Team:REC-CHENNAI/Description
PROJECT DESCRIPTION
The emergence of multi-drug resistance in food-borne bacteria is an increasing problem globally. Described by WHO, CDC and other International agencies as one of the world’s most pressing public health threats, antibiotic resistant infections from food-borne bacteria cause an estimated 430,000 illnesses each year in the USA alone. This issue thus has attracted significant attention towards Antimicrobial peptides (AMPs).
AMPs are gaining popularity due to their effectiveness against a wide range of pathogenic organisms that are resistant to conventional drugs. Since their mode of action, for the most part, exploits general but fundamental structural characteristics such as the bacterial cell membrane and in many cases they may have multiple targets within cells, the likelihood of the emergence of resistance is thought to be considerably reduced compared with that for many current antibiotics, which have more specific molecular targets (Håvard Jenssen et al).
For easy biosynthetic production, we decided to work with a small peptide, devoid of cysteine knots. In addition, we preferred that it not be easily degraded by proteases. Keeping these in mind, we looked for peptides that possess antimicrobial activity .Of the many that we came across, we were interested in working Lt2a.
Latarcin2a (commonly known as M-Zodatoxin) is a 26 amino acid peptide isolated from the venom of the Asian spider Lachesana tarabaevi. Latarcins (Lt-2a) adopt an amphipathic alpha helical structure in membrane-mimicking environments.
NMR structure of Latarcin2a
Amphipathicity corresponds to the segregation of hydrophobic and polar residues between the two opposite faces of the a-helix, a distribution well suited for membrane binding. The polar part is shown in magenta color, and hydrophobic part is shown in wheat color.
They produce lytic effects on both gram positive and gram negative bacteria via a carpet mechanism. As explained by Tamba and Yamazaki, this mechanism involves the external binding of the peptide to the membrane, followed by its critical destabilization.
Carpet model of antimicrobial-induced killing
In this model, the peptides disrupt the membrane by orienting parallel to the surface of the lipid bilayer and forming an extensive layer or carpet. Hydrophilic regions of the peptide are shown coloured red, hydrophobic regions of the peptide are shown coloured blue.
However, Lt-2a has a disadvantage; it is found to possess 20% haemolytic and cytotoxic activity. A study done by A.A. Polyansky et al./ FEBS 583(2009) showed that a mutation in the sequence of native Lt-2a resulted in multifold reduction of its cytotoxic and haemolytic effects. Hence, we chose to work with this mutant, Lt-2a F10K (Phe replaced by Lys) as well.
Position of the F10 in the peptide is shown in green color.
Our project involves genetically engineering E.coli DH5a cells to produce Latarcin and the mutant. We chose E.coli to be our chassis due to its higher transformation efficiency. However, the problem with making E.coli produce this peptide is the toxicity of this peptide to the chassis itself. In order to overcome this, a specially designed quorum sensing mechanism is employed.
Bacterial quorum sensing is defined as a cell-to-cell communication mechanism wherein gene expression is regulated by fluctuations in cell concentration. Bacteria that use quorum sensing mechanisms release certain signalling molecules known as autoinducers into their surroundings. Beyond a threshold limit, these autoinducers alter gene expression by binding to specific promoters within the genome. When a quorum sensing mechanism is incorporated into our project, Latarcin production will begin only after the cells reach a high concentration. Thus, large quantities of Latarcin can be synthesized.
The proposed genetic circuit for quorum sensing consists of three functional devices. The first of these three contains a constitutive promoter (Anderson promoter) coding for the gene for AHL synthesis (LuxI). The second device has another Anderson promoter that regulates the LuxR repressor gene. The last has a Lux promoter that is activated by the binding of AHL and LuxR. This regulates the expression of the mutant Latarcin.
At low cell density, the quantity of AHL produced is too little for it to bring about Latarcin production. As the cell density increases, the cells divide and simultaneously produce both AHL and LuxR. Without the AHL, LuxR is unstable and gets degraded shortly after production. But beyond a certain threshold level, AHL begins forming a complex with LuxR, preventing its degradation. This complex now binds to the Lux promoter that regulates Latarcin synthesis. On binding, the promoter immediately switches ON and begins to produce a large amount of Latarcin. A small portion of the synthesised Latarcin destroys the host, and the rest can now be isolated and purified.
It is estimated that food worth $1 trillion is wasted every year. This accounts for about one third of the world's food. Of this loss, 21% is caused due to microbial food spoilage. Thus finding a niche in the food preservation sector, we decided to develop a food wrapper that can extend the shelf life of mainly fruits and vegetables.
Inspired by 2012 iGEM Team Groningen, we propose a delivery system containing spores of genetically engineered Bacillus enclosed within two layers of PVDF membrane. (We chose to work with E.coli to carry out cloning experiments , due to its ease of handling. However, we propose that Bacillus spores be used in making the Food wrapper) The system is designed in such a way that only the PVDF membrane in contact with food will allow Latarcin to pass through. The purpose of the outer membrane(filter) is to prevent the entry of microorganisms, thus avoiding chances of further contamination.
To serve as a nutrient source for the bacteria, LB broth is contained within a compartment made of PVC, which is sandwiched between the two PVDF membranes. When mechanical stress is applied, the delicate PVC membrane disintegrates, allowing the bacteria to come in contact with the LB broth. When this happens, the dormant bacteria start multiplying. Once the bacteria reach a substantial number, due to the quorum sensing mechanism, the mutant latarcin will be synthesised.
This results in abundant production of Latarcin by the host bacteria. Latarcin diffuses through the inner PVDF membrane and reaches the food. On encountering bacteria present on the food, Latarcin effectively kills them via the carpet mechanism. Because of its half life of 10 hours, any unused Latarcin is therefore degraded, making the food safe for human consumption.
