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author:("Shi, wenchuan")
1.  Evaluation of bacteria-induced enamel demineralization using optical profilometry 
Objectives
Streptococcus mutans is considered a major causative of tooth decay due to it’s ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.
Methods
Optical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.
Results
After 72 hr of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average, (Ra), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, RRMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.
Significance
The strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.
doi:10.1016/j.dental.2009.07.012
PMCID: PMC3454478  PMID: 19732947
enamel erosion; optical profilometry; biofilm; Streptococcus mutans; enamel demineralization; citric acid; lactic acid; AFM
2.  Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation 
Journal of Bacteriology  2012;194(5):1127-1135.
We have previously characterized the interactions of the response regulator ComE from Streptococcus mutans and DNA binding sites through DNase I footprinting and electrophoretic mobility shift assay analysis. Since response regulator functions are often affected by their phosphorylation state, we investigated how phosphorylation affects the biochemical function of ComE. Unlike many response regulators, we found that the phosphorylation state of ComE does not likely play a role in DNA binding affinity but rather seems to induce the formation of an oligomeric form of the protein. The role of this oligomerization state for ComE function is discussed.
doi:10.1128/JB.06565-11
PMCID: PMC3294772  PMID: 22210762
3.  Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans▿† 
Journal of Bacteriology  2011;193(14):3642-3652.
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.
doi:10.1128/JB.00155-11
PMCID: PMC3133340  PMID: 21602345
4.  Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans▿  
Previously we reported a novel strategy of “targeted killing” through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) (R. Eckert et al., Antimicrob. Agents Chemother. 50:3651-3657, 2006; R. Eckert et al., Antimicrob. Agents Chemother. 50:1480-1488, 2006). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable “building-block” approach.
doi:10.1128/AAC.01391-09
PMCID: PMC2863653  PMID: 20211885
5.  The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis 
Microbiology (Reading, England)  2007;153(Pt 6):1799-1807.
In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC–comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.
doi:10.1099/mic.0.2007/005975-0
PMCID: PMC2062498  PMID: 17526837
6.  Coordinated bacteriocin production and competence development: a possible mechanism for taking up DNA from neighbouring species 
Molecular microbiology  2005;57(2):392-404.
It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced coordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral bio-film, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.
doi:10.1111/j.1365-2958.2005.04695.x
PMCID: PMC1262684  PMID: 15978073
7.  Competition and Coexistence between Streptococcus mutans and Streptococcus sanguinis in the Dental Biofilm 
Journal of Bacteriology  2005;187(21):7193-7203.
The human mucosal surface is colonized by the indigenous microflora, which normally maintains an ecological balance among different species. Certain environmental or biological factors, however, may trigger disruption of this balance, leading to microbial diseases. In this study, we used two oral bacterial species, Streptococcus mutans and Streptococcus sanguinis (formerly S. sanguis), as a model to probe the possible mechanisms of competition/coexistence between different species which occupy the same ecological niche. We show that the two species engage in a multitude of antagonistic interactions temporally and spatially; occupation of a niche by one species precludes colonization by the other, while simultaneous colonization by both species results in coexistence. Environmental conditions, such as cell density, nutritional availability, and pH, play important roles in determining the outcome of these interactions. Genetic and biochemical analyses reveal that these interspecies interactions are possibly mediated through a well-regulated production of chemicals, such as bacteriocins (produced by S. mutans) and hydrogen peroxide (produced by S. sanguinis). Consistent with the phenotypic characteristics, production of bacteriocins and H2O2 are regulated by environmental conditions, as well as by juxtaposition of the two species. These sophisticated interspecies interactions could play an essential part in balancing competition/coexistence within multispecies microbial communities.
doi:10.1128/JB.187.21.7193-7203.2005
PMCID: PMC1272965  PMID: 16237003

Results 1-7 (7)