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An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro.

ABSTRACT: The dpr gene is an antioxidant gene which was isolated from the Streptococcus mutans chromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant of Escherichia coli, and it was proven to play an indispensable role in oxygen tolerance in S. mutans. Here, we purified the 20-kDa dpr gene product, Dpr, from a crude extract of S. mutans as an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. Unlike E. coli Dps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 microM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein in S. mutans and may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.

SUBMITTER: Yamamoto Y

PROVIDER: S-EPMC135054 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro.

Yamamoto Yuji Y Poole Leslie B LB Hantgan Roy R RR Kamio Yoshiyuki Y

Journal of bacteriology 20020601 11

The dpr gene is an antioxidant gene which was isolated from the Streptococcus mutans chromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant of Escherichia coli, and it was proven to play an indispensable role in oxygen tolerance in S. mutans. Here, we purified the 20-kDa dpr gene product, Dpr, from a crude extract of S. mutans as an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr ...[more]

PMID: 12003933

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Project description:The Dps-like peroxide resistance protein (Dpr) is essential for H2O2 stress tolerance and aerobic growth of the oral pathogen Streptococcus mutans Dpr accumulates during oxidative stress, protecting the cell by sequestering iron ions and thereby preventing the generation of toxic hydroxyl radicals that result from the interaction of iron with H2O2 Previously, we reported that the SpxA1 and SpxA2 regulators positively regulate expression of dpr in S. mutans Using an antibody raised against S. mutans Dpr, we confirmed at the protein level the central and cooperative nature of SpxA1 and SpxA2 regulation in Dpr production. During phenotypic characterization of the S. mutans Δdpr strain, we observed the appearance of distinct colony variants, which sometimes lost the oxidative stress sensitivity typical of Δdpr strains. Whole-genome sequencing of these phenotypically distinct Δdpr isolates revealed that a putative iron transporter operon, smu995-smu998, was a genomic hot spot with multiple single nucleotide polymorphisms identified within the different isolates. Deletion of smu995 or the entire smu995-smu998 operon in the Δdpr background strain completely reversed the oxidative stress-sensitive phenotypes associated with dpr inactivation. Conversely, inactivation of genes encoding the ferrous iron transport system FeoABC did not alleviate phenotypes of the Δdpr strain. Preliminary characterization of strains lacking smu995-smu998, feoABC, and the iron/manganese transporter gene sloABC revealed the interactive nature of these three systems in iron transport but also indicated that there may be additional iron uptake systems in S. mutansIMPORTANCE The dental caries-associated pathogen Streptococcus mutans routinely encounters oxidative stress within the human plaque biofilm. Previous studies revealed that the iron-binding protein Dpr confers protection toward oxidative stress by limiting free iron availability, which is associated with the generation of toxic hydroxyl radicals. Here, we report the identification of spontaneously occurring mutations within Δdpr strains. Several of those mutations were mapped to the operon smu995-smu998, revealing a previously uncharacterized system that appears to be important in iron acquisition. Disruption of the smu995-smu998 operon resulted in reversion of the stress-sensitive phenotype typical of a Δdpr strain. Our data suggest that the Smu995-Smu998 system works along with other known metal transport systems of S. mutans, i.e., FeoABC and SloABC, to coordinate iron uptake.

Project description:Background: Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. mutans. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0.05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the yeast form of C. albicans and to inhibit extracellular polysaccharide production by S. mutans. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested.

