IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.     

Strategies for transformation of naturally-occurring amphibian antimicrobial peptides into therapeutically valuable anti-infective agents

The emergence of strains of pathogenic microorganisms with resistance to commonly used antibiotics has necessitated a search for novel types of antimicrobial agents. Many frog species produce amphipathic alpha-helical peptides with broad spectrum antimicrobial activity in the skin but their therapeutic potential is limited by varying degrees of cytolytic activity towards eukaryotic cells. Methods for development of such peptides into anti-infective drugs are illustrated by the example of temporin-1DRa (HFLGTLVNLAK KIL.NH(2)). Studies with model alpha-helical peptides have shown that increase in cationicity promotes antimicrobial activity whereas increases in hydrophobicity, helicity and amphipathicity promote hemolytic activity and loss of selectivity for microorganisms. Analogs of temporin-1DRa in which each amino acid is replaced by L-lysine and D-lysine were synthesized and their cytolytic activities tested against a range of microorganisms and human erythrocytes. Small changes in structure produced marked changes in conformation, as determined by retention time on reversed-phase HPLC, and in biological activity. However, peptides containing the substitutions (Val(7) -->L-Lys), (Thr(5)-->D-Lys) and (Asn(8)-->D-Lys) retained the high solubility and potent, broad spectrum antimicrobial activity of the naturally occurring peptide but were appreciably (up to 10-fold) less hemolytic. In contrast, analogs in which Leu(9) and Ile(13) were replaced by the more hydrophobic cyclohexylglycine residue showed slightly increased antimicrobial potencies (up to 2-fold) but a 4-fold increase in hemolytic activity. The data suggest a strategy of selective increases in cationicity concomitant with decreases in helicity and hydrophobicity in the transformation of naturally-occurring antimicrobial peptides into non-toxic therapeutic agents.     

Antibiotic activity of reversed peptides of alpha-helical antimicrobial peptide,P18

P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics.     

A novel pyrimidine derivatives with aryl urea, thiourea and sulfonamide moieties: synthesis, anti-inflammatory and antimicrobial evaluation

A series of novel 4-(3-(trifluoromethyl)phenylamino-6-(4-(3-arylureiodo/arylthioureido/arylsulfonamido)-pyrimidine derivatives of biological interest were prepared by the sequential Suzuki cross coupling, acid amination, reduction followed by reaction of resulting amine with different arylisocyantes or arylisothiocyantes or arylsulfonyl chlorides. All the synthesized compounds (1-25) were screened for their pro-inflammatory cytokines (TNF-α and IL-6) and antimicrobial activity (antibacterial and antifungal). Biological data revealed that among all the compounds screened, compounds 5, 6, 11, 12, 16 and 20 were found to have moderate to potent anti-inflammatory activity (up to 48-78% TNF-α and 56-96% IL-6 inhibitory activity) with reference to standard dexamethasone at 10 μM. The compounds 10, 12, 13, 18, 20, 22, 24 and 25 found to have promising antimicrobial activity against all the selected pathogenic bacteria and fungi.     

Identification of the primary antimicrobial domains in human neutrophil cathepsin G

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.     

Activation of clindamycin phosphate by human skin

AIMS: To investigate the relative antimicrobial activity of clindamycin phosphate (CP) and clindamycin (Cly) and to examine the effect of skin homogenates on the activity of CP. METHODS AND RESULTS: Minimum inhibitory concentrations (MIC) were determined against dermally relevant organisms and bactericidal activity was studied using time-kill methodology. The effect of skin homogenates on the antimicrobial activity of CP was studied by well-diffusion assay. The MIC of Cly was substantially lower than that of CP in all susceptible organisms. Clindamycin also showed greater bactericidal activity (rate of kill) than CP. Phosphatases in skin homogenates activated CP at pH 4-8 with a maximal activation at pH 4. CONCLUSION: Phosphatases within the skin have been shown to convert CP to the more potent form Cly. SIGNIFICANCE AND IMPACT OF THE STUDY: Conversion to Cly is a major determinant of antimicrobial activity in the skin layers following topical application of CP.     

