Dental caries is one of the three major non-communicable diseases with high incidence and more harmful. However, many people often develop deep tooth decay or even pulpitis when they see a doctor with toothache or holes in their teeth. Dental caries can cause specific pain during its treatment, and the patient may have to visit the hospital multiple times, and the treatment costs are very high. Therefore, we tried to achieve early detection and prevention of dental caries through synthetic biology.
The main pathogenic bacteria of dental caries is Streptococcus mutans(S.mutans), and the proposed project is based on the detection of S.mutans. The following three parts are designed as:
- The prevention system has be achieved by using antimicrobial peptide to inhibit S.mutans growth.
- The detection system has be designed based on the density sensing system of S.mutans to estimate the degree of dental caries.
- Suicide mechanism has be adopted to prevent biological escape.
Prevention
Antibacterial peptides have the advantages of good antibacterial activity, low cytotoxicity, and resistance to tolerance. Therefore, we screened antibacterial peptides with an excellent antibacterial effect on S.mutans to prevent dental caries . Meanwhile, we used two antimicrobial peptide expression strategies for expression.
Screening of antimicrobial peptides
Antimicrobial peptides are a distinct and diverse class of molecules in sequence, structure, and sources. Over 2800 peptides sequences have been reported to date. Based on various existing antibacterial peptides, we needed to screen and find which AMP can effectively kill S.mutans. The MIC of AMPs showed that tachyplesin 1(TP-1) had high antibacterial activity on S.mutans .Therefore, TP-1 was selected as part of the prevention of dental caries. To further improve the bactericidal effect of TP-1, a targeting sequence for S.mutans was added and a improvement part BTP-1 was constructed.
Expression strategy of AMPs
Considering that TP-1 contain 17 amino acid residues and BTP-1 contain 36 amino acid residues , which are not easy to biosynthesize directly, we used Glutamate locus（E）to construct an antimicrobial peptide polymer by repeating 4 times in tandem. After the AMP polymeris expressed, it will be hydrolyzed into active monomers by glutamate endopeptidase.
Two expression strategies of antimicrobial peptides
1. Fusion expression in E.coli
Although we constructed AMPM, the molecular weight was still so small that it was challenging to achieve biosynthesis. Moreover, AMPM does not have any tags that make it difficult to achieve purification and preparation. Therefore, we inserted AMPM into the fusion expression vector pGEX-4T-2 so that AMPM could be expressed as a fusion with GST protein. In this way, we realized the expression of AMP in E.coli, separation, and purification to prepare active antimicrobial peptide proteins, which were finally hydrolyzed into antimicrobial peptide functional monomers that could be used to prepare products.
(Figure 1. Protein purification process diagram)
2. Secretory expression in lactic acid bacteria
Since E.coli fusion expression requires complex protein purification to obtain the desired peptide, there are uncontrollable factors in the separation process (such as sample contamination). Fortunately, 2019 SZPT-CHINA had constructed the lactic acid bacteria expression system successfully. This year，we used this system as the chassis to directly secrete and express the antimicrobial peptide so that the complicated separation and purification process could be eliminated from the secretion expression system. Furthermore, research papers showed that probiotics play an essential role in maintaining oral health.
According to the lactic acid bacteria preferred codons, we redesigned a new antimicrobial peptide polymer sequence (NAMPM) based on the specific hydrolase cleavage site K, which is secreted by harmful oral bacteria, and introduced the secretion signal peptide SPusp45 for secretion expression. Finally, the chassis secretes and expresses NAMPM, and NAMPM is hydrolyzed by hydrolase to produce antibacterial peptides to prevent dental caries. In addition, because the environment is acidic when the oral cavity is in an unhealthy state, we chose the acid-inducible promoter P170 to regulate the expression of NAMPM.
(Figure 2. Gene circuit diagram of prevention system)
Chewing gum to prevent dental caries
To develop preventive products with AMP, our team conducted market research. Based on these results, we determined to make a chewing gum that can prevent dental caries with expressed AMP or synthetic AMP probiotics. In fact, chewing gum has gained increasing acceptance as a delivery system for active ingredients over the years. These researches have laid the foundation for the preparation of our products.
Detection
This year, our team constructed an E.coli detector to detect S.mutans. We fused the leader peptide pelB with the histidine kinase receptor protein comD and with the help of pelB comD would be carried to cell membrane to form the receptor membrane protein. After comD received the signal peptide CSP secreted and expressed by Streptococcus mutans, it was phosphorylated and activated. The intracellular response regulator protein comE expressed by the bacteria was phosphorylated and activated by activated comD. The activated comE regulated the nlmC promoter to initiate downstream gene sfGFP expression to achieve the purpose of detection.
(Figure 3. Gene circuit diagram of detection system)
(comA is an ATP transporter, comB is an auxiliary protein for csp processing and export, comE encodes a csp precursor, comD is a histidine kinase receptor, comE is an intracellular response regulatory protein, and nlmC is an inducible promoter.)
However, this method of detecting dental caries by fluorescence requires microplate readers or ultraviolet lamps and other equipment. Therefore, we replaced the sfGFP gene with the lacZ gene and hydrolyzed X-gal by the expressed β-galactosidase to produce blue color to observe the color change with the naked eye.
Suicide mechanism
Although the lactic acid bacteria we chose for the chassis were of GRS grade and the carrier we selected was the food-grade vector pMG36N, we have designed a set of controllable suicide mechanisms for bioethical considerations to prevent engineered bacteria from escaping into the environment. According to the human oral environment, there is a certain lysine concentration, but there is no lysine outside the oral cavity. We set up a suicide mechanism regulated by the lysine riboswitch. When the bacteria colonize the oral cavity, the riboswitch controlled by lysine is turned off. When the bacteria escape the oral environment, the riboswitch opens and expresses the toxic protein RalR. RalR is a non-specific endonuclease. The DNA can be cut to kill the chassis to avoid the bacterial escape.
(Figure 4. Gene circuit diagram of suicide mechanism)
References
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- Tianlei Liu, Shoubin Xue, Wenbo Cai, et al. ComCED signal loop precisely regulates nlmC expression in Streptococcus mutans. 2014, 64(1):31-38.
- Zhang Kai. Experimental study on the effect of density induction on the growth and biofilm formation of oral Streptococcus mutans[C]. 2007:165.
- Shi J, So LY, Chen F, et al. Influences of disulfide connectivity on structure and antimicrobial activity of tachyplesin I. J Pept Sci. 2018;24(6)
- Kumar, P., Kizhakkedathu, J., Straus, S. . Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules, 2018, 8(1), 4.
- Kaur K, Nekkanti S, Madiyal M, Choudhary P.Effect of chewing gums containing probiotics and xylitol on oral health in children: a randomized controlled trial. [Original Research]. J. Int. Oral Health .2018. 10 (5), 237–243
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- Guo Y, Quiroga C, Chen Q, et al. RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli. Nucleic Acids Res. 2014;42(10):6448-6462.
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