Enterococcal RseP as a target for new diagnostics and antibiotics

Vancomycinresistente enterokokker (VRE) er ansvarlige for ulike infeksjoner hos mennesker, og behandlingen av disse er blitt utfordrende fordi bakteriene har blitt motstandsdyktige mot de fleste antibiotika. I dette prosjektet har vi tatt utgangspunkt i et nylig oppdaget et antimikrobielt peptid/bakteriosin som heter enterocin K1. Dette bakteriosinet har en meget spesifikk og potent aktivitet mot VRE, spesielt mot Enterococcus faecium som utgjør 80-85% av enterokokkinfeksjoner. Enterocin K1 binder seg til det transmembrane proteinet RseP, som fungerer som reseptor, og dreper deretter målceller ved å ødelegge membranfunksjoner. Fordi enterocin K1 har en annen virkningsmekanisme enn antibiotika, er dette bakteriosinet også aktiv mot multi-resistente VRE. Et annet viktig aspekt med dette systemet er at reseptorproteinet, RseP, er et meget egnet målprotein for utvikling av nye antibiotika, siden dette proteinet også er viktig for enterokokkenes evne til å etablere infeksjoner. I dette prosjektet har vi utviklet en metode for å rense membranreseptoren og vi har studert i detalj hvordan bakteriosinet gjenkjenner og binder til reseptoren. Vi har brukt denne grunnleggende kunnskapen til å lage varianter av bakteriosinet som virker mot nye patogene bakterier (inkludert Staphylococcus haemolyticus) og til å vise hvordan bakteriosinet kan brukes til diagnostikk for å detektere enterokokker i urinprøver. Videre har vi også vist at bakteriosinet er lovede for behandling av infeksjonsmodeller i modellorganismer. Prosjektet har lagt til rette for videre arbeid for å utnytte bakteriosiner til infeksjonsbehandling, diagnostikk og andre anvendelser.

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.