Huichun Tong

Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition.

The oral microbial flora contains over 500 different microbial species that often interact as a means to compete for limited space and nutritional resources. Streptococcus mutans, a major caries‐causing pathogen, is a species which tends to interact competitively with other species in the oral cavity, largely due to its ability to generate copious quantities of the toxic metabolite, lactic acid. However, during a recent clinical study, we discovered a novel oral streptococcal species, Streptococcus oligofermentans, whose abundance appeared to be inversely correlated with that of S. mutans within dental plaque samples and thus suggested a possible antagonistic relationship with S. mutans. In this study, we used a defined in vitro interspecies interaction assay to confirm that S. oligofermentans was indeed able to inhibit the growth of S. mutans. Interestingly, this inhibitory effect was relatively specific to S. mutans and was actually enhanced by the presence of lactic acid. Biochemical analyses revealed that S. oligofermentans inhibited the growth of S. mutans via the production of hydrogen peroxide with lactic acid as the substrate. Further genetic and molecular analysis led to the discovery of the lactate oxidase (lox) gene of S. oligofermentans as responsible for this biological activity. Consequently, the lox mutant of S. oligofermentans also showed dramatically reduced inhibitory effects against S. mutans and also exhibited greatly impaired growth in the presence of the lactate produced by S. mutans. These data indicate that S. oligofermentans possesses the capacity to convert its competitors main ‘weapon’ (lactic acid) into an inhibitory chemical (H2O2) in order to gain a competitive growth advantage. This fascinating ability may be an example of a counteroffensive strategy used during chemical warfare within the oral microbial community.

SO-LAAO, a Novel l-Amino Acid Oxidase That Enables Streptococcus oligofermentans To Outcompete Streptococcus mutans by Generating H2O2 from Peptone

We previously demonstrated that Streptococcus oligofermentans suppressed the growth of Streptococcus mutans, the primary cariogenic pathogen, by producing hydrogen peroxide (H(2)O(2)) through lactate oxidase activity. In this study, we found that the lox mutant of S. oligofermentans regained the inhibition while growing on peptone-rich plates. Further studies demonstrated that the H(2)O(2) produced on peptone by S. oligofermentans was mainly derived from seven L-amino acids, i.e., L-aspartic acid, L-tryptophan, L-lysine, L-isoleucine, L-arginine, L-asparagine, and L-glutamine, indicating the possible existence of L-amino acid oxidase (LAAO) that can produce H(2)O(2) from L-amino acids. Through searching the S. oligofermentans genome for open reading frames with a conserved flavin adenine dinucleotide binding motif that exists in the known LAAOs, including those of snake venom, fungi, and bacteria, a putative LAAO gene, assigned as aao(So), was cloned and overexpressed in Escherichia coli. The purified protein, SO-LAAO, showed a molecular mass of 43 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and catalyzed H(2)O(2) formation from the seven L-amino acids determined above, thus confirming its LAAO activity. The SO-LAAO identified in S. oligofermentans differed evidently from the known LAAOs in both substrate profile and sequence, suggesting that it could represent a novel LAAO. An aao(So) mutant of S. oligofermentans did lose H(2)O(2) formation from the seven L-amino acids, further verifying its function as an LAAO. Furthermore, the inhibition by S. oligofermentans of S. mutans in a peptone-rich mixed-species biofilm was greatly reduced for the aao(So) mutant, indicating the genes importance in interspecies competition.

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 H2O2 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 H2O2 in the earlier growth phase and log phase, while Lox mainly contributed to H2O2 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 H2O2 can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H2O2 production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. 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 H2O2 formation so as to win the interspecies competition.

PerR-regulated manganese ion uptake contributes to oxidative stress defense in an oral streptococcus

ABSTRACT Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H2O2, whereas Dpr (an Fe2+-chelating protein)-dependent H2O2 protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H2O2-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl2, S. oligofermentans increased survival after H2O2 stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn2+-mediated H2O2 resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ∼0.4 μM Mn2+) unless it was supplemented with ≥2.5 μM MnCl2 and caused 82 to 95% reduction of the cellular Mn2+ level, while mntABC overexpression increased cellular Mn2+ 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn2+ transporter in S. oligofermentans. mntA mutation reduced the survival of H2O2-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H2O2-challenged survival 12-fold, indicating that MntABC-mediated Mn2+ uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H2O2 pulsing upregulated mntABC, while H2O2-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H2O2 can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H2O2 stress defenses and may be distributed across all oral streptococci lacking catalase.

