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Introduction of protein transduction domains to the N‐terminus of β‐defensins influences microbicidal activity
Author(s) -
Tai Kenneth Patrick,
Le Valerie V.,
Ouellette Andre J.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.117.7
Subject(s) - transduction (biophysics) , fusion protein , peptide , defensin , in vitro , antimicrobial peptides , cytosol , escherichia coli , intracellular , chemistry , cytotoxic t cell , cytotoxicity , beta defensin , recombinant dna , biochemistry , microbiology and biotechnology , biology , gene , enzyme
Protein transduction domains (PTDs) enable protein fusion partners to transmigrate the cell membrane of mammalian cell lines to the cytosol. To test whether conjugation of PTDs to α‐defensins enhances microbicidal activity by membrane disruptive mechanisms, we prepared recombinant α‐defensins modified with N terminal PTDs. Rhesus myeloid α‐defensin‐4 (RMAD‐4) and mouse Paneth cell α‐defensin cryptdin‐4 (Crp4) were expressed in E. coli , introducing TAT (YGRKKRRQRRR) and nona‐Arg (9R) at both peptide N‐termini. In in vitro bactericidal peptide assays against E. coli, L. monocytogenes, V. cholerae, S. Typhimurium and S. aureus , both PTD‐conjugated peptides exhibited markedly increased activities when compared to the parent molecules. For example, at 0.75 μM, TAT‐RMAD‐4 effectively killed 99.9% of S. Typhimurium CS022 in contrast to RMAD‐4 which had no microbicidal effect at this concentration. Measurements of induced permeabilization of live E. coli cells showed that PTD‐modified peptides were significantly more membrane disruptive. Cytotoxicity assays on RAW264.7 cells showed that PTD fusion to RMAD‐4 and Crp4 imparted cytotoxic activity to the peptides at levels >10 μM. β‐Defensins with N‐terminal PTDs have enhanced antibacterial activities, suggesting potential applications against intracellular infections. Supported by NIH grants AI059346 and DK044632.