Cysteine-rich antimicrobial peptides are abundant in animal and plant tissues involved in host defense. In insects, most are synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich peptide with three forms differing by amino-terminal truncation, and we named it hepcidin (Hepc) because of its origin in the liver and its antimicrobial properties. Two predominant forms, Hepc20 and Hepc25, contained 20 and 25 amino acid residues with all 8 cysteines connected by intramolecular disulfide bonds. Reverse translation and search of the data bases found homologous liver cDNAs in species from fish to human and a corresponding human genomic sequence on human chromosome 19. The full cDNA by 5 rapid amplification of cDNA ends was 0.4 kilobase pair, in agreement with hepcidin mRNA size on Northern blots. The liver was the predominant site of mRNA expression. The encoded prepropeptide contains 84 amino acids, but only the 20 -25-amino acid processed forms were found in urine. Hepcidins exhibited antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. Hepcidin may be a vertebrate counterpart of cysteinerich antimicrobial peptides produced in the fat body of insects.Innate immunity relies on a variety of effector mechanisms to defend against microbial invasion. Among them are the abundant and widely distributed disulfide-linked cationic antimicrobial peptides found in both the plant and animal kingdoms. Generally, these peptides exhibit a broad range of activity against bacteria, fungi, protozoa, and enveloped viruses. Plants produce many cysteine-rich antimicrobial peptides including thionins, plant defensins, and the cysteine rich Ib-AMP 1-4 (1-3). In insects, cysteine-rich antimicrobial peptides are produced in the fat body (functional homologue of the mammalian liver) and transcriptionally induced and released into the hemolymph in response to infection or injury. These include insect defensins, heliomicin, drosomycin, and thanatin (4 -7). Mollusks also produce cationic and cysteine-rich antimicrobial peptides such as mytilin, mytimicin, and myticin (8). In mammals, similar antimicrobial peptides include ␣-and ␤-defensins and protegrins (9, 10).Like the insect fat body, the vertebrate liver is also centrally involved in innate immune response to infection. The "acute phase" response to infection or inflammation is a pattern of increased hepatic synthesis of many secreted proteins involved in host defense and the selective suppression of synthesis of other secreted proteins. In contrast to the abundant fat bodyderived antimicrobial peptides of insects, no vertebrate antimicrobial peptides originating in the liver have been described to date. In this work, we report the discovery of a novel hepatic antimicrobial peptide, hepcidin, whose processed form is found in urine. MATERIALS AND METHODSPur...

Human neutrophils contain two structurally distinct types of antimicrobial peptides, ␤-sheet defensins (HNP-1 to HNP-4) and the ␣-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 g/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 ␣-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.

We used solid-state NMR spectroscopy to investigate the oligomeric structure and insertion of protegrin-1 (PG-1), a ␤-hairpin antimicrobial peptide, in lipid bilayers that mimic either the bacterial inner membrane [palmitoyloleoylphosphatidyl ethanolamine and palmitoyloleoylphosphatidylglycerol (POPE͞POPG) bilayers] or the red blood cell membrane [neutral palmitoyloleoylphosphatidylcholine (POPC)͞cholesterol bilayers]. 1 H spin diffusion from lipids to the peptide indicates that PG-1 contacts both the lipid acyl chains and the headgroups in the anionic membrane but resides far from the lipid chains in the POPC͞cholesterol bilayer. 19 F spin diffusion data indicates that 75% of the ␤-hairpins have homodimerized N strands and C strands in the anionic membrane. The resulting (NCCN) n multimer suggests a membrane-inserted ␤-barrel enclosing a water pore. The lipids surrounding the ␤-barrel have high orientational disorder and chain upturns, thus they may act as fillers for the pore. These results revise several features of the toroidal pore model, first proposed for magainin and subsequently applied to PG-1. In the POPC͞cholesterol membrane, the N and C strands of PG-1 cluster into tetramers, suggesting the formation of ␤-sheets on the membrane surface. Thus, the membrane composition plays a decisive role in defining the assembly and insertion of PG-1. The different oligomeric structures of PG-1 help to explain its greater toxicity for bacteria than for eukaryotic cells.membrane composition ͉ spin diffusion ͉ toroidal pores ͉ 19 FNMR ͉ protegrin-1

