Interaction of the Phage Endolysin PlyC with Model Membranes

Abstract

Endolysins are bacteriophage-encoded peptidoglycan hydrolases expressed during the late stages of a phage replication cycle that function to lyse the bacterial cell wall, thus enabling progeny phage release. When exogenously added, these enzymes lyse the peptidoglycan of Gram-positive pathogens, resulting in osmotic lysis and cell death. One particular endolysin, PlyC, has a distinctive ability to translocate eukaryotic membranes and retain killing activity in the intracellular environment, making it a particularly interesting target molecule for the development of novel therapeutic approaches. However, the protein-membrane interactions and the cell penetration processes remain unknown. Here, we investigate molecular-scale aspects of such membrane interactions using sparsely-tethered lipid bilayer membranes (stBLMs), a robust planar biomimetic lipid membrane model.Applying complementary surface-sensitive techniques such as surface plasmon resonance, electrochemical impedance and neutron reflectometry, we demonstrate the first steps towards a mechanistic understanding of how the PlyC binding domain, PlyCB, initiates membrane translocation. Our data reveals that while the interaction of PlyCB with purely zwitterionic membranes is negligible, the protein strongly interacts with anionic membranes that contain phosphatidylserine (PS) above a well-defined concentration threshold. In contrast, PlyCB affinity for other anionic lipids tested is low, suggesting specificity for PS rather than non-specific ionic interactions. Furthermore, the PlyCB point mutant R66E that lacks the ability to translocate membranes has likewise lost affinity for PS. With Neutron reflection we identified two distinct PlyCB membrane-association modes. Depending on PS membrane concentration, PlyCB is either peripherally associated or membrane-spanning. Because the outer leaflet of eukaryotic membranes is largely zwitterionic, our findings imply that PlyC induces and/or recognizes PS exposure during cellular uptake. In addition, these results show how lipid membrane surface charge density and composition play a critical role for PlyC internalization.