iTRAQ-based quantitative analysis reveals the mechanism underlying the changes in physiological activity in a glutamate racemase mutant strain of Streptococcus mutans UA159

2020 ◽  
Vol 47 (5) ◽  
pp. 3719-3733
Author(s):  
Jia-cheng Lin ◽  
Xiang-zhu Wang ◽  
Ting Shen ◽  
Jian-ying Zhang
Keyword(s):  
Quantitative Analysis ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Physiological Activity ◽  
Glutamate Racemase

scholarly journals Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8300
Author(s):  
Xiangzhu Wang ◽  
Chanchan Chen ◽  
Ting Shen ◽  
Jiangying Zhang
Keyword(s):  
Kinetic Parameters ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Structure Characterization ◽  
Multiple Sequence ◽  
Chromatography Method ◽  
Native Page ◽  
Aggregation State ◽  
Glutamate Racemase

Background Glutamate 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. Methods Structure 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. Results The 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 (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, 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. Conclusion The 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.

scholarly journals Correlation among mutans streptococci counts, dental caries, and IgA to Streptococcus mutans in saliva

2004 ◽  
Vol 18 (4) ◽  
pp. 350-355 ◽  
Author(s):  
Cristiane Yumi Koga-Ito ◽  
Clélia Aparecida de Paiva Martins ◽  
Ivan Balducci ◽  
Antonio Olavo Cardoso Jorge
Keyword(s):  
Young Adults ◽  
Quantitative Analysis ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Salivary Flow ◽  
Mutans Streptococci ◽  
Who Criteria ◽  
Aerobic Flora ◽  
Free Status ◽  
Rampant Caries

Two-hundred and forty individuals were studied, divided into five groups as follows: caries-free children, children with caries, children with rampant caries, young adults with and without caries. Whole stimulated saliva was collected and all individuals were investigated for DMFT/dmft according to the WHO criteria and the simplified oral hygiene index (OHI-S). Quantitative analysis of the total aerobic flora and mutans streptococci in saliva was performed. Also, the level of salivary anti-S. mutans IgA was determined by ELISA. Children with rampant caries showed the highest OHI-S value. The highest total counts of microorganisms were found in the group of children with caries. No statistically significant differences were observed for salivary flow, OHI-S and microorganism counts between the groups of young adults. No correlation between mutans streptococci counts and anti-Streptococcus mutans IgA levels was observed in the studied groups. A correlation between increased anti-Streptococcus mutans IgA levels and caries-free status was observed among young adults but not among children.

Role of the luxS Gene in Initial Biofilm Formation by Streptococcus mutans

2015 ◽  
Vol 25 (1) ◽  
pp. 60-68 ◽  
Author(s):  
Zhiyan He ◽  
Jingping Liang ◽  
Zisheng Tang ◽  
Rui Ma ◽  
Huasong Peng ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Type Strain ◽  
Laser Scanning ◽  
Wild Type Strain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Initial Biofilm

Quorum sensing (QS) is a process by which bacteria communicate with each other by secreting chemical signals called autoinducers (AIs). Among Gram-negative and Gram-positive bacteria, AI-2 synthesized by the LuxS enzyme is widespread. The aim of this study was to evaluate the effect of QS <i>luxS</i> gene on initial biofilm formation by <i>Streptococcus mutans</i>. The bacterial cell surface properties, including cell hydrophobicity (bacterial adherence to hydrocarbons) and aggregation, which are important for initial adherence during biofilm development, were investigated. The biofilm adhesion assay was evaluated by the MTT method. The structures of the 5-hour biofilms were observed by using confocal laser scanning microscopy, and QS-related gene expressions were investigated by real-time PCR. The <i>luxS</i> mutant strain exhibited higher biofilm adherence and aggregation, but lower hydrophobicity than the wild-type strain. The confocal laser scanning microscopy images revealed that the wild-type strain tended to form smaller aggregates with uniform distribution, whereas the <i>luxS</i> mutant strain aggregated into distinct clusters easily discernible in the generated biofilm. Most of the genes examined were downregulated in the biofilms formed by the <i>luxS</i> mutant strain, except the <i>gtfB </i>gene. QS <i>luxS</i> gene can affect the initial biofilm formation by <i>S. mutans.</i>

scholarly journals Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

1998 ◽  
Vol 66 (5) ◽  
pp. 2180-2185 ◽  
Author(s):  
Karsten R. O. Hazlett ◽  
Suzanne M. Michalek ◽  
Jeffrey A. Banas
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Rat Model ◽  
Binding Protein ◽  
Protein A ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT Glucan-binding protein A (GbpA) of Streptococcus mutanshas been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenicgbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutanswas grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. ThegbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB andgtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure.

