Multicellular organisms live, by and large, harmoniously with microbes. The cornea of the eye of an animal is almost always free of signs of infection. The insect flourishes without lymphocytes or antibodies. A plant seed germinates successfully in the midst of soil microbes. How is this accomplished? Both animals and plants possess potent, broad-spectrum antimicrobial peptides, which they use to fend off a wide range of microbes, including bacteria, fungi, viruses and protozoa. What sorts of molecules are they? How are they employed by animals in their defence? As our need for new antibiotics becomes more pressing, could we design anti-infective drugs based on the design principles these molecules teach us?

A family of peptides with broad-spectrum antimicrobial activity has been isolated from the skin of the African clawed frog Xenopus laevis. It consists of two closely related peptides that are each 23 amino acids and differ by two substitutions. These peptides are water soluble, nonhemolytic at their effective antimicrobial concentrations, and potentially amphiphilic. At low concentrations they inhibit growth of numerous species of bacteria and fungi and induce osmotic lysis of protozoa. The sequence of a partial cDNA of the precursor reveals that both peptides derive from a common larger protein. These peptides appear to represent a previously unrecognized class of vertebrate antimicrobial activities.Over the past several years my laboratory has utilized the Xenopus laevis oocyte system to study RNA expression in eukaryotes (1-5). Ovaries used in these studies were removed surgically from anesthetized adult females. Incisions were made through both the skin and the nonadherent muscular layer of the abdomen into the peritoneum. After removal of the ovaries, the muscular wall and the skin were separately repaired with sutures. Despite the nonsterile surgical procedure and the microbially contaminated water-filled tanks to which the animals were returned immediately after surgery, it was extremely rare for these surgical wounds to develop infection. Indeed, sutures dissolved after several weeks, and normal healing of the scar almost always occurred. Infections were not seen on the cut margins of the wound, at the sites of suture placement, or within the communicating subdermal space or peritoneum. Healing occurred with little gross evidence of inflammation or cellular reaction at the wound sites. The absence of infection under these conditions was medically remarkable.The manner in which wound healing occurs in this animal suggested that there might be a "sterilizing" activity in the skin. I report here the characterization of a family of potent antimicrobial peptides purified from female X. laevis skin.These peptides may be responsible for the extraordinary freedom from infection characteristic ofwound healing in this animal and appear to constitute a previously unrecognized antimicrobial host-defense system. MATERIALS AND METHODS Purification. The skin of one adult female X. laevis was used for each preparation. All procedures were performed at 4°C. The animal was anesthetized by immersion in 0.1% tricaine for about 15 min, and the skin from the ventral surface including that overlying abdomen, thorax, and legs was surgically removed. The animal was subsequently sacrificed. The skin was homogenized in a chilled blender containing 4 vol (based on initial skin weight) of 0.2 M sodium acetate (pH 4.0), 0.2% Triton X-100/pepstatin A at 50 ,ug/ml, leupeptin at 50 ,ug/ml, and 3 mM phenylmethylsulfonyl fluoride added just prior to homogenization (phenylmethylsulfonyl fluoride was dissolved in 100% ethanol, so buffers contained the corresponding ethanol concentrations). The foamy gray homo'genate was centrifuged ...

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification. We mapped FOP to chromosome 2q23-24 by linkage analysis and identified an identical heterozygous mutation (617G --> A; R206H) in the glycine-serine (GS) activation domain of ACVR1, a BMP type I receptor, in all affected individuals examined. Protein modeling predicts destabilization of the GS domain, consistent with constitutive activation of ACVR1 as the underlying cause of the ectopic chondrogenesis, osteogenesis and joint fusions seen in FOP.

A human bronchial xenograft model was used to characterize the molecular basis for the previously described defect in bacterial killing that is present in the cystic fibrosis (CF) lung. Airway surface fluid from CF grafts contained abnormally high NaCl and failed to kill bacteria, defects that were corrected with adenoviral vectors. A full-length clone for the only known human beta-defensin (i.e., hBD-1) was isolated. This gene is expressed throughout the respiratory epithelia of non-CF and CF lungs, and its protein product shows salt-dependent antimicrobial activity to P. aeruginosa. Antisense oligonucleotides to hBD-1 ablated the antimicrobial activity in airway surface fluid from non-CF grafts. These data suggest that hBD-1 plays an important role in innate immunity that is compromised in CF by its salt-dependent inactivation.

The airway surface is an important host defense against pulmonary infection. Secretion of proteins with antimicrobial activity from epithelial cells onto the airway surface represents an important component of this innate immune system. Defensins are the best characterized epithelial-derived peptide antibiotics. A member of another family of peptide antibiotics called cathelicidins recently was identified from human bone marrow. We show in this paper that this human peptide named LL-37͞hCAP-18 also may play a role in innate immunity of the human lung. In situ hybridization localized high levels of LL-37͞hCAP-18 RNA to surface epithelial cells of the conducting airway as well as serous and mucous cells of the submucosal glands. LL-37͞hCAP-18 peptide with antimicrobial activity was partially purified from airway surface f luid from human lung and a human bronchial xenograft model. The synthetic peptide LL-37 demonstrated antibiotic activity against a number of Gram-negative and Gram-positive organisms including Pseudomonas aeruginosa; bacterial killing of LL-37 was sensitive to NaCl and was synergistic with lactoferrin and lysozyme. In summary, we show that LL-37͞hCAP-18 is a peptide with broad antimicrobial activity that is secreted onto the airway surface from epithelial cells of the human lung.

Fibrodysplasia ossificans progressiva (FOP) is an autosomal dominant human disorder of bone formation that causes developmental skeletal defects and extensive debilitating bone formation within soft connective tissues (heterotopic ossification) during childhood. All patients with classic clinical features of FOP (great toe malformations and progressive heterotopic ossification) have previously been found to carry the same heterozygous mutation (c.617G>A; p.R206H) in the GS activation domain of activin A type I receptor/activin-like kinase 2 (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor. Among patients with FOP-like heterotopic NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ossification and/or toe malformations, we identified patients with clinical features unusual for FOP. These atypical FOP patients form two classes: FOP-plus (classic defining features of FOP plus one or more atypical features) and FOP variants (major variations in one or both of the two classic defining features of FOP). All patients examined have heterozygous ACVR1 missense mutations in conserved amino acids. While the recurrent c.617G>A; p.R206H mutation was found in all cases of classic FOP and most cases of FOP-plus, novel ACVR1 mutations occur in the FOP variants and two cases of FOP-plus. Protein structure homology modeling predicts that each of the amino acid substitutions activates the ACVR1 protein to enhance receptor signaling. We observed genotype-phenotype correlation between some ACVR1 mutations and the age of onset of heterotopic ossification or on embryonic skeletal development.

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin-the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

Previous studies have implicated the novel peptide antibiotic human ␤ -defensin 1 (hBD-1) in the pathogenesis of cystic fibrosis. We describe in this report the isolation and characterization of the second member of this defensin family, human ␤ -defensin 2 (hBD-2). A cDNA for hBD-2 was identified by homology to hBD-1. hBD-2 is expressed diffusely throughout epithelia of many organs, including the lung, where it is found in the surface epithelia and serous cells of the submucosal glands. A specific antibody made of recombinant peptide detected hBD-2 in airway surface fluid of human lung. The fully processed peptide has broad antibacterial activity against many organisms, which is salt sensitive and synergistic with lysozyme and lactoferrin. These data suggest the existence of a family of ␤ -defensin molecules on mucosal surfaces that in the aggregate contributes to normal host defense. ( J. Clin. Invest. 1998. 102:874-880.)