Project description:Genetic studies have shown that variations in enamel formation genes are associated with caries susceptibility. The aim of this study was to test in vitro whether variants in these genes are associated with dental enamel demineralization in a Streptococcus mutans biofilm model. DNA and enamel samples were obtained from 213 individuals. DNA was extracted from saliva, and 16 single nucleotide polymorphisms were analyzed. The physical and chemical properties of sound enamel samples and the mineral loss and the lesion depth of the demineralized enamel samples under cariogenic challenge were analyzed. Microhardness, enamel chemicals, mineral loss and demineralization depth were compared between different genotypes at each single nucleotide polymorphism. The GG genotype of TUFT1 (rs17640579) and the GT genotype of MMP20 (rs1612069) exhibited increased microhardness (p = 0.044 and 0.016, respectively). The GG genotype of AMBN (rs7694409) had a higher magnesium level, while the CT genotype of TFIP11 (rs2097470) had a lower magnesium level (p = 0.044 and 0.046, respectively). The GT genotype of MMP20 (rs1612069) had a higher calcium level (p = 0.034). The GG genotype of AMBN (rs13115627), the AG genotype of ENAM (rs12640848) and the AA genotype of MMP20 (rs2292730) had a lower phosphorus level (p = 0.012, 0.006, and 0.023, respectively). The GG genotype of AMBN (rs13115627) was also associated with a higher calcium-phosphorus ratio (p = 0.034). Individuals with the CC genotype of TFIP11 (rs134143) exhibited significantly more mineral loss (p = 0.011) and a deeper lesions (p = 0.042). Individuals with the TT genotype of TFIP11 (rs2097470) had more mineral loss (p = 0.018). Individuals with the GG genotype of TUFT1 (rs17640579) exhibited a shallower demineralization depth (p = 0.047). Individuals with the GT genotype of MMP20 (rs1612069) exhibited a shallower demineralization depth (p = 0.042). Individuals with the GG genotype of ENAM (rs12640848) exhibited less mineral loss (p = 0.01) and a shallower demineralization depth (p = 0.03). Genetic variations in TFIP11, TUFT1, MMP20, and ENAM influenced enamel demineralization in a Streptococcus mutans biofilm model.

Project description:A study was made of the interaction of plasma ascorbate and ascorbate free radical (AFR) with exogenously added iron. The quantitative determination of AFR has the advantage that transient increases in ascorbate oxidation can be directly monitored by e.p.r. spectroscopy. An AFR signal was found in the plasma of all donors and was unaffected by superoxide dismutase, catalase and the strong iron chelator deferoxamine. These findings and the rapid decrease in AFR under a nitrogen atmosphere suggest that plasma AFR is probably a result of air auto-oxidation. Iron loading of plasma did not affect the intensity of the AFR signal until the iron concentration approached or exceeded the plasma latent iron-binding capacity. In iron-overloaded plasma, the intensity of the AFR signal increased to about 10 times the normal level before decreasing rapidly to undetectable levels after 15-20 min. Determination of plasma ascorbate showed that the disappearance of AFR was due to a complete loss of the vitamin. When 50 microM-ascorbate was loaded with iron in iso-osmotic phosphate buffer there was an increase in the AFR signal, independent of the iron concentration, which was stable at least for 15 min. Thus the rate of ascorbate loss in the iso-osmotic phosphate buffer was considerably lower than in iron-overloaded plasma. The addition of different iron chelators produced comparable effects on the intensity of the AFR signal in both iron-overloaded plasma and ascorbate solution. These results suggest that the characteristic behaviour of plasma AFR after iron loading is due to its specific iron-binding capacity and to plasma ferroxidase activity. The ferroxidase activity of plasma is important to promote the transfer of Fe2+ into transferrin without a transient ascorbate oxidation. Spin-trapping studies with 5,5-dimethyl-1-pyrroline N-oxide and N-t-butyl-alpha-phenylnitrone revealed that iron-overloaded plasma was unable to produce spin-trap adducts even in the presence of 50-300 microM-hydrogen peroxide or 100 microM-azide. Evidence of OH. radical formation was obtained only after the addition of EDTA. Therefore, iron-overloaded plasma itself does not produce a Fenton reaction and, if ascorbate does indeed have a free-radical-mediated pro-oxidant role, it is not detectable in plasma by spin-trapping experiments.

Dataset Information

An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro.

Publications

An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro.

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