We want to estabilish a method/system, which could solve the problem of emergence of antibiotic resistance using the power of natural selection. Here, we have choosen a problem to prove the model.
E. coli O157:H7 and Salmonella typhimurium are gram -ve pathogens, which can infect a broad range of vertebrate hosts. These pathogens can also be present in food and can infect the organism feeding on it. Therefore, we make a system, which would be able to handle these pathogens.
Also, we would use the Lactococcus lactis strain as prebiotic to cure animals, humans and eliminate pathogens from food.
We will synthesize a bacterial system that:
- Senses the cell density of pathogenic bacteria
- Our system releases anti-microbial peptides which kill pathogens, when it has sensed high cell density
- As the population goes down we release a peptide that neutralizes the activity of anti-microbial peptides, resulting into a stress free environment.The stress-free environment would be favourable to the wild type compared to the mutants which have developed antibiotic resistance
Consequentially this will lead to a population of predominantly wild type bacteria. Again, as the population of wild type goes up, the same cycle of steps 1-3 is repeated. After few cycles, the pathogens should be eliminated.
The system we propose will use an anti microbial peptide Alyteserin, which has bactericidal effects. Alyteserin-1a has a lethal effect on gram negative bacteria. We have designed a novel short peptide sequence (which shall be called Naly from herein). Molecular Dynamics simulations of Alyteserin-1a and Naly have been performed under various conditions and conformations, and we have found that the two peptides interact favourably (check out the In-Silico section under project in the menu bar for animations).
The pathogen of interest, which has to be a gram negative bacterium in this case was chosen to be E. coli. We chose E. coli for its ease of availability in a laboratory setting, but Alyteserin-1a is a broad antimicrobial peptide and is effective against a wide range of gram negative bacteria including E. coli and Salmonella Typhi.
We chose the carrier of the AMP to be Lactococcus Lactis, for several reasons. L. lactis is a gram positive bacterium. This ensures that the AMP would not have lethal effects on L. lactis itself. Also, L. lactis is a non-pathogenic bacteria, and will not elicit an inflammatory response from the human immune system. Developing the genetic circuit for timed release of AMP and its inhibitory molecule inside L. lactis will provide more scope in the future for developement of a robust drug delivery system.
How does the circuit work?
Most pathogenic microbes secrete quorum sensing (QS) molecules like AHL, AI-2 when present in high cell density. QS molecules are sensed by receptor proteins on the cell surface of pathogens. These signaling molecules help them in regulating their communal activities.
In the lab, we will be using E. coli DH5α strain as pathogenic model. E. coli DH5α does not secrete quorum sensing molecules natively. Therefore, we will genetically modify E. coli DH5α strain to release Auto-Inducer-2, a signaling molecule. We do this so as to create a recombinant that mimics many pathogenic bacterium that secrete quorum sensing molecules.
We will be using Lactococcus lactis NZ9000 strain as a receiver. It has a simple circuit to detect high and low cell density of pathogens and release the appropriate molecules. L. lactis activates the expression of Alyteserin-1a (anti-microbial peptide) at high cell density and NAly (neutralizing anit-microbila peptide) at low cell density. The precise mechanism with the genes involved is explained below.
At low cell density, LuxP, LuxQ, LuxU receptor proteins act as kinases that results in the phosphorylation of LuxO (LuxO-P) that activates qrr1-5 sRNAs with the help of sigma54 RNAP subunit factor, qrr sRNAs degrade the mRNA of LuxR which has been found to activate and repress a number of gene when expressed in the cell.
While at high cell density, the same receptor proteins (LuxP, LuxQ, LuxU) acts as phosphatases which removes the phosphate from LuxO-P and this results into the higher expression of LuxR gene.
We are a team of undergraduates from the Department of Biotechnology, IIT Madras in iGEM 2015
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Department of Biotechnology, IIT Madras Sardar Patel Road, Chennai
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