Enterococcal RseP as a target for new diagnostics and antibiotics

Vancomycin-resistant enterococci (VRE) are responsible for several human infections, and their treatment has become challenging because the bacteria have become resistant to most antibiotics. In this project, we started from a recently discovered antimicrobial peptide/bacteriocin called enterocin K1. This bacteriocin has a very specific and potent activity against VRE, especially against Enterococcus faecium which accounts for 80-85% of enterococcal infections. Enterocin K1 binds to the transmembrane protein RseP, which acts as a receptor, and then kills target cells by disrupting membrane functions. Because enterocin K1 has a different mechanism of action than antibiotics, this bacteriocin is also active against multi-resistant VRE. Another important aspect of this system is that the receptor protein, RseP, is very suitable as a target protein for the development of new antibiotics, since this protein is also important for the enterococci's ability to establish infections. Enterocin K1 thus mediates a two-sided attack, inhibiting both membrane functions and virulence. In this project, we have developed a method to purify the membrane receptor and we have studied in detail how the bacteriocin recognizes and binds to the receptor. We used this basic knowledge to create variants of the bacteriocin that act against new pathogenic bacteria (including Staphylococcus haemolyticus) and to show how the bacteriocin can be used for diagnostics to detect enterococci in urine samples. Furthermore, we have also shown that the bacteriocin is promising for the treatment of infection models in model organisms. The project has facilitated further work to utilize bacteriocins for infection treatment, diagnostics and other applications.

The project has resulted in detailed insights into how leaderless bacteriocins (specifically enterocin K1) works and interacts with it’s receptor. The in vitro and in vivo results emerging from the project has demonstrated the potential of these bacteriocins to treat infections and to act as diagnostic probes. These results has set the basis for further projects to decipher the function of these peptides and to develop such bacteriocins for different applications, including infection treatment, diagnostics and food preservation, including funded projects BacPress and PrevEco. Furthermore, the project has contributed to build competence on different methods for the project participants and NMBU (including membrane protein purification, murine infection assays, peptide design, biofilm assays), two doctoral degrees at NMBU finished in 2023 and increased collaboration between NMBU and University of Copenhagen. The results from the project have been communicated both by publication of scientific papers (>10) and population science articles and interviews.

Vancomycin-resistant enterococci (VRE) are involved in diverse infections in humans and treatment of these infections has been very challenging in recent years because the bacteria have become resilient to most antibiotics. We have recently discovered an antimicrobial peptide/bacteriocin called enterocin K1 that has a very specific and potent activity against VRE, especially against Enterococcus faecium which accounts for 80-85% of enterococcal infections. The bacteriocin binds specifically to a transmembrane protein (called RseP) and kills target cells by membrane disruption. Because its mode of killing (membrane disruption) is different from traditional antibiotics, which often are enzyme inhibitors, enterocin K1 readily kills multi-antibiotic resistant VRE. Most importantly, this work on enterocin K1 has revealed RseP to be a novel and seemingly excellent drug target. RseP is involved in regulated intramembrane proteolysis (RIP) in pathways that are vital for enterococcal cells to develop virulence and establish infection in animal hosts. In a rabbit endocarditis model it has been shown that, when the gene encoding RseP is deleted, the affected pathogen is attenuated in the animal host and no virulence developed. Similarly, truncation or frame shift mutations within rseP confer enterococcal cells resistant to enterocin K1 but these cells do not survive in stress conditions. Thus, binding of enterocin K implies a powerful double-attack (membrane-disruption & causing inability to develop virulence), leaving the pathogen no chance to develop infection. In this project proposal we will study the interaction between enterocin K1 and RseP to assess the potential of enterocin K1 in therapeutic treatments. Furthermore, we will use the enterocin K1-RseP system to unravel features of ligand binding to RseP which could be used to develop or screen for non-bacteriocin-based compounds able to inhibit VRE and other RseP containing pathogens such as Staphylococcus aureus.