Ratiometric fluorescent probe for sensing Streptococcus mutans glucosyltransferase , a key factor in the formation of dental caries

a College of Pharmacy, Academy of Integrative Medicine, The National & Local Joint Engineering Research Center for Drug Development of Neurodegenerative Disease, Dalian Medical University, Dalian 116044, People’s Republic of China b State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China c Chinese Acad Sci, Natl Engn Lab TCM Standardizat Technol, Shanghai Res Ctr Modernizat Tradit Chinese Med, Shanghai Inst Mat Med, Shanghai 201203, China d Department of Chemistry, University of Bath, Bath BA2 7AY, United Kingdom

Similarly, the influence of amino acids on the fluorescence intensities of PENA and PENA-G and the glucosylation capability of GTFs were studied in the presence of various amino acids, such as Ala, Pro, Iso, Phe, Met, Gln, Try, Glu, Asp.
In order to estimate the kinetic parameters of PENA for GTFs, the reaction kinetics were performed.Briefly, PENA (0−40 μM) was incubated with GTFs for 30 min which ensured less than 20% of substrate was metabolized, and the formation rates of PENA-G were in relation to incubation time and protein concentration in the linear range.The PENA stock solution was prepared to be 10 mM in DMSO, which was added into the GTFs enzymatic reaction system with DMSO < 1%.The apparent Km and Vmax values were calculated from nonlinear regression analysis of experimental data according to the Michaelis-Menten equation. v= The Vmax represents the maximum rate and Km is the substrate concentration at the half-maximal rate.Kinetic constants were obtained using Origin 7.5 (origin Lap Corp. Northampton.MA.USA) and produced as the mean ± SD of the parameter estimate.

Confocal laser microscopic imaging of bacteria.
In our work, PENA was incubated with seven bacteria strains, including

Isolated of GTFs inhibitors from green tea.
500 g green tea was extracted with 70% ethanol in water refluxed for 2h.Then, after the evaporation of solvents, the extract was subjected to Waters preparative S10 HPLC with RP C-18 column for the bioactive fractions preparation.The mobile phase (methanol-water) was assigned as gradient elution for the pre-HPLC.The gradient elution was determined as 10%-20% methanol (0-10 min); 20%-30% methanol (10-40 min); 30%-40% methanol (40-50 min); 40%-100% methanol (50-55 min).For the bioactive fractions with inhibitory effects on GTFs, the main chemical constituents were purified using pre-HPLC again.The structures of isolated compounds were determined on the basis of 1 H NMR, 13 C NMR and ESI-MS analysis in combination with the literatures.The spectroscopic data of isolated compounds were submitted as followed.
Streptococcus mutans, Hemolytic streptococcus, Lactobacillus amylovorus, Streptococcus pasteurianus strain 080205, Bacillus cereus 994000168 LBK, Escherichia coli DH5alpha BRL, Staphylococcus aureus ssp.aureus DSM 3463, to get the fluorescence images by confocal laser scanning microscope.These bacteria were culutred in Luria-Bertani (LB) medium for 24 h to get enuogh bacteria cells with OD value 0.8.Then, PENA (50 μM) was subjected into the culture for a co-incubation with bacteria about 1h at 37 ºC.After the clean out of the medium, the bacterial cells were suspended in PBS solution, which were dropped on slide glasses for imaging experiment.The bacteria were imaged using Confocal Microscope with λex 405/λem 415 -465 nm, λex 405/λem 535 -585 nm.To study the enzyme specificity, the bacteria were pretreated with enzyme inhibitor EGCG (50 μM) and labeled with PENA for imaging studies.

Fig. S7 .
Fig. S7.The conversion rate of PENA glucosylation by GTFs in various PBS solutions with different pH conditions.

Fig. S10 .
Fig. S10.Fluorescence images of various bacteria blank control groups without

Fig. S12 .
Fig. S12.Dose-dependent inhibitory effect of CA on the glucosylation of PENA by GTFs.

Fig. S17 .
Fig. S17.Dose-dependent inhibitory effect of GC on the glucosylation of PENA by GTFs.

Fig. S21 .
Fig. S21.Inhibition kinetics of GCG on the glucsoylation of PENA mediated by GTFs.(a,b) Lineweaver-Burk plot of GCG's inhibition towards the activity of GTFs; (c) Dixon plot of GCG's inhibition towards the activity of GTFs; (d) determination of inhibition kinetic parameter (Ki) using the slopes from Lineweaver-Burk plot towards the concentration of GCG.The data points represent the mean value of duplicateexperiments.

Fig. S22 .
Fig. S22.Inhibition kinetics of EGCG on the glucsoylation of PENA mediated by GTFs.(a,b) Lineweaver-Burk plot of EGCG's inhibition towards the activity of GTFs; (c) Dixon plot of EGCG's inhibition towards the activity of GTFs; (d) determination of inhibition kinetic parameter (Ki) using the slopes from Lineweaver-Burk plot towards the concentration of EGCG.The data points represent the mean value of duplicate experiments.