Role of Membranes in Synergistic Activity of Antimicrobial Peptides

Antibiotic-resistant bacterial strains represent a global health problem with a strong social and economic impact. Because of increasing antibiotic resistance against conventional antibiotics, there is an urgent need for the development of antibiotics with novel mechanisms of action. Significant research efforts have been devoted to antimicrobial peptides, because they interfere physically with the cell membrane of bacteria and offer a fast killing, non-receptor-mediated defense against invading pathogens. Of particular interest is the observation of synergistic activity of antimicrobial peptides, i.e. application of mixtures of antimicrobial peptides shows larger effects than the sum of individual applications. However, a quantitative understanding of peptide synergism is lacking at large. The aim of the project is to derive the quantitative physicochemical foundations pertaining to this effect. A specific focus of the project is on the role of the bacterial membrane and its structural and dynamic coupling to peptides. Previous data suggests that synergism arises from a preconditioning of the membrane by one peptide, which helps the insertion and membrane perturbation by the second peptide. We will combine a unique complementary set of experimental and theoretical tools to master the aims of the project. In particular we will apply (i) X-ray/neutron scattering to derive the structural and elastic response of bacterial membrane mimics, including peptide induced membrane domains, (ii) solid-state NMR to determine accurately the location and orientation (topology) of these peptides in aligned membranes, including peptide aggregation and dynamics and (iii) molecular dynamics simulations to gain atomistic insight on the molecular interactions involved. The application involves the development of new methods, such as the deconvolution of the dynamics of membrane bound peptides, as well as correlating the motions of peptides and lipids in membrane environments using solid-state NMR spectroscopy. The combination of all data will provide a complete picture of the physical events and the underlying modes of action with methodological improvements that can be applied to lipid/peptide interactions in general. Specifically, we will study mixtures of PGLa and magainin 2, antimicrobial peptides from frog skin, as well as the naturally occurring heterodimer distinctin and homodimer homotarsinin. Further, we will study chemically linked PGLa and magainin 2. In doing so, we will address the differential effects of crosslinking of like or unlike peptide helices and their physical relation to peptide synergism. Further this allows us also to address the question whether or not peptide synergism is achieved through peptide pair formation. It is expected that this project will lead to ground breaking scientific knowledge that will enable us to establish molecular mechanism(s) for the synergistic activity of antimicrobial peptides as an indispensable prerequisite for the future design of new more potent therapeutic approaches against bacteria.

Antibiotic-resistant bacterial strains represent a global health problem with a strong social and economic impact. The situation may become worse because of the steady increase of pathogens becoming resistant to conventional antibiotics while the approval of new antibiotics declines. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. This project focused on the synergistic activity of two antimicrobial peptides, PGLa and magainin-2, isolated from frog skin, i.e. application of mixtures of these peptides shows larger effects than the sum of individual applications. Such a synergistic activity was only observed in model systems that mimic the cytoplasmic membrane of Gram-negative bacteria such as E. coli or P. aeruginosa. This finding indicates that the lipid composition of membranes plays an essential role. Thereby the main component phosphatidylethanolamine is the key molecule. This lipid, characterized by an inverted cone-like molecular geometry, forms a tight energetic barrier, which can be overcome more easily in the presence of both peptides allowing an insertion of the peptides in the membrane interface. In turn the membrane structure is strongly perturbed finally leading to cell death. Application of complementary biophysical approaches of the working groups at the University of Graz and Strasbourg, respectively, as well as of the group at CEITEC in Brno, Czech Republic, allowed getting a full picture of the process. Location and orientation (topology) of these peptides in aligned lipid membranes were determined primarily by solid-state NMR (Strasbourg). X-ray and neutron scattering experiments combined with a newly developed data analysis (Graz), which also included information from molecular dynamics simulation (Brno), yielded further details: In the synergistic concentration range and in the presence of magainin-2 PGLa penetrates less deeply into the membrane interface resulting in larger membrane defects and the formation of large multilamellar aggregates. Thereby, the peptides form a fibril-like structure, which is in agreement with NMR data. Our studies demonstrate that there is no need for the formation of stable peptide pores within the cell membranes for synergistic activity of PGLa and magainin-2, which contradicts frequent reports in literature. Elucidating the molecular mechanism of synergistic activity of antimicrobial peptides opens new avenues for novel strategies regarding the treatment of Gram-negative bacteria. In particular multiresistant Enterococci causing severe blood and urinary tract infections or Acinetobacter, especially prevalent in hospitals being resistant to almost all available antibiotics, are major challenges of clinicians.