| Popis: |
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, gains cellular entry and clears intracellular streptococci without compromising cell viability, implying that an unknown mechanism provides entry to the streptococci-specific endolysin into mammalian cells. Here, we investigate the molecular basis of PlyC binding domain, PlyCB, ability to bind and cross the mammalian plasma membrane using synthetic model membranes.Complementary surface-sensitive techniques showed membrane integrity during PlyCB exposure and quantified membrane-binding affinity. Surface plasmon resonance 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. Neutron reflection data showed that PlyCB binding to the membrane surface is followed by penetration into the hydrophobic membrane core, while impedance spectroscopy confirms that the membrane integrity is not affected. Those findings provide clues to a mechanistic understanding of how PlyCB binds and initiates membrane translocation. After the initial membrane interaction, PlyCB is known to subsequently internalize and be trafficked inside the cell where it eventually kills the streptococci. PlyC's entry point into the cell, PS is primarily located in the inner leaflet of the plasma membrane where it would not be available for binding external endolysin. However, in cells stressed by bacteria assault and inflammation, lipid asymmetry is compromised: PS may be exposed on the outer plasma membrane. |
