Difference between revisions of "Team:MIT"

Preliminary Project Overview

Dental caries or ‘tooth decay’ affects the large majority of the population and even spawned its own field of study, dentistry. As a chronic but non-lethal condition it is often overlooked as a target for modern therapeutics, and its quality of treatment has lagged behind more life-threatening diseases such as cancer. However, considering how dental caries is a life-long disease requiring specific self-treatment twice daily as well as (at minimum!) bi-annual visits to a specialist - it’s still prevalent. Our team seeks to create a new therapy for dental caries based on synthetic biology. Hit the road, plaque!

The primary bacteria responsible for tooth decay is Streptococcus mutans, a gram-positive microbe that thrives in the salivary microbiome. On teeth it forms biofilms, connected colonies of adherent cells held together by water-insoluble glucans (WIGs) synthesized from ingested sugars. Commonly referred to as dental plaque, this biofilm contains multiple species of Streptococci as well as other microorganisms of the mouth.

S. mutans release lactic acid as a byproduct of anaerobic respiration, and dense biofilm leads to especially increased extracellular acid because the bacteria have less access to oxygen for aerobic respiration. These high concentrations demineralize teeth and cause cavities to form. Although S. mutans is highly adapted to the biofilm lifestyle, it can also survive in a planktonic form in the mouth. The transition from planktonic to biofilm life is coordinated via quorum sensing.

Quorum sensing is a mode of communication employed by many bacteria to coordinate gene expression and behavior across a population or species. Most quorum-sensing systems involve small, diffusible peptides which are sent out as a signal by one bacteria and received by another. After the peptides are detected by a receptor on the cell membrane or in the cytoplasm, the cell initiates 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, the bacterias’ behavior shifts to perform a synchronized activity.

S. mutans employs three quorum-sensing systems: ComRS, LuxR, and ComCDE. Whereas LuxR is a well-defined inter-species signalling system, ComRS and ComCDE are related to biofilms. The ComCDE system is specifically used to initiate biofilm formation through signalling system involving two steps between the sensing of the small peptide and gene regulation, and is primarily made up of gene products of ComC, ComD, and ComE. The quorum-sensing peptide in this system is known as Competence Stimulating Peptide (CSP), transcribed from the ComC gene. Mature CSP is secreted from the cell using an ABC transporter, a form of efflux pump encoded by the ComA gene. Once outside the cell CSP diffuses into the environment where it can be detected by the ComD receptor kinases on S. mutans cell membranes. CSP is then recognized by the receptor, ComD is autophosphorylated, and its kinase domain has the ability to phosphorylate ComE. This system is known as a two-component signalling system (TCS), and similar signal transduction pathways are present in a wide variety of bacteria.

TCS is one of the simplest signal transduction pathways for gene regulation based on external stimuli. It consists of a receptor and a response regulator that binds DNA once its conformation has been changed by the activated receptor. The response regulator can be an inhibitor or activator and recognizes specific DNA sequences to regulate transcription. S. mutans’ response regulator is ComE. It activates the transcription of a variety of genes involved in biofilm formation as well as upregulating genes in the ComCDE system to create a positive feedback loop. For our project, we sought to transplant the sensing functionality of the ComCDE system into mammalian cells. Our goal was to enable human cells to sense CSP and respond with an anti-biofilm agent in order to prevent tooth decay.