Isolation and Characterization of Human μ-Defensin-3, a Novel Human Inducible Peptide Antibiotic

Abstract

The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human ␤-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor ␣ and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.

UV-independent induction of beta defensin 3 in neonatal human skin explants

“…Cui and coworkers reported that UV promoted transcriptional increases in pro-opiomelanocortin (POMC), the protein precursor for MSH, in a cell damage and p53-dependent manner in epidermal keratinocytes 9 , supporting the hypothesis that melanocytic MC1R responses are modified by intracutaneous UV-regulated mechanisms. Similarly, recent studies reported that UVB radiation caused an increase BD3 mRNA and protein levels both in vivo and in vitro 10 , either in a cell-autonomous, damage-dependent manner or in response to inflammatory mediators such as interleukin-1 (IL-1β) and tumor necrosis factor (TNFα) known to promote its induction 11, 12 . In an effort to understand the mechanisms of how BD3 production may be influenced by UV radiation, we determined its expression in freshly isolated human skin explants.…”

Innate defences against methicillin‐resistant Staphylococcus aureus (MRSA) infection

“…β-defensin Four β-defensins (hBD1-4) have been identified and found to be expressed in various epithelial tissues: the trachea, skin, and intestine; and in monocytes and dendritic cells [8,[60][61][62][63][64][65][66][67][68][69]. Like α-defensins, hBDs have three disulphide moieties of cysteine linking residues 1-5, 2-4, and 3-6. hBD1 was first identified in haemodialysate fluid [70] and then from urogenital tissues, skin, intestine, and other tissues [61][62][63][64][65][66][67][68][69]71].…”

“…Like α-defensins, hBDs have three disulphide moieties of cysteine linking residues 1-5, 2-4, and 3-6. hBD1 was first identified in haemodialysate fluid [70] and then from urogenital tissues, skin, intestine, and other tissues [61][62][63][64][65][66][67][68][69]71]. In vitro experiments show that hBD1 is constitutively expressed and that its production is not influenced by bacterial exposure, while other hBDs are inducible when exposed to bacteria [67,[72][73][74][75]. In particular, hBD2, first identified from skin [8], is highly responsive to bacteria, pro-inflammatory stimuli (IL−1β, TNFα, LPS), and phorbol myristate acetate [63,72,74,[76][77][78][79].…”

“…Dinulos et al demonstrated that in keratinocytes from human neonatal skin, hBD2 expression is induced by bacterial contact including S. aureus, E. coli, and P. aeruginosa, while S. pyogenes induced expression poorly [73]. Harder et al reported that hBD3 is slightly induced in keratinocytes by S. aureus [67]. Because the total analysis of expression of anti-microbial peptides has not been performed in keratinocytes, we investigated the expression of hBD1-3 and LL37 in keratinocytes derived from skin exposed to S. aureus cells [74].…”

“…Since we had demonstrated the expression of hBD1-3 and LL37 in keratinocytes, we synthesized the four anti-microbial peptides in their mature forms, purified them, and then evaluated antibacterial activity against S. aureus [74]. Generally, two antibacterial assays were used for evaluation [67,74,102,119,124]. One was the minimum inhibitory concentrations (MICs) under growing conditions and the other was to measure the killing rate (%) under non-growing conditions using 10 mM phosphate buffer.…”