iGEM Paris Bettencourt 2018: STAR CORES - Protein scaffolds for star-shaped AMPs

 iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

iGEM Paris Bettencourt Team (2018) group photo. Left to right: Maksim Baković, Juliette Delahaye, Annissa Améziane, Santino Nanini, Elisa Sia (Team leader), Antoine Levrier, Jake Wintermute (Secondary P.I.), Ariel Lindner (Primary P.I.), Darshak Bhatt (Team leader), Oleksandra Sorokina (Advisor). Bottom row: Anastasia Croitoru, Camille Lambert, Naina Goel. Missing from the photo are Shubham Sahu, Alexis Casas, Haotian Guo (Mentor), Ana Santos (Mentor), and Gayetri Ramachandran (Mentor)

Microorganisms such as bacteria and yeasts are fascinating! They are both beneficial and harmful to us. Over the decades, we have been using antibiotics to kill such harmful, disease-causing bacteria. With time, over-prescription and misuse of these drugs have made bacteria resistant to them; thus, evolving into what we call “superbugs”. This is a global health crisis that we currently face where simple and treatable bacterial infections have become incurable.
Recent statistics have shown that antibiotic resistance is responsible for an estimate of 25,000 deaths per year in the European Union (EU). More importantly, it is predicted to be responsible for up to 700,000 deaths each year, which is expected to rise – overtaking cancer by 2050. Not only does it take many lives but it also has a huge economic impact. In 2009, the cost of treating multidrug-resistant bacterial infections amounted to € 1.5 million in the EU alone. Likewise, according to a CDC report in 2013 entitled, “Antibiotic Resistance Threats in the United States”, antibiotic resistance was responsible for $20 billion in direct health-care costs in the United States.

In order to fight this catastrophe, many strategies have been developed but are primarily focused on humans. Thus, the World Health Organization (WHO) has come up with a more holistic approach to deal with this problem - One health concept. This states that the dispersal of resistance genes is not only limited to human species but it also spread through animals and the environment. Given the complex interactions between different sectors, one has to expand our focus to other areas like animal farming, agricultural industries, hospitals, urban and rural sectors to curb the spread of this man-made problem.
    
In response, the iGEM Paris Bettencourt 2018 team has decided to concentrate on animal-husbandry. According to Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES), the pig and pork industry consumed the highest amount of farm antibiotics in the year 2015 (129 kg/PCU), making the situation worse, while the European Food and Safety Authority (EFSA) stated that it is time to Reduce, Replace, and Re-think the use of antimicrobials given to animals. Thus, our team chose to work for a possible replacement for antibiotics for reared pigs, along with engaging the public to increase awareness of the misuse of antibiotics.

After doing some intensive research, we came across a promising alternative to antibiotics which is called antimicrobial peptides (AMPs), a diverse class of naturally occurring proteins. AMPs have a broad host range and are highly efficient: since they target the bacterial membranes, resistance to AMPs evolves at a much slower rate. Despite their great potential, there are some limitations for clinical usage including their potential toxicity, susceptibility to protease degradation, and high cost of production.

Considering the prospects of using AMPs and to overcome their drawbacks, we have proposed to employ an E. coli-based cell-free expression platform to produce naturally occurring or artificially designed AMPs (Fig. 1, step 2). These AMPs have been fused to self-assembling scaffold proteins to improve their bactericidal efficiency. We started with screening the best possible AMPs and scaffold protein complexes, in terms of their bactericidal property and biocompatibility (Fig. 1, step 1), which will followed by their production in cell-free systems. Then, we would test their mechanism of action by liposome leakage assay and microscopy. In addition, we want to mathematically model the influence of the charge distribution on the efficacy of the AMPs. To achieve this aim, we have generated 12,000 variants from 5 native sequences which were suitable candidates for designing our library. Lastly, the selected AMPs fused with the scaffold proteins would be tested for their efficacy via killing kinetics experiment in which the minimum inhibitory concentration was also determined (Figure 1, Step 3). We also check for any resistance development after exposure to the controls and our experimental product. Finally, we would determine their toxicity on mammalian cell lines.

 Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Figure 1. The three core components of the project. Step 1: Screening of the AMPs and scaffold protein complexes. Step 2: Production of the selected AMPs and scaffold protein via E. coli-based cell-free expression. Step 3: Test for the efficacy and safety of the AMPs fused with the scaffold protein produced via cell-free synthesis.

Our battle against antibiotic resistance bugs has just started, and if you would like to join us on our journey to save the planet, please do follow us on our social media portals (Facebook, Instagram, Twitter, and our YouTube channel). Feel free to message us via email or any of our social media accounts. We are open to questions, suggestions, collaborations, and monetary support.