title: A bacteriophage endolysin that eliminates intracellular streptococci.
abstract: PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, United States.
authors.forename: Marilia
authors.initials: M
authors.lastname: Barros
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, United States.
authors.forename: Tarek
authors.initials: T
authors.lastname: Vennemann
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.|National Institute of Standards and Technology, Gaithersburg, United States.
authors.forename: D Travis
authors.initials: DT
authors.lastname: Gallagher
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Linden
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Heselpoth
authors.affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, United States.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Spencer
authors.affiliation: Animal Biosciences and Biotechnology Lab, Agricultural Research Service, USDA, Beltsville, United States.
authors.forename: David M
authors.initials: DM
authors.lastname: Donovan
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.|Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Rockville, United States.
authors.forename: John
authors.initials: J
authors.lastname: Moult
authors.affiliation: Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, United States.
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Fischetti
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, United States.|Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Heinrich
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, United States.|Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, United States.|Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States.
authors.forename: Mathias
authors.identifier: 0000-0001-6666-916X
authors.initials: M
authors.lastname: Lösche
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, United States.|Department of Veterinary Medicine, University of Maryland, College Park, College Park, United States.
authors.forename: Daniel C
authors.identifier: 0000-0003-3248-4831
authors.initials: DC
authors.lastname: Nelson
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism / Q000382:microbiology; D050296:Microbial Viability / Q000187:drug effects*; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010718:Phosphatidylserines / Q000378:metabolism; D021381:Protein Transport; D017108:Streptococcus Phages / Q000201:enzymology*; D013297:Streptococcus pyogenes / Q000187:drug effects*
chemical_list: D010718:Phosphatidylserines; D010450:Endopeptidases; C001143:endolysin
keywords: <i>s. pyogenes</i>; bacteriophage; biochemistry; biophysics; endolysin; human; membrane protein; structural biology; structure/function