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Team:Groningen/Template/MODULE/Project/detection
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Detection
Detection of infected wounds
Why a detection system?
Resistance of anti-microbials, especially antibiotics, cause a major problem in the modern medicine world. The World Health Organization state’s “A post-antibiotic era—in which common infections and minor injuries can kill—far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century” (April, 2014)1. Therefore we will not use antibiotics against the infections but rather use a set of Infection Preventing Molecules (IPMs). Little resistance has been shown for these molecules (Source, resistantie against Nisin) and are therefore excellent targets for our fight against infections. More about these IPMs can be read in our secretion page. To further lower the chance of resistance we integrate a detection system in our LactoAid. This detection system prevents unnecessary production of the IPMs and thus lowering the chance of resistance development in bacteria.
What is LactoAid going to detect?
Our lactoAid focusses primarily on the treatment of burn wounds. The most occurring infections with burn wounds are caused by Staphylococcus aureus and Pseudonomas aeruginosa. Both these pathogenic (i.e. capable of causing disease) bacteria are opportunistic bacteria. Meaning that they hardly infect healthy people, but when immunity is lowered or the skin is damaged they can cause infection. Only when the density of the pathogen is above a certain threshold it starts to transcribe pathogenic genes and produces toxins 2.
The reason a single cell or low density population will not start to produce these toxins is because they cannot produce enough toxins to kill surrounding cells in order to start an infection. If a pathogen starts to produce these toxins in a low density population, it would waste energy. Therefore the pathogens wait until the density is high enough to inflict damage by simultaneously producing the toxins. So how do the bacteria know when the optimal density is reached? They secrete signal molecules.The pathogens know how big their population is by secreting those signaling molecules. This signaling is called quorum sensing (QS)2. We designed our LactoAid in such a way that it is able to detect the QS of both staphylococcus aureus and Pseudonomas aeruginosa.
Detection of Staphylococcus aureus
Quorum sensing pathway in Staphylococcus aureus
The quorum sensing system in Staphylococcus aureus consist of four genes (Figure 1). These genes are controlled by a leaky inducible promoter (P2), and encode for two membrane proteins (AgrB and AgrC), one regulator protein (AgrA) and a precursor peptide (AgrD). The membrane protein AgrB cleaves the precursor peptide AgrD so the mature signaling peptide is formed. The mature signal peptide is called autoinducing peptide (AIP). After the AIP is secreted from the cell it is able to bind to the second membrane protein AgrC. After binding the AgrC phosphorylates AgrA, AgrA is then able to induce the P2 promoter which leads to increased production of the AIPs.
Figure 1:
Quorum sensing system of Staphylococcus aureus The precursor peptide, AgrD (1), is modified to AIP by the membrane protein AgrB (2) and translocated over the membrane. The AIP binds to AgrC (3) which in turn phosphorylates AgrA (4) the phosphorylated ArgA induces the P2 promoter.
How LactoAid detects S. aureus
In our LactoAid we introduced the DNA encoding for the membrane protein AgrC and the regulator protein ArgA. When Staphylococcus aureus starts to produce the AIPs, the LactoAid should be able to sense the molecules through the same system used by S. aureus. In figure 2 the designed biobrick for the detection of S. aureus is shown. Here we also show the P2 promoter which will be induced. More about the secretion system can be found at the sectretion page
Figure 2:
Overview of the desinged detection system against Staphylococcus aureus. The proteins AgrA and AgrC are present in the LActoAid. After detection of the AIPs by AgrC the AgrA will be phosphorylated and induce the P2 promoter. The details about the activated secretion system can be found at the sectretion page.
Figure 3:
Staphylococcus aureus picture adopted from center for disease control and prevention
