Dental caries, commonly known as tooth decay, is a transmissible bacterial disease in the oral cavity with high morbidity in all age groups. According to the World Health Organization (WHO), 60-90% of children and the majority of adults in developed countries suffer from dental caries. The WHO has listed it as one of the three major human priorities along with cancer and cardiovascular diseases, affecting around 2 billion cases of permanent teeth and 520 million cases of primary teeth worldwide. In China, more than half of children aged 3-6 have dental caries, with a prevalence rate of 59.5% in Tianjin and 63.1% in Guangzhou.
Affected by several aspects such as saliva, biofilm on the tooth surface, tooth mineral composition, genetic influence, and demineralization, the disease is mainly caused by a series of cariogenic bacteria, mostly led by Streptococcus mutans. A key feature and difficulty in destroying S. mutans is its formation of biofilm, which provides a defensive habitat for cariogenic bacteria in the oral cavity. Biofilms have high resistance against antibiotics, making them very hard to wipe out.
Most modern treatments against dental caries surround the usage of fluoride and going to the dentist. The former creates large amounts of pollution, affecting both the environment and human health, while the latter is very expensive, consuming around 5-10% of the healthcare budget in developed countries according to the WHO. Therefore, we aim to create a new kind of agent against dental caries that is environmentally friendly, less expensive, and more effective, through the techniques of biosynthesis.
ClyR is a chimeric lysin that is actively against numerous S. mutans serotypes, being able to eliminate S. mutans colonies without enticing antibodies, making it an effective and safe agent for killing S. mutans. Yet, its effect is limited due to its inability to wipe out and inhibit biofilms. Meanwhile, this problem can be solved by DexA70, the truncated form of DexA, a dextranase produced by S. mutans. Studies show that DexA70 can actively disrupt biofilms and inhibit the formation of biofilms, being able to expose and make it easier for antimicrobial agents to diffuse into the biofilm and attack bacteria.
Phage lyase is a phage-encoded glycosidic hydrolase with the function of digesting the cell wall of bacterial host cells. Arabinose can be used to induce the expression of phage lyase. Dexanase can inhibit biofilms by hydrolyzing A-1, 6-glycosidic bonds. Dexanase A (DexA) can inhibit biofilm formation and destroy preformed biofilms in vitro.
Since a combination of DexA70 and antimicrobial agents will perform a better treatment, we transformed and expressed both ClyR and DexA70 into Escherichia coli strain Nissle 1917 in order to create an agent that is able to kill S. mutans, destroy existing biofilms, and inhibit the formation of biofilms, thus provide a more effective solution towards curing dental caries.
First, we downloaded the gene sequence of DexA100-733 from the NCBI database, and designed the fusion gene sequence of DexA100-733, after codon optimization of the gene sequence, it was synthesized into the pUC57 plasmid by a gene synthesis company.
Then, Escherichia coli strain Nissle 1917 cells were transferred to obtain recombinant strain A. Glucanase secretion and activity were determined by SDS-PAGE and reducing sugar method.
Next, the fermentation supernatant of genetically engineered bacteria A was used to test the ability of inhibiting the biofilm formation and breaking down of Streptococcus mutans.
After that, genetically engineered strain B was obtained by transferring pBAD-Myc-HESa-OmPA-PaqC1-Clyr-6His-Paqc1 DNA into engineered strain A. The engineered strain B was induced by adding arabinose. The supernatant was used to test the removal of Streptococcus mutans biofilm and bactericidal effect. Co-culture with Streptococcus mutans after arabinose induction.
1. Successfully construct plasmidpuc57-kan-mini-J23101-OmpA-PaqC1-smDexA-PaqC1-TT.dna.
2. The expressed peptide can achieve the dual role of removing biofilm barrier and lysis of dental caries.