scholarly journals RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
C. J. Kovacs ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Critical Function ◽  
Content Type ◽  
The Impact

ABSTRACT Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans. IMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

scholarly journals Quantitative Correlation of Salivary Streptococcus Mutans Count Amongst Siblings and their Mothers

2017 ◽  
Vol 05 (02) ◽  
pp. 090-096
Author(s):  
Paramdeep Singh ◽  
Avninder Kaur ◽  
Neerja Kakkar ◽  
Manjeet Kaur ◽  
Shivesh Acharya
Keyword(s):  
Quantitative Analysis ◽  
Streptococcus Mutans ◽  
Positive Association ◽  
Older Children ◽  
Significant Positive Association ◽  
Plaque Score ◽  
Quantitative Correlation ◽  
The Family ◽  
Related Individuals ◽  
Highly Correlated

Abstract Aim: The present study was planned to analyze quantitative correlation of salivary Streptococcus Mutans (S. Mutans) in siblings and their mothers. Materials and Methods: Quantitative analysis of S. Mutans in saliva was performed using Dentocult SM strip mutans kit (Orion Diagnostica, Helsinki, Finland) in closely related members of the family i.e. siblings along with their mothers. Results: S. Mutans count between the siblings showed positive correlation which was statistically highly significant. The younger childrens’ S. Mutans count was very highly correlated (rs = 0.711) with the mothers’ as compared to that of the older children (rs = 0.412). The S. Mutans count was found to be associated with caries score and was statistically significant. A statistically highly significant positive association was also found with the plaque score. Conclusion: The correlation between the S. Mutans count of related individuals has been reaffirmed. S. Mutans is positively associated with dental caries.

scholarly journals Expression of Pyridoxal 5′-Phosphate-Independent Racemases Can Reduce 2-Aminoacrylate Stress inSalmonella enterica

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Kelsey M. Hodge-Hanson ◽  
Allison Zoino ◽  
Diana M. Downs
Keyword(s):  
Mutant Strain ◽  
Metabolic Stress ◽  
Reactive Metabolites ◽  
Methanococcus Maripaludis ◽  
Content Type ◽  
Direct Quenching ◽  
Glutamate Racemase ◽  
Independent Mechanism ◽  
Domains Of Life

ABSTRACTThe RidA protein (PF01042) fromSalmonella entericais a deaminase that quenches 2-aminoacrylate (2AA) and other reactive metabolites. In the absence of RidA, 2AA accumulates, damages cellular enzymes, and compromises the metabolic network.In vitro, RidA homologs from all domains of life deaminate 2AA, and RidA proteins from plants, bacteria, yeast, and humans complement the mutant phenotype of aridAmutant strain ofS. enterica. In the present study, a methanogenic archaeon,Methanococcus maripaludisS2, was used to probe alternative mechanisms to restore metabolic balance.M. maripaludisMMP0739, which is annotated as an aspartate/glutamate racemase, complemented aridAmutant strain and reduced the intracellular 2AA burden. The aspartate/glutamate racemase YgeA fromEscherichia coliorS. enterica, when provided intrans, similarly restored wild-type growth to aridAmutant. These results uncovered a new mechanism to ameliorate metabolic stress, and they suggest that direct quenching by RidA is not the only strategy to quench 2AA.IMPORTANCE2-Aminoacrylate is an endogenously generated reactive metabolite that can damage cellular enzymes if not directly quenched by the conserved deaminase RidA. This study used an archaeon to identify a RidA-independent mechanism to prevent metabolic stress caused by 2AA. The data suggest that a gene product annotated as an aspartate/glutamate racemase (MMP0739) produces a metabolite that can quench 2AA, expanding our understanding of strategies available to quench reactive metabolites.

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