We have previously described the antibacterial capacity of protegrin-1 (PG-1), a cysteine-rich, cationic peptide from porcine leukocytes, against Neisseria gonorrhoeae. We now report genetic and biochemical evidence that gonococcal susceptibility to the lethal action of PG-1 and other structurally unrelated antibacterial peptides, including a peptide (LL-37) that is expressed constitutively by human granulocytes and testis and inducibly by keratinocytes, is modulated by an energy-dependent eff lux system termed mtr. These results indicate that such eff lux systems may enable mucosal pathogens like gonococci to resist endogenous antimicrobial peptides that are thought to act during infection.

Protegrin-1 (PG-1) is a broad-spectrum beta-sheet antimicrobial peptide found in porcine leukocytes. The mechanism of action and the orientation of PG-1 in lipid bilayers are here investigated using (2)H, (31)P, (13)C, and (15)N solid-state NMR spectroscopy. (2)H spectra of mechanically aligned and chain-perdeuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers indicate that PG-1 at high concentrations destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region is significantly altered, as seen from the (31)P spectra of POPC and the (2)H spectra of headgroup-deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine. These observations indicate that PG-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks, where a significant fraction of lipids is located in the edges of the disks with a distribution of orientations. These edges allow the lipid bilayer to bend back on itself as in toroidal pores. Interestingly, this loss of bilayer orientation occurs only in long-chain lipids such as POPC and not in shorter chain lipids such as 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC). To understand the mode of binding of PG-1 to the lipid bilayer, we determined the orientation of PG-1 in DLPC bilayers. The (13)CO and (15)N chemical shifts of Val-16 labeled PG-1 indicate that the beta-strand axis is tilted by 55 degrees +/- 5 degrees from the bilayer normal while the normal of the beta-sheet plane is 48 degrees +/- 5 degrees from the bilayer normal. This orientation favors interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. This is the first time that the orientation of a disulfide-stabilized beta-sheet membrane peptide has been determined by solid-state NMR.

The minimum surface tension and respreadability of a surfactant monolayer is limited by a two to three dimensional instability called collapse. Liquid-condensed or solid phase monolayers collapse via fracture followed by loss of material. Liquid-expanded phase monolayers collapse by solubilization into the subphase. Monolayers that retain a continuous liquid-expanded phase network surrounding islands of liquid-condensed or solid phase collapse at low surface tensions via a localized, large amplitude buckling. The buckled regions coexist with the flat monolayer, remain attached to the interface, and reversibly reincorporate into the monolayer upon expansion. [S0031-9007(98)06943-9]

Human bone marrow expresses a pseudogene that encodes an antimicrobial peptide homologous to rhesus monkey circular minidefensins ( -defensins). We prepared the putative ancestral human peptide by solid-phase synthesis and named it ''retrocyclin.'' Retrocyclin did not cause direct inactivation of HIV-1, and its modest antibacterial properties resembled those of its rhesus homologs. Nevertheless, retrocyclin had a remarkable ability to inhibit proviral DNA formation and to protect immortalized and primary human CD4 ؉ lymphocytes from in vitro infection by both T-tropic and M-tropic strains of HIV-1. Confocal fluorescent microscopy studies performed with BODIPY-FL-labeled RC-101, a close analog of retrocyclin, showed that the peptide formed patch-like aggregates on the surface of CD4 ؉ cells. These findings suggest that retrocyclin interferes with an early stage of HIV-1 infection and that retrocyclin-like agents might be useful topical agents to prevent sexually acquired HIV-1 infections.

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substratespecific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel ␤-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.