A Preliminary Study of Biological Characteristics of Streptococcus oligofermentans in Oral Microecology

This study was designed to explore the biological characteristics of a new oral streptococcus species Streptococcus oligofermentans. Plaque samples were collected from caries-free and caries-active subjects. S. oligofermentans was selectively grown on Mitis salivarius agar plates and identified by using 2-step PCR and was isolated from 38% of 18 subjects. Isolates were found mostly on healthy tooth surfaces. S. oligofermentans had weaker abilities in acid production and demineralizing hydroxyapatite. When Streptococcus mutans was coincubated with S. oligofermentans, total number, acid production and calcium release were significantly inhibited. In conclusion, inhibition of S. mutans could be a beneficial biological characteristic of S. oligofermentans in oral microecology.

Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pH-cycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.

Complete Genome Sequence of an Oral Commensal, Streptococcus oligofermentans Strain AS 1.3089

ABSTRACT Streptococcus oligofermentans, an oral commensal, inhibits the growth of the dental caries pathogen Streptococcus mutans by producing large amounts of hydrogen peroxide. Therefore, it can be a potential probiotic for oral health. Here we report the complete genome sequence of S. oligofermentans strain AS 1.3089.

Role of operon aaoSo-mutT in antioxidant defense in Streptococcus oligofermentans.

Previously, we have found that an insertional inactivation of aaoSo, a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aaoSo and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aaoSo and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aaoSo insertional mutant, indicating that gene polarity derived from the inactivation of aaoSo attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H2O2 (min·mg protein)–1]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aaoSo mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant’s growth by 11%, indicating that the aaoSo mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aaoSo. Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aaoSo mutant. This demonstrated that inactivation of aaoSo caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aaoSo-mutT operon in antioxidant defense in S. oligofermentans.

CcpA-Dependent Carbohydrate Catabolite Repression Regulates Galactose Metabolism in Streptococcus oligofermentans

Streptococcus oligofermentans is an oral commensal that inhibits the growth of the caries pathogen Streptococcus mutans by producing copious amounts of H(2)O(2) and that grows faster than S. mutans on galactose. In this study, we identified a novel eight-gene galactose (gal) operon in S. oligofermentans that was comprised of lacABCD, lacX, and three genes encoding a galactose-specific transporter. Disruption of lacA caused more growth reduction on galactose than mutation of galK, a gene in the Leloir pathway, indicating that the principal role of this operon is in galactose metabolism. Diauxic growth was observed in cultures containing glucose and galactose, and a luciferase reporter fusion to the putative gal promoter demonstrated 12-fold repression of the operon expression by glucose but was induced by galactose, suggesting a carbon catabolite repression (CCR) control in galactose utilization. Interestingly, none of the single-gene mutations in the well-known CCR regulators ccpA and manL affected diauxic growth, although the operon expression was upregulated in these mutants in glucose. A double mutation of ccpA and manL eliminated glucose repression of galactose utilization, suggesting that these genes have parallel functions in regulating gal operon expression and mediating CCR. Electrophoretic mobility shift assays demonstrated binding of CcpA to the putative catabolite response element motif in the promoter regions of the gal operon and manL, suggesting that CcpA regulates CCR through direct regulation of the transcription of the gal operon and manL. This provides the first example of oral streptococci using two parallel CcpA-dependent CCR pathways in controlling carbohydrate metabolism.

Molecular Insights into Hydrogen Peroxide Sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses

Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn2+ content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn2+ and H2O2 sensors in mediating H2O2 resistance by an oral streptococcus. H2O2 disrupted So-MntR binding to Mn2+ transporter mntABC promoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in H2O2-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished H2O2-induced disulfide-linked dimers and weakened H2O2 damage on So-MntR binding, indicating that H2O2 inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to H2O2 oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon H2O2 pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in H2O2-pulsed cells, confirming Cys-11 and Cys-156 contributed to H2O2-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of mntABC and cellular Mn2+ decreased, but H2O2 susceptibility increased. In the C123S mutant, increased mntABC expression, cellular Mn2+ content, and manganese-mediated H2O2 survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.