title: Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.
abstract: Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, Korea.
authors.forename: Aeyung
authors.initials: A
authors.lastname: Kim
authors.forename: Tae-Hwan
authors.initials: TH
authors.lastname: Shin
authors.forename: Seung-Min
authors.initials: SM
authors.lastname: Shin
authors.forename: Chuong D
authors.initials: CD
authors.lastname: Pham
authors.forename: Dong-Ki
authors.initials: DK
authors.lastname: Choi
authors.forename: Myung-Hee
authors.initials: MH
authors.lastname: Kwon
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000818:Animals; D017104:Bacteriophage M13 / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D016466:CHO Cells; D021941:Caveolae / Q000378:metabolism; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D004926:Escherichia coli / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism*; D006367:HeLa Cells; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D002966:Clathrin; D015696:Gene Products, tat; D019812:Heparan Sulfate Proteoglycans; D011956:Receptors, Cell Surface