Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice

Defensins comprise a family of cationic antimicrobial peptides that are characterized by the presence of six conserved cysteine residues. We identified two novel human beta-defensin (hBD) isoforms by mining the public human genomic sequences. The predicted peptides conserve the six-cysteine motif identical with hBD-4, termed hBD-5 and hBD-6. We also evaluated the characteristics of the mouse homologs of hBD-5, hBD-6, and HE2beta1, termed mouse beta-defensin (mBD)-12, mBD-11, and mouse EP2e (mEP2e). The mBD-12 synthetic peptide showed salt-dependent antimicrobial activity. We demonstrate the epididymis-specific expression pattern of hBD-5, hBD-6, mBD-11, mBD-12, and mEP2e. In situ hybridization revealed mBD-11, mBD-12, and mEP2e expression in the columnar epithelium of the caput epididymis, contrasting with the predominant expression of mBD-3 in the capsule or septum of the whole epididymis. In addition, the regional specificity of mBD-11, mBD-12, and mEP2e was somewhat overlapping, but not identical, in the caput epididymis, suggesting that specific regulation may work for each member of the beta-defensin family. Our findings indicated that multiple beta-defensin isoforms specifically and cooperatively contribute to the innate immunity of the urogenital system.     

N-Terminus Three Residues Deletion Mutant of Human Beta-Defensin 3 with Remarkably Enhanced Salt-Resistance

In this study, we designed and synthesized three N-terminal deletion analogs of human beta-defensin 3 (hBD-3), namely, hBD-3Δ4, hBD-3Δ7, and hBD-3Δ10, to determine the effect of N-terminal residues on the antibacterial activity and salt resistance of these peptides. The antibacterial activities and salt resistance of hBD-3 and its analogs were tested against a broad range of standard and clinically isolated strains. The deletion of nine N-terminal residues significantly reduced the antibacterial activity of hBD-3 against most of tested strains, particularly Klebsiella pneumonia. Compared with hBD-3 and other analogs, the analog with a deletion of three residues, hBD-3Δ4, exhibited significantly higher antimicrobial activity against almost all the tested strains, especially Escherichia coli and Enterococcus faecium, at high NaCl concentrations. Given its broad spectrum of antimicrobial activity and high salt resistance, hBD-3Δ4 could serve as a promising template for new therapeutic antimicrobial agents.     

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.     

Engineering disulfide bonds of the novel human beta-defensins hBD-27 and hBD-28: differences in disulfide formation and biological activity among human beta-defensins

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.     

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the beta-defensin family, human beta-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of beta-defensins is mediated by at least three different mechanisms.     

Identification of a novel, multifunctional β-defensin (human β-defensin 3) with specific antimicrobial activity

Previous studies have shown the implication of beta-defensins in host defense of the human body. The human beta-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human beta-defensin, called human beta-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-alpha (TNF-alpha), hBD-3 expression is increased particularly after stimulation by interferon-gamma. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human beta-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.     

Antibacterial activities of synthetic peptides corresponding to the carboxy-terminal region of human beta-defensins 1-3

The antibacterial activities of synthetic human beta-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human beta-defensins (HBD-1-3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca(2+) and Mg(2+). The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human beta-defensins could be of help in understanding facets of antimicrobial activity of beta-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.     

Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism

beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired. We have determined the cysteine connectivities of a corresponding synthetic hBD-1(Ser35) peptide, investigated the structure by circular dichroism spectroscopy, and assayed the in vitro antimicrobial activity. Despite a different arrangement of the disulfides, hBD-1(Ser35) proved as active as hBD-1 against the microorganisms tested. This activity likely depends on the ability of hBD-1(Ser35) to adopt an amphipathic conformation in hydrophobic environment, similar to the wild type peptide, as suggested by CD spectroscopy.     

Structure determination of human and murine β-defensins reveals structural conservation in the absence of significant sequence similarity

Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.     

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

Isolation of human β-defensin-4 in lung tissue and its increase in lower respiratory tract infection

Background

Human β-defensin-4 (hBD-4), a new member of the β-defensin family, was discovered by an analysis of the genomic sequence. The objective of this study was to clarify hBD-4 expression in human lung tissue, along with the inducible expression in response to infectious stimuli, localization, and antimicrobial activities of hBD-4 peptides. We also investigated the participation of hBD-4 in chronic lower respiratory tract infections (LRTI) by measuring the concentrations of hBD-4 peptides in human bronchial epithelial lining fluid (ELF).

