Dental caries is a common disease. It not only directly affects human oral health, but also often causes adverse symptoms in other parts of the body. Global disease statistics in 2016 show that the incidence of dental caries in the population is ranking second among common diseases. Studies have shown that the formation of dental plaque is the result of the joint action of a variety of bacteria, including Streptococcus mutans, Lactobacillus, Actinomycetes, etc. Phage lyase ClyR (combined from different bacteriophage lytic enzymes) has a broad bactericidal spectrum, especially the only one reported to be extremely strong against Streptococcus mutans and Streptococcus mulberry. The enzyme is promising to kill these two kinds of streptococci are the main cause of dental caries.
This project designed a genetically engineered probiotic (Escherichia coli strain Nissle 1917 (EcN)), which can be induced and expressed of lysate enzyme ClyR upon exposed to monosaccharides (arabinose) which released from food arabinoxylan in the oral (Figure 1).
Figure 1 Schematic diagram of ClyR in the prevention or treatment of dental caries.
Genetically engineered probiotics bacteria which produce and secret ClyR are constructed. Construct puc57-kan-mini-J23101-OmpA-arab-TT secretes arabinosidase to hydrolyze polysaccharides into monosaccharides; construct B pBAD-Myc-HisA-OmpA-ClyR-6His-TT is induced by arabinose and produce the ClyR enzyme. Construct C pBAD-Myc-HisA-OmpA-amilGFP-TT is used to test the function of the system. All the constructs were confirmed by DNA sequencing.
Figure 2. Schemtic maps of plasmids A, B, and C..
(1) AmilGFP expression in cells transformed with plasmid C
In the cells transformed with plasmid C, the fluoroscopic data of cells increased with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with the prolongation of incubation time.
Figure 3. The fluoroscopic data of cells transformed with plasmid C with the addition of arabinose.
(2) AmilGFP expression in cells transformed with plasmids A and C
In the cells transformed with constructs A and C, the fluoroscopic data of cells increased with the addition of araboxylan. Meanwhile, the fluoroscopic data become larger with the prolongation of incubation time (Figure 4).
Figure 4. The fluoroscopic data of cells transformed with plasmids A and C.
(3) ClyR expression
Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by electroporation. The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression. For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.
The protein concentration was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the average of the A595 data (Table 1). The A595 of the culture supernatant of E. coli with plasmid A and B suggested the possible expression of ClyR (Table 2). The A595 data at “0 %” was referred as blank, and the protein concentration could be calculated referred to the standard formula.
Table 2. The protein concentration of the culture supernatant of E. coli with plasmids A and B.
As can be seen from figure 5, in the transformed bacteria with plasmid A and B, the concentration of the reducing sugar increased with the elongation of the incubation time. During the expression process, bacteria lysis occurred with the elongation of the incubation time. But the ClyR band could obviously be obtained as is shown in figure 5.
Figure 5. The SDS-PAGE of supernatant.
(4) In vitro activity assay of ClyR
E. coli Nissle 1917 transferred with plasmid A and B were culture with Gram-positive bacteria Lactobacillus casei subsp. casei (ATCC334). The value of OD600 was monitored using Multiscan Spectrum (BioTek). The OD600 of the L. casei subsp. casei cell suspension gradually reduced with the addition of supernatant (Figure 6).
Figure 6. The in vitro assay of ClyR.
AmilGFP was successfully expressed, suggesting the feasibility and possibility of the inducible secretory expression of ClyR. We have successfully obtained the ClyR protein in the culture supernatant of cells carrying plasmids with araB and ClyR genes. The concentration of ClyR was low, so more efforts are still needed in future. It is worth to note that the expression of ClyR protein may cause the host to suicide, which can be supported by the obvious cell lysis and the presence of flocculent precipitate.