Dataset Information

Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans.

ABSTRACT: The enzyme alanine racemase (Alr) has been a new target for the development of antibacterial drugs based on the involvement of D-Ala in bacterial cell wall biosynthesis. Our previous study noted that Alr is essential for the growth and interspecies competitiveness of S. mutans, the major causative organism of dental caries. However, physiological activity and cariogenicity of S. mutans affected by Alr remains unknown. The current study examined the biofilm biomass, biofilm structure, extracellular polysaccharide (EPS) synthesis, glucosyltransferase (gtf) gene expression, acid production and acid tolerance in the alr-mutant strain. We found that biofilm formation, biofilm structure, and EPS synthesis was in a D-Ala dose-dependent manner. Biofilm structure was loose in alr-mutant group and the ratio of EPS/bacteria was also elevated. Additionally, the expression levels of multiple gtfs were up-regulated, and acid tolerance was decreased. We also established in vivo models of dental caries and found that the incidence and severity of the caries were decreased in the alr-mutant group in comparison to the parental S. mutans group. Our in vivo and in vitro experiments demonstrate that Alr is essential for the cariogenicity of S. mutans and that Alr might be a potential target for the prevention and treatment of caries.

SUBMITTER: Liu S

PROVIDER: S-EPMC5899142 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans.

Liu Shiyu S Wei Yuan Y Zhou Xuedong X Zhang Keke K Peng Xian X Ren Biao B Chen Vivian V Cheng Lei L Li Mingyun M

Scientific reports 20180413 1

The enzyme alanine racemase (Alr) has been a new target for the development of antibacterial drugs based on the involvement of D-Ala in bacterial cell wall biosynthesis. Our previous study noted that Alr is essential for the growth and interspecies competitiveness of S. mutans, the major causative organism of dental caries. However, physiological activity and cariogenicity of S. mutans affected by Alr remains unknown. The current study examined the biofilm biomass, biofilm structure, extracellul ...[more]

PMID: 29654290

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Project description:Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

Project description:Radiation caries is one of the most common complications of head and neck radiotherapy. A shift in the oral microbiota is the main factor of radiation caries. A new form of biosafe radiation, heavy ion radiation, is increasingly being applied in clinical treatment due to its superior depth-dose distribution and biological effects. However, how heavy ion radiation directly impacts the oral microbiota and the progress of radiation caries are unknown. Here, unstimulated saliva samples from both healthy and caries volunteers and caries-related bacteria were directly exposed to therapeutic doses of heavy ion radiation to determine the effects of radiation on oral microbiota composition and bacterial cariogenicity. Heavy ion radiation significantly decreased the richness and diversity of oral microbiota from both healthy and caries volunteers, and a higher percentage of Streptococcus was detected in radiation groups. In addition, heavy ion radiation significantly enhanced the cariogenicity of saliva-derived biofilms, including the ratios of the genus Streptococcus and biofilm formation. In the Streptococcus mutans-Streptococcus sanguinis dual-species biofilms, heavy ion radiation increased the ratio of S. mutans. Next, S. mutans was directly exposed to heavy ions, and the radiation significantly upregulated the gtfC and gtfD cariogenic virulence genes to enhance the biofilm formation and exopolysaccharides synthesis of S. mutans. Our study demonstrated, for the first time, that direct exposure to heavy ion radiation can disrupt the oral microbial diversity and balance of dual-species biofilms by increasing the virulence of S. mutans, increasing its cariogenicity, indicating a potential correlation between heavy ions and radiation caries. IMPORTANCE The oral microbiome is crucial to understanding the pathogenesis of radiation caries. Although heavy ion radiation has been used to treat head and neck cancers in some proton therapy centers, its correlation with dental caries, especially its direct effects on the oral microbiome and cariogenic pathogens, has not been reported previously. Here, we showed that the heavy ion radiation directly shifted the oral microbiota from a balanced state to a caries-associated state by increasing the cariogenic virulence of S. mutans. Our study highlighted the direct effect of heavy ion radiation on oral microbiota and the cariogenicity of oral microbes for the first time.

Project description:BackgroundGlutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome.MethodsStructure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS-PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures.ResultsThe glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (K m, d-Glu?l-Glu = 0.3631 ± 0.3205 mM, V max, d-Glu?l-Glu = 0.1963 ± 0.0361 mM min-1, k cat, d-Glu?l-Glu = 0.0306 ± 0.0065 s-1, k cat/K m, d-Glu?l-Glu = 0.0844 ± 0.0128 s-1 mM-1, with d-glutamate as substrate; K m, l-Glu?d-Glu = 0.8077 ± 0.5081 mM, V max, l-Glu?d-Glu = 0.2421 ± 0.0418 mM min-1, k cat , l - Glu?d-Glu = 0.0378 ± 0.0056 s-1, k cat/K m, l-Glu?d-Glu = 0.0468 ± 0.0176 s-1 mM-1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0.ConclusionThe findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

Dataset Information

Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans.

Publications

Function of alanine racemase in the physiological activity and cariogenicity of Streptococcus mutans.

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