Methods

The antimicrobial activity of synthetic hBD-4 peptides against E. coli and P. aeruginosa was measured by radial diffusion and colony count assays. We identified hBD-4 in homogenated human lung tissue by reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay (RIA). Localization of hBD-4 was studied through immunohistochemical analysis (IHC). We investigated the effects of lipopolysaccharide (LPS) on hBD-4 expression and its release from small airway epithelial cells (SAEC). We collected ELF from patients with chronic LRTI using bronchoscopic microsampling to measure hBD-4 concentrations by RIA.

Results

hBD-4 exhibited salt-sensitive antimicrobial activity against P. aeruginosa. We detected the presence of hBD-4 peptides in human lung tissue. IHC demonstrated the localization of hBD-4-producing cells in bronchial and bronchiolar epithelium. The levels of hBD-4 peptides released from LPS-treated SAECs were higher than those of untreated control cells. ELF hBD-4 was detectable in 4 of 6 patients with chronic LRTI, while the amounts in controls were all below the detectable level.

Conclusion

This study suggested that hBD-4 plays a significant role in the innate immunity of the lower respiratory tract.     

Rhinovirus increases human beta-defensin-2 and -3 mRNA expression in cultured bronchial epithelial cells

Human beta-defensins (hBDs) are antimicrobial peptides that play important roles in host defense against infection, inflammation and immunity. Previous studies showed that micro-organisms and proinflammatory mediators regulate the expression of these peptides in airway epithelial cells. The aim of the present study was to investigate the modulation of expression of hBDs in cultured primary bronchial epithelial cells (PBEC) by rhinovirus-16 (RV16), a respiratory virus responsible for the common cold and associated with asthma exacerbations. RV16 was found to induce expression of hBD-2 and -3 mRNA in PBEC, but did not affect hBD-1 mRNA. Viral replication appeared essential for rhinovirus-induced beta-defensin mRNA expression, since UV-inactivated rhinovirus did not increase expression of hBD-2 and hBD-3 mRNA. Exposure to synthetic double-stranded RNA (dsRNA) molecule polyinosinic:polycytidylic acid had a similar effect as RV16 on mRNA expression of these peptides in PBEC. In line with this, PBEC were found to express TLR3, a Toll-like receptor involved in recognition of dsRNA. This study shows that rhinovirus infection of PBEC leads to increased hBD-2 and hBD-3 mRNA expression, which may play a role in both the uncomplicated common cold and in virus-associated exacerbations of asthma.     

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Antimicrobial activity of human β-defensin 4 analogs: Insights into the role of disulfide linkages in modulating activity

Human β-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1–3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid β-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.     

Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S

The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.     

Roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions

Cationic antimicrobial peptides (CAPs) occur as important innate immunity agents in many organisms, including humans, and offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane lysis and eventually cell death. In this work, we studied the biophysical and microbiological characteristics of designed CAPs varying in hydrophobicity levels and charge distributions by a variety of biophysical and biochemical approaches, including in-tandem atomic force microscopy, attenuated total reflection-FTIR, CD spectroscopy, and SDS-PAGE. Peptide structural properties were correlated with their membrane-disruptive abilities and antimicrobial activities. In bacterial lipid model membranes, a time-dependent increase in aggregated β-strand-type structure in CAPs with relatively high hydrophobicity (such as KKKKKKALFALWLAFLA-NH(2)) was essentially absent in CAPs with lower hydrophobicity (such as KKKKKKAAFAAWAAFAA-NH(2)). Redistribution of positive charges by placing three Lys residues at both termini while maintaining identical sequences minimized self-aggregation above the dimer level. Peptides containing four Leu residues were destructive to mammalian model membranes, whereas those with corresponding Ala residues were not. This finding was mirrored in hemolysis studies in human erythrocytes, where Ala-only peptides displayed virtually no hemolysis up to 320 μM, but the four-Leu peptides induced 40-80% hemolysis at the same concentration range. All peptides studied displayed strong antimicrobial activity against Pseudomonas aeruginosa (minimum inhibitory concentrations of 4-32 μM). The overall findings suggest optimum routes to balancing peptide hydrophobicity and charge distribution that allow efficient penetration and disruption of the bacterial membranes without damage to mammalian (host) membranes.