2-Heteroarylimino-5-benzylidene-4-thiazolidinones analogues of 2-thiazolylimino-5-benzylidene-4-thiazolidinones with antimicrobial activity: synthesis and structure-activity relationship

2-Heteroarylimino-5-benzylidene-4-thiazolidinones, unsubstituted or carrying hydroxy, methoxy, nitro and chloro groups on the benzene ring, were synthesised and assayed in vitro for their antimicrobial activity against gram positive and gram negative bacteria, yeasts and mould. The antimicrobial activity of the 2-benzo[d]thiazolyl- and of the 2-benzo[d]isothiazolyl-imino-5-benzylidene-4-thiazolidinones is, on the whole, lower in comparison with the high activity detected for the derivatives of the 2-thiazolylimino-5-benzylidene-4-thiazolidinone class. Nevertheless most of the benzo[d]thiazole analogues display good inhibition of the growth of gram positive bacilli and staphylococci, including methicillin-resistant Staphylococcus strains. Among the 2-benzo[d]isothiazole analogues a few derivatives show a strong and selective activity against bacilli. Moreover, it is worth noting that the replacement of the thiazole nucleus for the benzo[d]thiazole bicyclic system in the parent 2-(benzo[d]thiazol-2-ylimino)thiazolidin-4-one leads to significant antifungal properties against both yeasts and moulds, properties not shown by the analogous 2-thiazolyl- and 2-benzo[d]isothiazolyl-imino)thiazolidin-4-ones. The structure-activity relationship of 33 analogues possessing the 2-heteroarylimino-4-thiazolidinone structure is analysed through QSAR models.     

Exploring relationships between mimic configuration, peptide conformation and biological activity in indolizidin-2-one amino acid analogs of gramicidin S.

Indolizidin-2-one amino acids (I2aas, 6S- and 6R-1) possessing 6S- and 6R-ring-fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution-phase and solid-phase techniques were used to synthesize three analogs with I2aa residues in place of the d-Phe-Pro residues at the turn regions of GS: [(6S)-I2aa4-5,4'-5']GS (2), [Lys2,2',(6S)-I2aa4-5,4'-5']GS (3) and [(6R)-I2aa4-5,4'-5']GS (4). Although conformational analysis of [I2aa4-5,4'-5']GS analogs 2-4 indicated that both ring-fusion stereoisomers of I2aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)-I2aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)-I2aa analog 4, suggesting a greater propensity for the (6S)-diastereomer to adopt the beta-turn/antiparallel beta-pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)-I2aa analog 2 exhibited significantly better potency than the (6R)-I2aa diastereomer 4. Relative to GS, [(6S)-I2aa4-5,4'-5']GS (2) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)-I2aa diastereomer. The synthesis and evaluation of GS analogs 2-4 has furnished new insight into the importance of ring-fusion stereochemistry for turn mimicry by indolizidin-2-one amino acids as well as novel antimicrobial peptides.     

Short KR‐12 analogs designed from human cathelicidin LL‐37 possessing both antimicrobial and antiendotoxic activities without mammalian cell toxicity

KR‐12 (residues 18–29 of LL‐37) was known to be the smallest peptide of human cathelicidin LL‐37 possessing antimicrobial activity. In order to optimize α‐helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR‐12 analogs. Highest hydrophobic analogs KR‐12‐a5 and KR‐12‐a6 displayed greater inhibition of lipopolysaccharide (LPS)‐stimulated tumor necrosis factor‐α production and higher LPS‐binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR‐12 analogs, KR‐12‐a2, KR‐12‐a3 and KR‐12‐a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL‐37. KR‐12‐a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL‐37. Also, these KR‐12 analogs (KR‐12‐a2, KR‐12‐a3, KR‐12‐a4 and KR‐12‐a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 μM) against methicillin‐resistant Staphylococcus aureus. Taken together, these KR‐12 analogs have the potential for future development as a novel class of antimicrobial and anti‐inflammatory therapeutic agents. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Burkholderia thailandensis outer membrane vesicles exert antimicrobial activity against drug-resistant and competitor microbial species

Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.     

Preservation of antimicrobial properties of complement peptide C3a, from invertebrates to humans

The human anaphylatoxin peptide C3a, generated during complement activation, exerts antimicrobial effects. Phylogenetic analysis, sequence analyses, and structural modeling studies paired with antimicrobial assays of peptides from known C3a sequences showed that, in particular in vertebrate C3a, crucial structural determinants governing antimicrobial activity have been conserved during the evolution of C3a. Thus, regions of the ancient C3a from Carcinoscorpius rotundicauda as well as corresponding parts of human C3a exhibited helical structures upon binding to bacterial lipopolysaccharide permeabilized liposomes and were antimicrobial against gram-negative and gram-positive bacteria. Human C3a and C4a (but not C5a) were antimicrobial, in concert with the separate evolutionary development of the chemotactic C5a. Thus, the results demonstrate that, notwithstanding a significant sequence variation, functional and structural constraints imposed on C3a during evolution have preserved critical properties governing antimicrobial activity.     

