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Latest revision as of 01:58, 18 October 2018
Background
As a chronic but non-lethal condition, dental caries are often overlooked as a target for modern therapeutics, and the quality of
dental caries treatment has lagged behind that of more life-threatening diseases such as cancer. However, it is a well established
fact that oral health affects overall health--for example, severe cavities can lead to systemic illnesses such
as heart disease. Therefore, dental caries is still a prevalent and critical issue.
The process of cariogenesis is highly facilitated by the growth of dense, sticky biofilm on the surface of teeth. This bioflim
cuts off the bacteria's access to oxygen, thereby forcing them to revert to anaerobic respiration. Lactic acid is released as a
byproduct of bacterial respiration, and leads to demineralization of the enamel and cavity formation.
Biofilm Formation
In its natural, non-pathogenic state, S. mutans lives planktonically (free-floating).
The transition from the planktonic to biofilm state requires expression of virulence genes, which is coordinated via
quorum sensing: a mode of communication employed by many bacteria to coordinate gene expression
and synchronous behavior across a population or species. In the case of S. mutans, quorum sensing results in the expression of
genes coding for extracellular glucosyltransferase (GTF) enzymes. These enzymes synthesize adhesive glucans, which are used by
the bacteria to adhere both to each other and to the enamel of the tooth.
Most quorum-sensing systems involve small, diffusible peptides which are sent out as a signal by one bacteria and
received by another. The peptides are detected by a receptor on the membrane or in the cytoplasm of other bacteria, which then
initiate a signal transduction pathway that upregulates the quorum-sensing peptide, as well as other genes related to the coordinated
behavior of the population. Once enough of the signal has been received by the majority of the population, bacterial behavior
shifts to perform a synchronized activity.Biofilm formation is one of the synchronized behaviors initiated by quorum sensing.
During this process, S. Mutans create and perpetuate the Competence Stimulating Peptide (CSP), a small molecule that is used to
initiate the formation of dental plaque. The pathway through which the cells sense and respond to CSP is referred to the ComCDE pathway.
Quorum Sensing in S. mutans
Competence Stimulating Peptide (CSP)
CSP is an 18 amino acid long peptide that binds specifically to ComD, leading to activation of the TCS pathway.
ComD
ComD is the transmembrane histidine kinase receptor and homo-dimeric protein. It has 6 transmembrane segments with three extracellular loops and 1 intracellular kinase domain.1 ComD sense the S.mutans density based on the concentration of CSP (competence signaling peptide) that S.mutans population secrets into the environment. When CSP gets to a critical concentration, ComD trans-autophosphorylates between two dimer’s kinase domain histidine and then transfers the phosphate group from histidine to an aspartic acid on ComE (the response regulator protein).2
ComE
ComE is the response regulator protein of the TCS pathway. When ComD is activated by CSP binding, a phosphate group is transferred from ComD to ComE. This phosphorylation activates ComE, enabling it to drive a ComE specific promoter. The phosphorylation likely contributes to ComE activation by enabling oligomerization of the protein, which may assist in facilitating DNA binding activity or DNA bending to allow access to RNA polymerase.3,4
Genetic Response
In S.mutans, comE binds to multiple genes that are involved in genetic competence development, biofilm formation, and bacteriocin secretion.5Among the downstream genes of comE, gtfC is one of the proteins that is involved in biofilm formation by catalyzing the reaction that transforms glucose to adhesive glucan.
(hover over each element to learn about its role)
Our Synthetic Circuit
Our goal is to port the ComCDE system into Human Embryonic Kidney (HEK) cells. This would allow our engineered cells
to sense the presence of CSP and therefore detect when biofilm formation is about to occur. ComD and ComE will be
expressed constitutively, while our three outputs (ScFv's P126 and P136, and Kappa casein) will be expressed under the ComE
promoter, which is activated by the ComE response regulator protein as described above.
Output
Upon activation of our biofilm-sensing system, our cells will output proteins that have been shown to
reduce the biofilm-forming capabilities of S. mutans.
Our actuation protein of choice is kappa casein, a protein derived from bovine milk. It interferes with the adhesive power of the
glucans post synthesis through a combination of hydrophilicity and negative charge.6