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The emergence of multidrug 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 Latarcin2a.
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 the production of Lt2a in genetically modified organisms. We chose E.coli DH5a to be our chassis due to its multiple mutations that correspond to its distinct characteristics. These mutations allow for blue-white screening for recombinants and ensure high plasmid transfer rates by lowering endonuclease activity.
A major issue that was to be tackled during the course of our project was Host toxicity. Latarcin being antimicrobial would naturally be lethal to the chassis itself. In order to overcome this, a specially designed quorum sensing mechanism is employed.
