Periodic Reporting for period 1 - BACtheWINNER (Bacteriocins from interbacterial warfare as antibiotic alternative)
Berichtszeitraum: 2023-04-01 bis 2025-09-30
Antimicrobial resistance (AMR) is an ongoing global crisis exacerbated by lack of discovery of novel antimicrobials. New alternatives that are target-specific and do not cause collateral damage to the microbiome would revolutionise human and animal health. Bacteriocins are small antibacterial peptides produced by bacteria that are gene-encoded and can be narrow or broad spectrum. They have the potential for the development of new antimicrobial molecules through discovery and protein engineering resulting in potent, targeted antimicrobials. They have many possible applications including treatment of gut and topical infections and microbiome editing. However, basic research issues stand in the way of their exploitation including low production levels often in unsuitable hosts, resistance development in bacterial targets, and inadequate potency/inhibition spectrum. The main aim of BACtheWINNER is to develop bacteriocins as novel antimicrobials by solving these challenges through combining and innovating state-of-the-art technologies in peptide bioengineering and molecular genetics, which will lay the foundation for the generation of this new family of therapeutics that target WHO and CDC priority pathogens and undesirable microbiota. The immediate outputs of BACtheWINNER will create the foundations to bridge the current gap between scientific discovery of bacteriocins in the laboratory and their actual use as a new family of biotherapeutics in modern medicine. But BACtheWINNER will go far beyond its immediate scientific goals. Indeed, by tapping into the very molecules that bacteria themselves use for competitive survival, BACtheWINNER will establish bacteriocins as essential biological tools to control and improve the microbial communities on our planet.
The project's technical and scientific activities have encompassed a comprehensive strategy for the discovery, characterization, and initial optimization of novel bacteriocins. This involved extensive in silico screening of genomic databases, leading to the identification of numerous potential bacteriocin-encoding genes, particularly within the Nisin-like, leaderless, and circular bacteriocin classes. A significant achievement was the identification of a substantial number of leaderless bacteriocin core peptides within the Actinomycetota phylum, a previously underexplored source. Functionality was assessed through culturing selected strains and employing MALDI-TOF mass spectrometry to confirm bacteriocin production. Several Nisin-like lanthipeptides were heterologously expressed and demonstrated antimicrobial activity. The production of novel bacteriocins, including Streptocyclin BTW and the leaderless peptides Arcanocin and Arachnicin, was confirmed. Antimicrobial testing against CDC and WHO priority pathogens revealed promising activity for certain candidates, such as Actifensin and Capidermicin against VRE. Genetic characterization involved whole-genome sequencing and bioinformatic analysis of bacteriocin-producing isolates, enabling the identification of biosynthetic gene clusters. Purification and initial characterization efforts yielded purified forms of several novel bacteriocins, including circular and leaderless types, with structural insights gained through NMR analysis. The mechanisms of action of Actifensin and Capidermicin were elucidated through collaborations, revealing cell wall targeting and pore-forming activity, respectively. Initial cytotoxicity assays provided encouraging safety profiles for lead candidates. Furthermore, novel methodologies were developed and implemented across discovery, production, and characterization, including enhanced bioinformatic tools, direct synthesis of leaderless bacteriocins, and the establishment of E. coli and P. pastoris as a production platform. The project also explored novel microbiome sources, such as isolating producers from exotic zoo animals, resulting in the identification of novel antimicrobials which were varying in their structural complexity. Inter-disciplinary collaborations significantly advanced the understanding of Actifensin and Capidermicin's mechanisms and structural properties. Knowledge transfer was facilitated through research mobility between institutions. The outcomes of these actions have resulted with a biobank of novel bacteriocin candidates, where detailed characterization of their properties and mechanisms is ongoing, and promising leads for future therapeutic and biotechnological applications including microbiome editing.
The project has yielded several results that represent substantial advancements beyond the current state of the art in bacteriocin research. The extensive in silico screening and subsequent characterization have significantly expanded the known diversity of bacteriocins, particularly within the leaderless class and in previously underexplored phyla like Actinomycetota. The discovery and characterization of five novel circular bacteriocins, including the first reported from Lactococcus raffinolactis, and the identification of Paenibacins with an unprecedented seven lanthionine rings, represent significant breakthroughs in their respective fields. The unexpected isolation of a Paenibacillus strain with activity against a honeybee pathogen and the findings related to methane reduction and subclinical mastitis diagnosis highlight the broad and potentially transformative impact of this research. The elucidation of the distinct mechanisms of action of Actifensin and Capidermicin provides crucial knowledge for rational drug design. In addition, the characterization of orphan bacteriocin immunity mechanisms within certain bacteria are particularly significant since it could compromise the clinical efficacy. The characterization of orphan bacteriocin immunity mechanisms within certain bacteria are particularly significant since it could compromise clinical efficacy. Key needs to ensure further uptake and success include continued research into the structure-function relationships and mechanisms of action of the novel bacteriocins, demonstration of their efficacy in relevant pre-clinical models, securing access to markets and finance for commercialization, robust intellectual property rights support, fostering international collaborations, and engaging with regulatory bodies to establish supportive frameworks. An overview of the results indicates a significant expansion of the bacteriocin landscape, the development of novel tools and methodologies for the identification of promising candidates with activity against critical pathogens and in novel applications, and the establishment of valuable inter-disciplinary collaborations and knowledge transfer pathways. These outcomes provide a strong foundation for future translational efforts aimed at addressing challenges in human and animal health, food safety, and environmental sustainability.