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−	<p><b>Figure 3.</b> Dose-dependent and time-dependent lytic activity of ClyR. A. Variation curve of OD<sub>600</sub> of <i>S. mutans</i> UA159 treated with different ClyR concentrations. B. Decrease in OD<sub>600</sub> of <i>S. mutans</i> UA159 treated with different concentrations ClyR. The error bars indicate standard error (SEM) of three independent replications.</p>	+	<p><b>Figure 3.</b> Dose-dependent and time-dependent lytic activity of ClyR. <b>A.</b> Variation curve of OD<sub>600</sub> of <i>S. mutans</i> UA159 treated with different ClyR concentrations. <b>B.</b> Decrease in OD<sub>600</sub> of <i>S. mutans</i> UA159 treated with different concentrations ClyR. The error bars indicate standard error (SEM) of three independent replications.</p>
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	<p>Besides, we also detected the viable counts of ClyR treated <i>S. mutans</i>. PBS resuspended <i>S. mutans</i> UA159 was treated with ClyR solution in 2 ml EP tubes for 10 min. At the end of the reaction, serial dilutions of each tube were plated on BHI agar and incubated at 37 ℃ overnight. The results obtain from triple independent experiments are shown in <b>Figure 4.</b></p>		<p>Besides, we also detected the viable counts of ClyR treated <i>S. mutans</i>. PBS resuspended <i>S. mutans</i> UA159 was treated with ClyR solution in 2 ml EP tubes for 10 min. At the end of the reaction, serial dilutions of each tube were plated on BHI agar and incubated at 37 ℃ overnight. The results obtain from triple independent experiments are shown in <b>Figure 4.</b></p>

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Revision as of 08:24, 27 October 2020

Chimeric lysin ClyR with activity against Streptococcus mutans.

ClyR is a chimeric lysin with extended streptococcal host range and has been demonstrated high lytic activity against Streptococcus mutans. It’s composed of a catalytic domain from PlyC and a cell-wall binding domain from PlySs2.

Usage and Biology

This chimeric lysin can bind to S. mutans by a cell-wall binding domain and then lysis its cell wall from outside by a catalytic domain. ClyR has no activity for Gram-negative bacteria and can be expressed in Escherichia coli. With the guidance of appropriate signal peptide, ClyR can be secreted to the outside of the cells, killing S. mutans in the environment.

Sequence and Features

Functional Parameters

To obtain the ClyR protein, we transferred pET-28a(+)-ClyR (with His-tag) to E. coli BL21(DE3). The E. coli strain was cultured to OD₆₀₀~0.6, induced with 0.2 mM IPTG, and allowed to grow overnight at 16 ℃. The harvested bacteria were resuspended with a Binding buffer containing 10 mM imidazole, and then the cell was lysis by ultrasonication. Purification was performed following the instructions of Ni-NTA Sefinose^TM Resin (Sangon Biotech, Shanghai, China). By SDS-PAGE analysis, we can demonstrate that ClyR can be expressed as a soluble protein in E. coli at the condition described above (Figure 1.). Purified ClyR was desalted and concentrated by Amicon® Ultra-4 Centrifugal Filter Units (Millipore). After quantitation by Bradford assay, the ClyR solution was stored at -20 ℃.

Figure 1. SDS-PAGE analysis of ClyR expression.

S. mutans UA159 was used as a test strain to verify the activity of purified ClyR protein. Overnight cultured S. mutans UA159 was resuspended in PBS and then treated with 120 μg/mL ClyR. The same volume of PBS was added to another tube as control. After shaking 1 h at 37℃, a significant decrease of turbidity was observed in the ClyR treated group (Figure 2).

Figure 2. Turbidity change of ClyR treated S. mutans UA159.

To determine the dose-dependent and time-dependent lytic activity of ClyR, PBS resuspended S. mutans UA159 was adjusted to an OD₆₀₀ of ~0.8. In the 96-well plates, 20 μl ClyR solution at different concentrations and 140 μl cell suspension were added in each well. The Synergy H1 microplate reader was used to measure the drop of OD₆₀₀ at 37 ℃ for 1 h. The results obtain from triple independent experiments are shown below (Figure 3).

Figure 3. Dose-dependent and time-dependent lytic activity of ClyR. A. Variation curve of OD₆₀₀ of S. mutans UA159 treated with different ClyR concentrations. B. Decrease in OD₆₀₀ of S. mutans UA159 treated with different concentrations ClyR. The error bars indicate standard error (SEM) of three independent replications.

Besides, we also detected the viable counts of ClyR treated S. mutans. PBS resuspended S. mutans UA159 was treated with ClyR solution in 2 ml EP tubes for 10 min. At the end of the reaction, serial dilutions of each tube were plated on BHI agar and incubated at 37 ℃ overnight. The results obtain from triple independent experiments are shown in Figure 4.

Figure 4. Viable counts of ClyR treated S. mutans. The error bars are obtained from three independent experiments.

Reference

[1] Yang, Hang, et al. "A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method." Scientific reports 5 (2015): 17257.
[2] Yang, Hang, et al. "Antibiofilm activities of a novel chimeolysin against Streptococcus mutans under physiological and cariogenic conditions." Antimicrobial agents and chemotherapy 60.12 (2016): 7436-7443.
[3] Xu, Jingjing, et al. "Activity of the chimeric lysin ClyR against common Gram-positive oral microbes and its anticaries efficacy in rat models." Viruses 10.7 (2018): 380.