Cloning And Expression Of Human (3-defensin 3 And Its Up-regulated Expression In Skin Infections

Department of Burn Surgery, Xijing Hospital, Xi'an 710032Bum wound sepsis remains a common and serious complication of major bum injury. Use of topical agents and systemic antibiotics temporarily controls spread of infection, but has been dramatically challenged with increasing emergence of highly resistant strains, leading to a high morbidity and mortality and thus making it more important to find new types of antibiotics that will cause no drug-resistance. Recent studies have demonstrated that eukaryotic cells can produce a great number of antimicrobial peptides that have potent bactericidal activity against a broad spectrum of pathogens, and thus comprising an important pathway of host defense. Since these proteins are natural peptides and cause no drug-resistance, they can be developed to be an ideal new type antibiotic and provide a better solution for the present situation of extensive antibiotics-resistance.Defensins represent a large group of antimicrobial peptides. These small cationic molecules have a broad spectrum of antimicrobial activities against pathogens like bacteria, fungi and many other micro-organisms without drug-resistance. All the known defensins can be divided into three families, namely plant defensins, insect defensins and vertebrate defensins. Vertebrate defensins fall into two structural subfamilies, namelya-defensins and p-defensins depending on the specific pattern of their cysteine spacing and disulfide connections, a-defensins are mainly found in immune cells like neutrophils, while(3-defensins are synthesized and secreted in epithelial cells. Three human(3-defensins have been identified so far andtermed humanp-defensin 1 (hBDl) , humanp-defensin 2 (hBD2) and humanp-defensin 3 (hBD3) .The genes are mapped to chromosome 8 in a less than 1Mb region of 8p22-8p23 and encode 36, 65 and 45 amino acid peptide respectively. Each peptide has 6 cysteine residues in disulfide linkages and is rich in arginine. hBDl is constitutively expressed in kidney tubes and to a lesser extent in the pancreas, lung, breast, salivary gland, ocular anterior segment and other epithelial sites. hBD2 and hBD3 expressions are induced upon epithelial stimulation in skin and mucosal surfaces, indicating their strong relationship with infections, notably those ensue bum wounds.Like most antimicrobial peptides, defensins are cationic molecules with spatially separated hydrophobic and charged regions. This arrangement allows them to insert themselves into phospholipid membranes so that their hydrophobic regions are buried within the oily membrane interior and their cationic regions interact with anionic phospholipid head groups and water, hi the membrane, some defensins assemble into multimeric pores. The defensins preferentially disrupt bacterial membranes that are rich in negatively charged phospholipids such as in bacteria, fungi, and even some enveloped viruses. Irreversible cell injury occurs as more defensins and normally excluded molecules (ions, other peptides) enter the cell, whereas essential minerials, ions and metabolites leak out, with resultant cell death. However, conversely, the lower anionic phospholipid content of the cell membranes of higher animals may provide relative protection from collateral damage.hBD3 was first isolated from human lesional psoriatic scales and cloned from keratinocytes by J Harder et al in 2000. This new small peptide demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic either gram positive or negative microbes and showed even higher sensitivity to gram positive bacterium including multiresistant S. aureus. Recombinant hBD3 and chemically synthesized hBD3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity andbiochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supematants revealed kera...