Peptidator P-800: Pathogens, you've been terminated!

Pathogens, you've been terminated!

This year, we chose to design a novel synthetic platform for high throughput isolation and characterization of peptides with antimicrobial properties (AMPs) to serve as The Terminator for multi-resistant bacterial pathogens. We used the available genomic sequencing data for Carcharodon carcharias (great white shark) as a source of novel peptide sequences that can be used instead of antibiotics. To identify such elements, we established a bioinformatic approach based on the tBLASTn algorithm and known antimicrobial peptides as quarries. We identified nine putative AMPs that successfully fulfilled our criteria for successful heterologous expression in E.coli - 10-30 amino acids in length, monomeric molecules, no unnatural amino acids, no C/N-terminus modifications. We designed expression constructs for them that consist of a T7 promotor, a strong RBS, an ATG start codon, the AMP-coding sequence, a TAA stop codon and a T7 terminator. These constructs were synthesised as gBlocks and cloned into pSB1C3 vector. We used KRX cells kindly provided by Promega, because they allow efficient cloning and T7-driven protein expression without the need for subcloning. Next, we tested the activity of these putative AMPs using different techniques and indicator strains. Meanwhile, we designed an overexpression cassette for AMP production that utilizes the DAMP4 protein fusion partner, masks toxicity and allows cheap and easy purification of the peptide of interest. In addition, we developed a design for a future second version of that cassette that has EDDIE instead of DAMP4 and allows the production of peptides with an intact N-terminus.

Project Inspiration

It all begins with a cause, right? For the longest time, we were at a loss... we couldn’t find the right battle to fight, because there are just way too many great causes one could support. The team was split between causes, most were fighting deadlines in university and studying for exams. We were all plain confused as to how to come to an agreement. All of a sudden, one of our team members, Ivan, who studies Medicine, summoned us all excited, to share “the most important thing you’ll hear all week”. He claimed that to be “a possible project idea” and, just like that, we were hooked. He went on about his day and what he learned in the hospital. According to him, the first-ever confirmed clinical strain of a vancomycin-resistant Enterococcus faecalis in Bulgaria was isolated in the very hospital he works in. A mere year later, 5 more cases of similar infections had popped up on the map in various clinical establishments around the country. Such resistance against one of humanity’s biggest weapons against pathogens calls for immediate action! This is what made us realize the seriousness of antibiotic resistance and the dire need for a new alternative to modern-day antibiotics.

After numerous discussions with clinical microbiologists, we found ourselves once again scared by the facts they laid onto us. Supposedly, vancomycin resistance is not uncommon, nor is it an isolated phenomenon. More and more pathogens are becoming resistant against the most common treatments. The worse than average regulation of antibiotic usage in rural agriculture in Eastern Europe further exacerbates the problem.

If no action is taken, multiresistant bacteria could cause 10 million deaths each year by 2050 according to the UN Interagency Coordination Group (IACG) on Antimicrobial Resistance. We found such data to be rather concerning and, as young, aspiring scientists decided to tackle the problem heads-on!