Periodic Reporting for period 1 - MicroREvolution (MicroREvolution - a high-throughput drop-based microfluidic platform for rapid screening of highly diverse antimicrobial peptide mutant libraries generated via directed evolution.)
Période du rapport: 2021-10-01 au 2023-09-30
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.
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.
A custom-built microscope serving as a base for all the microfluidic technologies used in the MicroREvolution platform was engineered and assembled. Subsequently, a platform that enables the encapsulation of DNA-encoded antimicrobial peptide (AMP) mutant library at a single template per droplet encapsulation efficiency and subsequent in-drop amplification was established. Later, a picoinjection platform that enables the introduction of cell-free protein synthesis mix into the droplets with amplified template DNA was established. During this step, AMP mutants are synthesised inside the droplets. The same platform later was used to introduce bacterial cells with viability dye into the droplets with synthesised mutants of antimicrobial peptides. The microfluidic droplet sorting platform that enables a high-throughput (1 000 000 droplets per hour) separation of droplets containing AMP mutants with strong bacteriocidal activity from the droplets with AMP mutants exhibiting weak bacteriocidal activity was established.
Finally, all individual technologies were combined into a single platform, MicroREvolution (Figure 1), enabling one to screen large DNA-encoded antimicrobial peptide libraries in a cost and time-efficient way. The platform was introduced to the public during the BPS conference in San Diego, USA in 2023.
Additionally, state-of-the-art technologies developed in the host laboratory were employed to study the mechanisms of action of cell-penetrating AMPs. We discovered that AMPs have a lot in common with the biomolecules that undergo liquid-liquid phase separation (LLPS), which is emerging as a key principle controlling subcellular organisation, and was shown to be involved in a variety of biological processes including RNA metabolism, ribosome biogenesis, DNA damage response and signal transduction in cells across kingdoms of life. Using machine learning-based sequence analysis, we discovered that a significant number of AMPs have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. Then we demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics. The findings were published in Nature Communications (Targeting Nucleic Acid Phase Transitions as a Mechanism of Action for Antimicrobial Peptides, 10.1038/s41467-023-42374-4) and also presented at three conferences (EMBO | EMBL Symposium: Cellular mechanisms driven by phase separation; Biomolecular Condensates 2.0 Symposium; Biomolecular Condensate Collaborative Symposium: Liquid-liquid phase separation of biopolymers).
Finally, all individual technologies were combined into a single platform, MicroREvolution (Figure 1), enabling one to screen large DNA-encoded antimicrobial peptide libraries in a cost and time-efficient way. The platform was introduced to the public during the BPS conference in San Diego, USA in 2023.
Additionally, state-of-the-art technologies developed in the host laboratory were employed to study the mechanisms of action of cell-penetrating AMPs. We discovered that AMPs have a lot in common with the biomolecules that undergo liquid-liquid phase separation (LLPS), which is emerging as a key principle controlling subcellular organisation, and was shown to be involved in a variety of biological processes including RNA metabolism, ribosome biogenesis, DNA damage response and signal transduction in cells across kingdoms of life. Using machine learning-based sequence analysis, we discovered that a significant number of AMPs have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. Then we demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics. The findings were published in Nature Communications (Targeting Nucleic Acid Phase Transitions as a Mechanism of Action for Antimicrobial Peptides, 10.1038/s41467-023-42374-4) and also presented at three conferences (EMBO | EMBL Symposium: Cellular mechanisms driven by phase separation; Biomolecular Condensates 2.0 Symposium; Biomolecular Condensate Collaborative Symposium: Liquid-liquid phase separation of biopolymers).
Shortly, we expect to present a set of AMP variants with improved characteristics that we have found using the MicroREvolution platform developed during this project. The introduction of such a high-throughput platform that drastically facilitates the screening of DNA-encoded AMP mutant libraries will have a drastic impact on the development of new peptide-base antimicrobials, and, hopefully, will contribute to the discovery of AMPs with high pharmaceutical potential. The discovery of a new mechanism of action of antimicrobial peptides that we made will hopefully open a new route for the development of effective AMPs.