Role of hydrogen peroxide in competition and cooperation between Streptococcus gordonii and Actinomyces naeslundii

Abstract

In dental plaque alpha-haemolytic streptococci, including Streptococcus gordonii, are considered beneficial for oral health. These organisms produce hydrogen peroxide (H(2)O(2)) at concentrations sufficient to kill many oral bacteria. Streptococci do not produce catalase yet tolerate H(2)O(2). We recently demonstrated that coaggregation with Actinomyces naeslundii stabilizes arginine biosynthesis in S. gordonii. Protein arginine residues are sensitive to oxidation by H(2)O(2). Here, the ability of A. naeslundii to protect S. gordonii against self-produced H(2)O(2) was investigated. Coaggregation with A. naeslundii enabled S. gordonii to grow in the absence of arginine, and promoted survival of S. gordonii following growth with or without added arginine. Arginine-replete S. gordonii monocultures contained 20-30 microM H(2)O(2) throughout exponential growth. Actinomyces naeslundii did not produce H(2)O(2) but synthesized catalase, removed H(2)O(2) from coaggregate cultures and decreased protein oxidation in S. gordonii. On solid medium, S. gordonii inhibited growth of A. naeslundii; exogenous catalase overcame this inhibition. In coaggregate cultures, A. naeslundii cell numbers were >90% lower than in monocultures after 24 h. These results indicate that coaggregation with A. naeslundii protects S. gordonii from oxidative damage. However, high cell densities of S. gordonii inhibit A. naeslundii. Therefore, H(2)O(2) may drive these organisms towards an ecologically balanced community in natural dental plaque.

Fig. 1

Enhanced survival of Streptococcus gordonii by coaggregation with Actinomyces naeslundii. Monocultures or coaggregate cultures in CDMΔarg (left panel) or CDM (right panel) were incubated aerobically and viable counts of S. gordonii cells were determined at the times indicated. Data shown are mean values of triplicate measurements and SDs from triplicate measurements. Data represent one of two experiments that gave similar results.

Fig. 2

Effects of coaggregation on H₂O₂ concentration and protein oxidation in Streptococcus gordonii. Monocultures or coaggregate cultures in CDM (0.5 mM arginine) were incubated aerobically. Turbidity (a) and extracellular H₂O₂ concentrations (b) were determined at intervals. Symbols represent S. gordonii monocultures (■), coaggregate cultures containing S. gordonii and Actinomyces naeslundii (○) and A. naeslundii monocultures (◆). After 3 h incubation, samples were removed and proteins extracted. (c) Equal amounts (1 μg) of proteins were separated using SDS-PAGE and oxidized proteins containing carbonyl groups were detected by immunostaining with the Oxyblot kit. Approximately 85–90% of proteins in the sample from coaggregate cultures originated from S. gordonii. Experiments were repeated three times with similar results.

Fig. 3

Inhibition of Actinomyces naeslundii growth by Streptococcus gordonii-produced H₂O₂. (a) A colorimetric assay, based on conversion of an indicator, ABTS, in the presence of HRP and H₂O₂ was used to detect H₂O₂ in cells grown on THB agar. The purple colour on and around S. gordonii cells indicates the presence of H₂O₂. (b) Streptococcus gordonii (left) and A. naeslundii (right) were spotted in close proximity on THB agar without (upper panel) or with (lower panel) prior addition of 10 U catalase to the S. gordonii cells.

Fig. 4

Inhibitory properties of Streptococcus gordonii towards Actinomyces naeslundii in CDM (0.5 mM arginine) (a) and THBYE medium (b). Viable A. naeslundii cells in aerobic monocultures or coaggregate cultures containing S. gordonii were enumerated on selective agar. Experiments were performed twice with similar results. Data are means and SDs from triplicate measurements of one representative experiment.