Function of the pyruvate oxidase-lactate oxidase cascade in interspecies competition between Streptococcus oligofermentans and Streptococcus mutans

Abstract

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H(2)O(2) production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H(2)O(2) in the earlier growth phase and log phase, while Lox mainly contributed to H(2)O(2) production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H(2)O(2) can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H(2)O(2) production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H(2)O(2). In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H(2)O(2) formation so as to win the interspecies competition.

Fig 1

Hydrogen peroxide production by various S. oligofermentans strains growing in BHI cultures. Overnight cultures of S. oligofermentans wild type and various mutant strains were diluted at a 1:40 ratio into fresh BHI medium. Subsequently, cultures were sampled every hour and then measured for optical density at 600 nm and H₂O₂ production upon shaking at 200 rpm/min for 20 min. (A) Growth curve of various S. oligofermentans strains expressed as OD₆₀₀. (B) Hydrogen peroxide production throughout the growth phase. Data are expressed as H₂O₂ concentration (mM) per optical density at 600 nm. ♦, wild type; ▵, lox mutant; □, pox mutant; ●, pox lox double mutant. Data are representative of three independent experiments.

Fig 2

Expression profiles and enzymatic activities of pyruvate oxidase and lactate oxidase in S. oligofermentans. Cells were collected at different growth phases and then assayed for luciferase, pyruvate oxidase, and lactate oxidase activities as described in Materials and Methods. (A) Luciferase activities of Ppox-luc and Plox-luc fusions throughout the growth phase. Data are expressed as the relative light units (RLU) per optical density at 600 nm. □, Ppox-luc; ♦, Plox-luc. (B) Pyruvate oxidase (□) and lactate oxidase (♦) activities. Results are expressed as the means ± standard deviations of three independent experiments.

Fig 3

Time course of lactate production by S. oligofermentans. Overnight cultures of S. oligofermentans were diluted at a 1:40 ratio into fresh BHI medium and incubated as a static culture; samples were taken every hour to measure optical density at 600 nm and lactate concentration. ●, growth expressed as OD₆₀₀; □, lactate concentration (mM). Results are expressed as means ± standard deviations of three independent experiments.

Fig 4

Lactate oxidase activities of S. oligofermentans wild type and pox and lox mutants. Lactate concentration (A) and H₂O₂ production (B) were measured in the stationary-phase cultures before (black bar) and after (gray bar) shaking at 200 rpm/min for 20 min. Data are expressed as the means ± standard deviations of three independent experiments. ∗∗∗, data are statistically significant in comparison to values before shaking, as verified by Student's t test (P < 0.05).

Fig 5

pox and lox mutant strains of S. oligofermentans lost antagonism against S. mutans in the two-species biofilms formed in BHI-sucrose. Overnight cultures of various S. oligofermentans strains and S. mutans UA140 were adjusted to the same optical density at 600 nm (∼1.0) and then were inoculated (1:10) into a 12-well plate containing fresh BHI broth supplemented with 0.5% sucrose to form mono- or mixed-species biofilms. After 8 h of incubation, the biofilms were harvested and cell numbers enumerated by plating. (A) CFU of S. mutans in mixed cultures with various S. oligofermentans strains (bars 1 to 4) and in a monoculture (bar 5). Bars: 1, with wild type; 2, with pox mutant; 3, with lox mutant; 4, with pox lox double mutant. (B) CFU of various S. oligofermentans strains in mixed cultures with S. mutans (black bar) and in monocultures (gray bar). Data are expressed as the means ± standard deviations of three independent experiments.