃SH were reduced by approximately 65% and 25%, respectively. Western Blot analysis of cytosolic proteins revealed a more than 50% reduction of CDHase expression in F. nucleatum grown in the presence of 17 µg/ml ZnCl₂. In contrast, no significant reduction in the expression of METase was observed (up to 42.6 µg/ml ZnCl₂). Metabolic activity of F. nucleatum was not affected at the above ZnCl₂ concentrations. Conclusion: Based on results obtained from this study, ZnCl₂ may control human halitosis by specifically suppressing the expression of CDHase leading to reduced levels of H₂S production. (Research supported by University of Illinois at Chicago, College of Dentistry)