Endolysins are a class of bacteriophage-encoded peptidoglycan hydrolases expressed during the late stages of a phage replication cycle that function to lyse the bacterial host cell wall in order to release progeny phage. Significantly, when these enzymes are purified and added exogenously to susceptible Gram-positive pathogens which have exposed peptidoglycan, degradation of the cell wall leading to osmotic lysis and subsequent bacterial cell death can result in seconds. As a result, these enzymes offer a unique antimicrobial alternative and are therefore referred to as enzybiotics. One particular enzybiotic, PlyC, has a distinctive ability to translocate eukaryotic membranes and retain killing activity in an intracellular environment. This makes it a particularly interesting target molecule for developing a treatment against bacterial species that can occupy intracellular niches to survive traditional antimicrobial prophylaxis. However, the initial interaction of PlyC at the membrane-fluid interface and the cell penetration processes that take place remain unknown. Investigation of molecular-scale aspects of such membrane interactions can be achieved using sparsely-tethered lipid bilayer membranes (stBLMs), a robust planar biomimetic lipid membrane model.
In this study, we present first steps towards a mechanistic understanding of how the binding domain PlyCB initiates membrane translocation. Our approach combines complementary surface-sensitive techniques to probe membrane integrity during PlyCB exposure (electrochemical impedance spectroscopy), membrane-binding affinity and binding kinetics of PlyCB (surface plasmon resonance) and the structure of the PlyCB/membrane complex (neutron reflectometry). While the interaction of PlyCB with purely zwitterioninc lipids is low, the protein interacts strongly with anionic membranes that contain phosphatidylserine (PS) above threshold concentration. Neutron reflection data show that PlyCB binding to the membrane surface is followed by penetration into the hydrophobic membrane core. Because eukaryotic membranes present largely uncharged plasma membrane to their environment, our results imply that PlyC more likely uses a surface cell receptor for cellular entry.