In process of tooth decay, two kinds of bacteria dominate at different times. In our daily life, after we brush our tooth, some kinds of primary bacteria that do not harm our teeth will start to adhere to them. In the early stage of tooth decay, adhesion of Streptococcus species will form plaque and start to destroy enamel. One species, Streptococcus mutans, is the main bacteria. In a later period, Lactobacilli will play a leading role in destroying the dentin.
CSP (Competence-Stimulating Peptide)
- Streptococcus mutans
Streptococcus mutans is a gram negative bacteria that is the main cause of cavities. After we brush our teeth, some bacteria will again attach to the tooth and become primary colonizers, after which S. mutans adheres to the primary colonizers by cell-cell interaction. To survive and grow into a colony, S. mutans will secret a short-length peptide, CSP (Competence-Stimulating Peptide). This peptide will direct S. mutans to secret a biotoxin, driving other bacteria out and forming a biofilm. This situation is called quorum sensing. When the biofilm becomes thick and soft, we call it plaque. Therefore, we detect the amount of CSP as an indicator of the biofilm[4,7].
Figure 1 Streptococcus mutans
- How did we detect CSP?
We use the CSP-comDE two-component system from S. mutans and transform it into our host bacteria Bacillus subtilis (B. subtilis strain MW12).
We used two different plasmids for transformation. One of them, which we called plasmid comDE, carries genes encoding comD and comE. It will product membrane-bound histidine kinase receptor, comD, and cytoplasmic response regulator comE. When CSP from the S. mutans triggers comD on our host B. subtilis, comD will autophosphorylate the receptor comE. Activated comE directly activates the other plasmid which carries the gene encoding GFP (green fluorescent protein) which we detect by measuring the strength of green light, which is correlated with the amount of CSP.
Lactobacilli will later become the dominant bacteria, when tooth decay becomes serious enough to damage dentin. The main reason is that Lactobacilli secrete lactic acid during the fermentation process. Lactic acid makes the environment of the tooth more acid. More acid surroundings make calcium, which is the main component of teeth, more soluble in saliva. This called decalcification. In the early stages of tooth cavity, S. mutans also uses lactic acid to damage teeth.
Detecting Lactobacilli is more difficult than detecting S. mutans because it is not a specific bacteria but a group of bacteria. Therefore, we decided to detect lactic acid rather than detect Lactobacilli directly. Another reason is that lactic acid directly causes tooth decay.
Figure 2 Lactobacillus casei
- How do we detect lactic acid?
We use the LldPRD operon from Escherichia coli with some modification. We transformed our host E. coli (E. coli strain DH5α), using only one plasmid for transformation. This plasmid carries genes encoding Lldr (the LldPRD operon) and GFP (green fluorescent protein). The Lldr gene will continue to produce Lldr until it reaches equilibrium. When Lldr is bound on the O1 binding site and O2 binding site, the LldPRD operon will be shut off and GFP can’t be produced. When L-lactate enters our host E. coli it triggers the LldPRD operon, so Lldr will be removed and the LldPRD operon can work temporarily, producing GFP until the operon is shut off again.
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