|SHEN, YANG - University Of Maryland|
|BARROS, MARILIA - Carnegie Mellon University|
|VENNEMANN, TAREK - Carnegie Mellon University|
|GALLAGHER, D. TRAVIS - University Of Maryland|
|YIN, YIZHOU - University Of Maryland|
|LINDEN, SARA - University Of Maryland|
|HESELPOTH, RYAN - University Of Maryland|
|SPENCER, DENNIS - Rockefeller University|
|MOULT, JOHN - University Of Maryland|
|FISCHETTI, VINCENT - Rockefeller University|
|HEINRICH, FRANK - Carnegie Mellon University|
|LOSCHE, MATHIA - Carnegie Mellon University|
|NELSON, DANIEL - University Of Maryland|
Submitted to: Stem Cells and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2015
Publication Date: 3/5/2016
Citation: Shen, Y., Barros, M., Vennemann, T., Gallagher, D., Yin, Y., Linden, S.B., Heselpoth, R.D., Spencer, D.J., Donovan, D.M., Moult, J., Fischetti, V.I., Heinrich, F., Losche, M., Nelson, D.C. 2016. PlyC, a bacteriophage endolysin that is internalized by epithelial cells and retains bacteriolytic activity against intracellular streptococci. Stem Cells and Development. doi: 10.7554/eLife.13152.
Interpretive Summary: There is a need to develop antimicrobials that will treat infections where bacteria enter mammalian cells; strep throat is an example of such an infection and is caused by the bacterium Streptococcus (S.) pyogenes. Our research demonstrated that an enzyme (PlyC) produced by bacterial viruses (bacteriophage) to infect S. pyogenes and kill the bacterium by breaching its cell wall integrity. PlyC was also successful in treating the infection of animal cells ex vivo (maintained in artificial growth systems in the lab) and subsequent eradication of S. pyogenes residing within the animal cell. This finding was followed up with in depth analyses to identify elements on the outer surface of the animal cell that interact with specific amino acids within the PlyC enzyme to permit entrance of PlyC into the cell. Assays that removed the PlyC amino acids that enabled the PlyC uptake into the mammalian cell confirmed how infection occurs. This is the first report demonstrating that the bacterial virus enzymes have a natural mechanism to enter mammalian cells. Furthermore, the results support the need for additional studies to examine the use of PlyC as an antimicrobial agent in lieu of classical antibiotics to eradicate bacteria residing both outside and within mammalian cells to reduce the incidence of disease.
Technical 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 mutants. This is the first report that a native endolysin can translocate epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extra and intracellular milieu and as a scaffold for engineering other functionalities.