Fluoride and organic weak acids as respiration inhibitors for oral streptococci in acidified environments

Abstract

Oxygen metabolism (respiration) of Streptococcus mutans GS-5 involving NADH oxidases, mainly of the H(2)O-producing type, was found to be acid sensitive, as was NADH oxidase activity of cell extracts. Respiration of intact cells in acidified media was also highly sensitive to fluoride, with a 50% inhibitory concentration of about 0.02 mM at pH 4. In contrast, NADH oxidases in cell extracts were fluoride insensitive. Fluoride inhibition of respiration of intact cells was related to weak-acid effects leading to enhanced proton permeability of cells, cytoplasmic acidification and resultant acid inhibition of NADH oxidases and glycolysis. Organic weak acids, such as indomethacin and benzoate, were also effective inhibitors. H(2)O(2) production by intact cells of Streptococcus sanguis NCTC 10904, a peroxide producer, was similarly inhibited by fluoride or organic weak acids in acidified media. Thus, weak acids act as respiratory inhibitors for oral streptococci indirectly by acidifying the cytoplasm rather than acting as direct inhibitors of NADH oxidases.

Mutation of the NADH Oxidase Gene ( nox ) Reveals an Overlap of the Oxygen- and Acid-Mediated Stress Responses in Streptococcus mutans

“…It has been shown that Nox activity in S. mutans decreases in model biofilms, which are highly acidic, and in the presence of proton donors, such as organic weak acids and sodium fluoride (43,48). However, we observed that Nox activity was abundant in extracts of cells grown in the chemostat at pH 5 or 7, indicating that while the enzyme is inhibited at low pH, assay of the enzyme in buffer at pH 7 restores enzymatic activity.…”

“…The results of the enzyme's activity are thought to be a key mechanism in the creation of an acidic, highly reduced environment in dental plaque, establishing a pathogenic niche on tooth surfaces (37). However, studies have also shown that the NADH oxidase is inhibited at low pH in model biofilms (43) and that weak acids are capable of reducing respiration by S. mutans (48).…”

“…Sod activity was measured by the cytochrome c-xanthine-xanthine oxidase method described by McCord and Fridovich (40) and Phan et al (48). Briefly, reactions were carried out at 25°C using 1-ml mixtures that contained 50 mM potassium phosphate buffer (pH 7), 10 M cytochrome c, 50 M xanthine, and various amounts of cell extract.…”

“…However, studies have shown that acidic biofilm cultures of S. mutans exhibit reduced NADH oxidase activity compared to planktonic cultures (43). Furthermore, internal acidification of S. mutans cells, using membrane-permeative weak acids, also inhibits NADH oxidase activity (48), suggesting that in the acidic environment of dental plaque, NADH oxidase activity could be diminished. Thus, a question has been posed about the role of NADH oxidase in the pathophysiology of S. mutans during growth at low pH (37).…”

Two Spx Proteins Modulate Stress Tolerance, Survival, and Virulence inStreptococcus mutans

“…Protein concentration of the cell lysates was determined by the method of Bradford. All assay methods have been previously published, so only a brief description was included below (9,43,46). The mixture used to determine NADH oxidase activity (46) contained 50 g of protein lysate in 100 mM potassium phosphate buffer, pH 7, with 0.3 mM EDTA.…”