Membrane Effects of Antimicrobial Peptides

Abstract

Antimicrobial resistance is becoming an increasing challenge that threatens the human health system. The overuse of antibiotics has led to an acceleration in the emergence of antibiotic-resistant bacteria, the result of bacterial adaption to evolutionary pressure when they encounter antibiotics. Thus, bacteria constantly evolve resistance genes in order to survive, which makes it hard if not impossible to find an effective antibiotic that does not induce any antibiotic resistance at all. Chemical modification has helped to extend the useful lifetime of clinically used antibiotics that encountered resistance. Yet, this likely only postpones the inevitable clinical failure of a whole class of antibiotics in the future. Hence, it is important to discover new antibiotics and study their modes of action. This thesis is centered on studying the mode of action (MoA) of antimicrobial peptides (AMPs). These AMPs form a large family of peptides that have broad spectrum antibiotic activity against bacteria including antibiotic-resistant bacteria. They are regarded as potential candidates to tackle the rising antimicrobial resistant problem. Chapter 1 introduces the bacterial cell wall synthesis pathway and its central precursor Lipid II as well as the family of lantibiotics, a large family of post-translationally modified AMPs. Those AMPS have the potential to tackle the antibiotic-resistant bacteria problem. In Chapter 2, we briefly describe the regulation of intracellular pH homeostasis, ion homeostasis and ATP synthesis, three interrelated phenomena, in bacteria. The intracellular proton gradient (∆pH), electrical potential (∆ψ) and ATP synthesis/hydrolysis are actively or passively regulated by bacteria when under outside stresses such as antibiotic treatment. This is especially relevant for AMPs that target the bacterial membrane as the membrane plays a central role in all these processes. Monitoring the changes of ∆pH, ∆ψ and ATP level thus provides important insight into the MoAs of these AMPs. Chapter 3 introduces epilancin 15X and the first steps to unravel its mode of action. Here, 5 probes are used to study the membrane effects of epilancin 15X and to compare these to those caused by nisin and the chemical variant of teixobactin, [R4L10]-teixobactin, in the gram-positive bacteria Staphylococcus simulans, Micrococcus flavus and Bacillus megaterium. Chapter 4 is zooming in on the mode of action of epilancin 15X and the possible involvement of Lipid II. Clear interaction between epilancin 15X and Lipid II was observed by antagonism-based experiments. However, epilancin’s interaction with Lipid II did not lead to membrane effects. While it remained uncertain if Lipid II is the target of epilancin 15X, depolarization assays did point to the involvement of a target within a polyisoprene-based biosynthesis pathway, as clear effects could be observed on the activity of epilancin 15X by compounds that acted specifically on these pathways. Yet, unfortunately, the exact target of epilancin 15X remains obscure so far. Chapter 5 studies the mode of action of brevibacillins. They belong to a novel class of non-ribosomally produced lipo-tridecapeptides and exhibit activity towards antimicrobial resistant pathogens. Binding to Lipid II and membrane permeabilization were thus considered as 2 independent modes of action of brevibacillins.