Influence of a model human defensive peroxidase system on oral streptococcal antagonism

Abstract

Streptococcus is a dominant genus in the human oral cavity, making up about 20 % of the more than 800 species of bacteria that have been identified, and about 80 % of the early biofilm colonizers. Oral streptococci include both health-compatible (e.g. Streptococcus gordonii and Streptococcus sanguinis) and pathogenic strains (e.g. the cariogenic Streptococcus mutans). Because the streptococci have similar metabolic requirements, they have developed defence strategies that lead to antagonism (also known as bacterial interference). S. mutans expresses bacteriocins that are cytotoxic toward S. gordonii and S. sanguinis, whereas S. gordonii and S. sanguinis differentially produce H(2)O(2) (under aerobic growth conditions), which is relatively toxic toward S. mutans. Superimposed on the inter-bacterial combat are the effects of the host defensive mechanisms. We report here on the multifarious effects of bovine lactoperoxidase (bLPO) on the antagonism between S. gordonii and S. sanguinis versus S. mutans. Some of the effects are apparently counterproductive with respect to maintaining a health-compatible population of streptococci. For example, the bLPO system (comprised of bLPO+SCN(-)+H(2)O(2)) destroys H(2)O(2), thereby abolishing the ability of S. gordonii and S. sanguinis to inhibit the growth of S. mutans. Furthermore, bLPO protein (with or without its substrate) inhibits bacterial growth in a biofilm assay, but sucrose negates the inhibitory effects of the bLPO protein, thereby facilitating adherence of S. mutans in lieu of S. gordonii and S. sanguinis. Our findings may be relevant to environmental pressures that select early supragingival colonizers.

Fig. 1.

Competitive host peroxidase and streptococcal defence mechanisms in the supragingival region of the oral cavity. (A) Cariogenic S. mutans produces bacteriocins (mutacins) that are known to be cytotoxic to other oral streptococci. (B) Oral health-compatible S. gordonii and S. sanguinis differentially produce H₂O₂ under aerobic growth conditions, thereby promoting an ecological advantage. (C) The human supragingival peroxidase system (consisting of the enzyme hSPO or hMPO, the substrate SCN⁻ and the oxidant H₂O₂) produces the antimicrobial hypothiocyanite (OSCN⁻). (D) The peroxidase proteins themselves are known to be antimicrobial. (E) Environmental pressures, which may be biotic or abiotic in origin, could affect the above defensive mechanisms.

Fig. 2.

Effect of the bLPO system on the growth of S. mutans and S. gordonii. (a) S. mutans was inoculated first and grown aerobically (5 % CO₂) for 16 h at 37 °C on BHI agar with (+SCN⁻) or without 10 mM SCN⁻ and with (+bLPO) or without 20 μg bLPO per 8 μl of inoculum. S. gordonii was then inoculated next to the pioneer colonizer (with or without bLPO in the inoculum), and the plates were incubated for 16 h and then photographed. The columns labelled Sm and Sg are reference cultures grown in spatial isolation. (b) The same protocol was followed, except that S. gordonii was the pioneer colonizer and S. mutans was the competing colonizer. (c) The same protocol was followed as for (a) and (b), except that S. gordonii was both the pioneer colonizer and the competing colonizer.

Fig. 3.

Effect of catalase on the growth of S. gordonii and S. mutans. The same protocol was followed as for Fig. 2, except that catalase was employed instead of bLPO.

Fig. 4.

Rate of decomposition of an initial concentration of OSCN⁻ of 500 μM at pH 7.4 and 19 °C in the presence of 50 % BHI (•). A first-order fit is illustrated (k=9.5±1.4×10⁻³ min⁻¹; t_1/2=73 min). H₂O₂ (initial concentration=500 μM) is stable under the same conditions (▪).

Fig. 5.

Quantification of attached biomass using a microtitre plate biofilm assay. Biofilm formation of S. mutans (UA140, white), S. gordonii (DL1, dark grey) and S. sanguinis (SK36, light grey) on microtitre plates was quantified with CV staining. Values were normalized to the BHI control, which was set to 100 %. (a) Biofilm biomass of cultures in BHI medium, BHI plus bLPO (1 mg ml⁻¹), BHI plus SCN⁻ (10 mM) and BHI plus bLPO (1 mg ml⁻¹) and SCN⁻ (10 mM). (b) Biofilm biomass of cultures supplemented with 0.5 % sucrose and 0.5 % sucrose plus bLPO (1 mg ml⁻¹). Data presented are the means±sd from three independent experiments. Asterisks indicate statistical significance.

Fig. 6.

Dose-dependent effect of bLPO protein (no SCN⁻) upon biofilm growth of S. mutans (UA140, white), S. gordonii (DL1, dark grey) and S. sanguinis (SK36, light grey) using the 96-well plate assay. Cultures were grown in BHI medium containing different concentrations of LPO (ranging from 0.125 to 2 mg ml⁻¹). Values were normalized to the BHI control, which was set to 100 %. Data presented are the means±sd from three independent experiments. Asterisks indicate statistical significance.

Fig. 7.

Biofilm architecture of cultures grown with and without LPO. CLSM images of S. mutans, S. sanguinis and S. gordonii biofilms grown in the Lab-TekII Chamber Slide system. Cells were stained with the LIVE/DEAD viability fluorescent stain. Bar, 100 μm.

Fig. 8.

Feedback limitation of H₂O₂ production by pyruvate oxidase (POX)-catalysed oxidation of pyruvate (Pyr) through inhibition of the upstream glycolytic enzymes hexokinase (HK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) by host peroxidase (PO=hSPO and hMPO)-catalysed production of OSCN⁻. G6P, glucose 6-phosphate; GADP, glyceraldehyde 3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate.