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A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

ABSTRACT: Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans within a seven species panel. This phenotype was common amongst all assayed isolates of S. mutans , but was specific to a single strain of S. mitis , ATCC 49456. The growth inhibitory factor was not effectively carried in spent supernatants of S. mitis . However, we documented ATCC 49456 to accumulate 4-5 times more hydrogen peroxide (H ₂ O ₂ ) than other species tested, and 5-18 times more than other S. mitis strains assayed. The S. mutans biofilm formation inhibitory phenotype was reduced when grown in media containing catalase or with a S. mitis mutant of pyruvate oxidase ( spxB ; pox ), confirming that SpxB-dependent H ₂ O ₂ production was the main antagonistic factor. Addition of S. mitis within hours after S. mutans inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms. Transcriptome analysis revealed responses for both S. mitis and S. mutans , with several S. mutans differentially expressed genes following a gene expression pattern previously described, while others being unique to the interaction with S. mitis . Finally, we show that S. mitis also affected coculture biofilm formation of several other commensal streptococci. Our study shows that strains with abundant H ₂ O ₂ production are effective at inhibiting initial growth of caries pathogens like S. mutans , but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.

SUBMITTER: Williams I

PROVIDER: S-EPMC11326308 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

Williams Isabella I Tuckerman Jacob S JS Peters Daniel I DI Bangs Madisen M Williams Emily E Shin Iris J IJ Kaspar Justin R JR

bioRxiv : the preprint server for biology 20240806

Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans within a seven species panel. This phenotype was common amongst all assayed isolates of S. mutans, but was specific to a single strain of S. mitis, ATCC 49456. The growth inhibitory factor was not effectively carried in spent superna ...[more]

PMID: 39149263

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Project description:Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans compared to other oral streptococci. This phenotype was common among all isolates of S. mutans, but was specific to a single strain of S. mitis, ATCC 49456. We documented ATCC 49456 to accumulate four to five times more hydrogen peroxide (H2O2) than other Streptococcus species tested, and 5-18 times more than other S. mitis strains assayed. S. mutans biofilm formation inhibition was dependent on cell contact/proximity and reduced when grown in media containing catalase or with a S. mitis mutant of pyruvate oxidase (spxB; pox), confirming that SpxB-dependent H2O2 production was a major antagonistic factor. Addition of S. mitis within hours after S. mutans inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms in an SpxB-dependent manner. Transcriptome analysis revealed responses for both S. mitis and S. mutans, with several S. mutans differentially expressed genes following a gene expression pattern we have previously described, while others being unique to the interaction with S. mitis. Finally, we show that S. mitis also affected coculture biofilm formation of several other commensal streptococci as well as cariogenic Streptococcus sobrinus. Our study shows that strains with abundant H2O2 production are effective at inhibiting initial growth of caries pathogens like S. mutans, but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.IMPORTANCEAntagonistic properties displayed by oral bacteria have been sought as therapeutic approaches against dental caries pathogens like Streptococcus mutans. An emergent theme has been the ability of select strains that produce high amounts of hydrogen peroxide to effectively inhibit the growth of S. mutans within in vitro and in vivo models. Our study builds on these previous findings by determining that Streptococcus mitis ATCC 49456 is a high hydrogen peroxide producer, compared to other Streptococcus species as well as additional strains of S. mitis. In addition to S. mutans, we show that ATCC 49456 also affects biofilm formation of other oral streptococci, a non-desirable trait that should be weighed heavily for strains under consideration as probiotics. Further phenotypic characterization of strains like S. mitis ATCC 49456 in mixed-species settings will allow us to hone in on qualities that are optimal for probiotic strains that are intended to prevent the emergence of odontopathogens.

Project description:BackgroundIt is cleared that some probiotic strains inhibit biofilm formation of oral bacteria, but its mechanisms are not clearly understood yet. It is proposed that one of the mechanisms can be biosurfactant production, a structurally diverse group of surface-active compounds synthesized by microorganisms. Hence, this study focused on the evaluation of the anti-biofilm and antiadhesive activities of the L. rhamnosus derived-biosurfactant against Streptococcus mutans and its effect on gtfB/C and ftf genes expression level.Materials and methodsIn this in vitro study Lactobacillus rhamnosus ATCC7469 overnight culture was used for biosurfactant production. The biosurfactant effect on the surface tension reduction was confirmed by drop collapse method. Chemical bonds in the biosurfactant were identified by Fourier transform infrared (FTIR). Anti-biofilm and antiadhesive activities of the biosurfactant were determined on glass slides and in 96-well culture plates, respectively. The effect of the biosurfactant on gtfB/C and ftf genes expression level was also investigated after biofilm formation, total RNA extraction, and reverse transcription by quantitative real-time reverse transcriptase polymerase chain reaction (PCR) assay (quantitative PCR). The data were assessed by one-way analysis of variance in the Tukey-Kramer postdeviation test for all pairs. P < 0.05 was considered statistically significant.ResultsThe FTIR results of biosurfactant showed that it was protein rich. It also showed anti-biofilm formation activity on the glass slide and antiadhesive activity till 40% on microtiter plate wells. It also showed a significant reduction (P < 0.05) in gtfB/C and ftf genes expression level.ConclusionL. rhamnosus-derived biosurfactant exhibits a significant inhibitory effect on biofilm formation ability of S. mutans due to downregulation of biofilm formation associated genes, gtfB/C and ftf. L. rhamnosus-derived biosurfactant with substantial antiadhesive activity is suitable candidates for use in new generations of microbial antiadhesive agents.

Project description:Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2 However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments.IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2 This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

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A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

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A Strain of <i>Streptococcus mitis</i> Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

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A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

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A Strain of &lt;i&gt;Streptococcus mitis&lt;/i&gt; Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

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A Strain of <i>Streptococcus mitis</i> Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.