Probing the Internalization Mechanism of a Bacteriophage-encoded Endolysin that can Lyse Extracellular and Intracellular Streptococci

dc.contributor.advisorNelson, Daniel Cen_US
dc.contributor.authorShen, Yangen_US
dc.contributor.departmentMolecular and Cell Biologyen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2013-10-03T05:33:24Z
dc.date.available2013-10-03T05:33:24Z
dc.date.issued2013en_US
dc.description.abstractBacteriophage-encoded peptidoglycan hydrolases, or endolysins, have been investigated as an alternative to antimicrobials due to their ability to lyse the bacterial cell wall upon contact. However, pathogens are often able to invade epithelial cells where they can repopulate the mucosal surface after antibiotic or endolysin prophylaxis. Thus, there is growing interest in endolysins that can be engineered, or inherently possess, a capacity to internalize in eukaryotic cells such that they can target extracellular and intracellular pathogens. Previously, one streptococcal specific endolysin, PlyC, was shown to control group A Streptococcus localized on mucosal surfaces as well as infected tissues. To further evaluate the therapeutic potential of PlyC, a streptococci/human epithelial cell co-culture model was established to differentiate extracellular vs. intracellular bacteriolytic activity. We found that a single dose (50 μg/ml) of PlyC was able to decrease intracellular streptococci by 96% compared to controls, as well as prevented the host epithelial cells death. In addition, the internalization and co-localization of PlyC with intracellular streptococci was captured by confocal laser scanning microscopy. Further studies revealed the PlyC binding domain alone, termed PlyCB, with a highly positive-charged surface, was responsible for entry into epithelial cells. By applying site-directed mutagenesis, several positive residues (Lys-23, Lys-59, Arg-66 and Lys-70&71) of PlyCB were shown to mediate internalization. We then biochemically demonstrated that PlyCB directly and specifically bound to phosphatidic acid, phosphatidylserine and phosphatidylinositol through a phospholipid screening assay. Computational modeling suggests that two cationic residues, Lys-59 and Arg-66, form a pocket to help secure the interaction between PlyC and specific phospholipids. Internalization of PlyC was found to be via caveolae-mediated endocytosis in an energy-dependent process with the subsequent intracellular trafficking of PlyC regulated by the PI3K pathway. To the best of our knowledge, PlyC is the first endolysin reported that can penetrate through the eukaryotic lipid membrane and retain biological binding and lytic activity against streptococci in the intracellular niche.en_US
dc.identifier.urihttp://hdl.handle.net/1903/14513
dc.subject.pqcontrolledMolecular biologyen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pqcontrolledMicrobiologyen_US
dc.subject.pquncontrolledBacteriophageen_US
dc.subject.pquncontrolledEndolysinen_US
dc.subject.pquncontrolledEnzyme-based therapyen_US
dc.subject.pquncontrolledGroup A Streptococcusen_US
dc.subject.pquncontrolledInternalization mechanismen_US
dc.subject.pquncontrolledIntracellular pathogenen_US
dc.titleProbing the Internalization Mechanism of a Bacteriophage-encoded Endolysin that can Lyse Extracellular and Intracellular Streptococcien_US
dc.typeDissertationen_US

Files

Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Shen_umd_0117E_14474.pdf
Size:
3.33 MB
Format:
Adobe Portable Document Format