The extensive overuse of antibiotics in medicine and the food industry resulted in the emergence of multidrug-resistant bacteria strains, putting at risk the lives of millions of patients worldwide. Therefore, the discovery and development of new antibiotics is vitally important. Finding new antibiotics, however, is a highly challenging and continuous process as, eventually, bacteria develop resistance to specific antibiotic(s). So, unfortunately, once new antibiotics are marketed, they are often treated as "last-line" drugs to combat severe bacterial infections.
Antimicrobial peptides (AMPs) are a part of the innate host defence system that counteract microbial infections. AMPs exhibit a broad range of antimicrobial activity covering Gram-positive and Gram-negative bacteria, fungi and viruses. Due to the latter feature, AMPs are very appealing as potential pharmacological agents that could be used as an alternative to conventional antibiotics. Moreover, AMPs have complicated mechanisms of action, often involving interactions with more than one target in microorganisms, meaning that multiple mutations within pathogens are needed to develop resistance to AMPs. While it is generally thought that AMPs primarily target cell membranes via distinct mechanisms, many AMPs can cross the bacterial membrane without altering its integrity and interact with cytosolic targets such as proteins and nucleic acids. Crucially, however, little is known about the intracellular mechanisms of action of AMPs.
From a vast number of known AMPs, only a few are approved for clinical use to date. This is mainly due to undesirable characteristics that some of them exhibit, including (i) poor stability; (ii) toxicity to eukaryotic cells; (iii) relatively high concentrations upon which direct antimicrobial effects of natural AMPs are visible; (iv) high sensitivity to environmental conditions resulting in discrepancies between in vitro vs. in vivo efficacy. Undesired characteristics can be overcome by evolving AMPs. In nature, the development of biomolecules is driven by evolution, while in a laboratory, this process can be mimicked by using directed evolutionary approaches to generate billions of variations of genetic sequences (DNA-encoded libraries) that encode myriad peptides or proteins some of which will possess desired characteristics. One of the main experimental challenges in directed evolution studies is the relatively large size of the DNA-encoded libraries that have to be screened. In the context of AMP evolution studies, an additional challenge is the time-consuming nature of the conventional assays used to assess the antimicrobial activity of AMPs. Another factor contributing to preventing the widespread implementation of AMPs in clinical use is the relatively limited knowledge of intracellular mechanisms of action of AMPs. Thus, there is a need for an ultrahigh-throughput platform which would enable screening large libraries of AMP mutants in a time- and cost-effective, as well as new approaches that would enable profound studies of the mechanisms of action of AMPs.
The main goal of this project was to contribute to fighting the antimicrobial resistance crisis by establishing a next-generation platform enabling ultrahigh-throughput studies of the directed evolution of AMPs, and employing state-of-the-art tools to better understand how AMPs exert their antibacterial effects.
During this project, a high-throughput microfluidics-based platform enabling screening of large DNA-encoded AMP mutant libraries was established and is now being put to the test. Additionally, a new mechanism by which AMPs exert their antibacterial effects was discovered.
