Introduction of human β-defensin-3 into cultured human keratinocytes and fibroblasts by infection of a recombinant adenovirus vector.
Cultured epidermal autografts and cultured skin substitute are vulnerable to infection. Human beta defensin (HBD)-3 is an antimicrobial peptide that exhibits a wide-spectrum antimicrobial activity against gram-positive/negative bacteria and fungi. This study determined whether normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity. An adenovirus vector with an HBD-3 cDNA inserted downstream of the CMV promoter (ADhBD3) was created. The HBD-3 gene was introduced into NHKs and HDFs via ADhBD3 infection. HBD-3 gene expression in each type of transfected cells was evaluated by RT-PCR. The presence of HBD-3 peptide in the culture supernatants of each type of transfected cells was evaluated by Western blotting. The antimicrobial activities of the culture supernatants of each type of transfected cells against several bacterial strains were also measured. Both NHKs and HDFs infected with ADhBD3 expressed the HBD-3 gene and secreted HBD-3 peptide into culture supernatants. These supernatants exhibited a strong bacteriocidal activity against a Staphylococcus aureus reference strain and methicillin-resistant S. aureus (MRSA). NHKs and HDFs transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity against S. aureus and MRSA.
Suzuki
Yosuke
Y
Department of Emergency and Critical Care Medicine, Tokai University School of Medicine, Kanagawa, Japan. y.suzuki@is.icc.u-tokai.ac.jp
Inokuchi
Sadaki
S
Takazawa
Kensuke
K
Umezawa
Kazuo
K
Saito
Takeshi
T
Kidokoro
Masako
M
Tanaka
Makiko
M
Matsuzawa
Hideyuki
H
Inoue
Shigeaki
S
Tuchiya
Izumi
I
Ando
Kiyoshi
K
D000256:Adenoviridae; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D004926:Escherichia coli / Q000187:drug effects; D005347:Fibroblasts / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000187:drug effects; D014162:Transfection / Q000379:methods; D023083:beta-Defensins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C422950:DEFB103A protein, human; D023083:beta-Defensins