Lipophilin, a novel heterodimeric protein of human tears.

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; pI, 9.47) was homologous to the C1 and C2 components of prostatein ('estramustine-binding protein'), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; pI, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid-regulated and steroid-binding molecules.

Activity of protegrins against yeast-phase Candida albicans.

We used a two-stage radial diffusion assay to perform a structure-activity study of the antifungal effects of protegrin-1 (PG-1) on yeast-phase Candida albicans. While doing so, we computed MICs from the radial diffusion assay data by three methods and compared the respective values with results from colony count and broth microdilution assays. This allowed us to identify several technical modifications that improved the sensitivity and accuracy of radial diffusion assays. We found that both PG-1 and enantiomeric PG-1 (composed exclusively of D-amino acids) were potently fungicidal for yeast-phase C. albicans. The protegrins PG-2, -3, and -5, but not PG-4, were as effective as PG-1. At least one intramolecular disulfide bond was required to retain optimal candidacidal activity at physiological NaCl concentrations. Truncated variants of PG-1 that lacked its first four residues showed decreased candidacidal activity, although their activity against bacteria was substantially intact. Altering the beta-turn region (residues 9 to 12) of PG-1 or its variants further decreased candidacidal activity. These studies suggest that only 12 residues are needed to endow protegrin molecules with strong antibacterial activity and that at least 4 additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin PG-2 likely represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi.

Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils.

Human neutrophils contain two structurally distinct types of antimicrobial peptides, beta-sheet defensins (HNP-1 to HNP-4) and the alpha-helical peptide LL-37. We used radial diffusion assays and an improved National Committee for Clinical Laboratory Standards-type broth microdilution assay to compare the antimicrobial properties of LL-37, HNP-1, and protegrin (PG-1). Although generally less potent than PG-1, LL-37 showed considerable activity (MIC, <10 microgram/ml) against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, and vancomycin-resistant enterococci, even in media that contained 100 mM NaCl. Certain organisms (methicillin-resistant S. aureus, Proteus mirabilis, and Candida albicans) were resistant to LL-37 in media that contained 100 mM NaCl but were susceptible in low-salt media. Burkholderia cepacia was resistant to LL-37, PG-1, and HNP-1 in low- or high-salt media. LL-37 caused outer and inner membrane permeabilization of E. coli ML-35p. Chromogenic Limulus assays revealed that LL-37 bound to E. coli O111:B4 lipopolysaccharide (LPS) with a high affinity and that this binding showed positive cooperativity (Hill coefficient = 2.02). Circular dichroism spectrometry disclosed that LL-37 underwent conformational change in the presence of lipid A, transitioning from a random coil to an alpha-helical structure. The broad-spectrum antimicrobial properties of LL-37, its presence in neutrophils, and its inducibility in keratinocytes all suggest that this peptide and its precursor (hCAP-18) may protect skin and other tissues from bacterial intrusions and LPS-induced toxicity. The potent activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it or related molecules might have utility as topical bronchopulmonary microbicides in cystic fibrosis.

Evaluation of the inactivation of infectious Herpes simplex virus by host-defense peptides.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.