Antimicrobial Evaluation of 1-Methyl-3-Nitro-1-Nitrosoguanidine

1-Methyl-3-nitro-1-nitrosoguanidine was shown to have in vitro antimicrobial activity against fungi and gram-positive and gram-negative bacteria; it was also shown to have in vivo activity in mice experimentally infected with Staphylococcus aureus or Escherichia coli comparable to that observed with penicillin or streptomycin sulfate.     

Structure-activity relationship of HP (2-20) analog peptide: enhanced antimicrobial activity by N-terminal random coil region deletion

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is a 19-aa antimicrobial peptide derived from N-terminus of Helicobacter pylori Ribosomal protein L1 (RpL1). In the previous study, several analogs with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, substitutions of Gln(16) and Asp(18) with Trp (Anal 3) for hydrophobic amino acid caused a dramatic increase in antibiotic activity without a hemolytic effect. HP-A3 is a potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and extended C-terminal regular alpha-helical region (residues 6-20). To obtain the short and potent alpha-helical antimicrobial peptide, we synthesized a N-terminal random coil deleted HP-A3 (A3-NT) and examined their antimicrobial activity and mechanism of action. The resulting 15mer peptide showed increased antibacterial and antifungal activity to 2- and 4-fold, respectively, without hemolysis. Confocal fluorescence microscopy studies showed that A3-NT was accumulated in the plasma membrane. Flow cytometric analysis revealed that A3-NT acted in salt- and energy-independent manner. Furthermore, A3-NT causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy. Circular dichroism (CD) analysis revealed that A3-NT showed higher alpha-helical contents than the HP-A3 peptide in 50% TFE solution. Therefore, the cell-lytic efficiency of HP-A3, which depended on the alpha-helical content of peptide, correlated linearly with their antimicrobial potency.     

Leukotriene B4 induces release of antimicrobial peptides in lungs of virally infected mice

Leukotriene B(4) (LTB(4)) is a lipid mediator of inflammation that was recently shown to exert antiviral activities. In this study, we demonstrate that the release of antimicrobial proteins by neutrophils contribute to an early host defense against influenza virus infection in vitro as well as in vivo. Daily i.v. treatments with LTB(4) lead to a significant decrease in lung viral loads at day 5 postinfection in mice infected with influenza A virus compared with the placebo-treated group. This reduction in viral load was not present in mice deficient in the high-affinity LTB(4) receptor. Viral clearance in lungs was associated with up-regulated presence of antimicrobial peptides such as beta-defensin-3, members of the mouse eosinophil-related RNase family, and the mouse cathelicidin-related antimicrobial peptide. Our results also indicate that neutrophils are important in the antiviral effect of LTB(4). Viral loads in neutrophil-depleted mice were not diminished by LTB(4) administration, and a substantial reduction in the presence of murine cathelicidin-related antimicrobial peptide and the murine eosinophil-related RNase family in lung tissue was observed. Moreover, in vitro treatment of human neutrophil cultures with LTB(4) led rapidly to the secretion of the human cathelicidin LL-37 and eosinophil-derived neurotoxin, known as antiviral peptides. Pretreatment of cell cultures with specific LTB(4) receptor antagonists clearly demonstrate the implication of the high-affinity LTB(4) receptor in the LTB(4)-mediated activity. Together, these results demonstrate the importance of neutrophils and the secretion of antimicrobial peptides during the early immune response mediated by LTB(4) against a viral pathogen.     

Functional characterization of starvation-induced lysosomal activity in Saccharomyces cerevisiae

Starvation induces significant alterations in lysosomal enzymes, and reduced concentrations of glucose increases the activity of several lysosomal enzymes. Therefore, to evaluate the lysosomal antimicrobial activity under starvation conditions, we added 0, 5, 10, 20, or 40 g/l of glucose (0%, 0.5%, 1%, 2%, or 4% glucose) supplemented YP medium to cultured Saccharomyces cerevisiae, and lysosomal fractions were isolated from S. cerevisiae grown under the various culture conditions. The lysosomes isolated from each condition exhibited increased antimicrobial activity against Escherichia coli as determined by a decrease in glucose concentration. In addition, a starvation-dependent increase in lysosomal activity coincided with increased lysosome intensity at the cytosol and distinct protein expression from lysosomes in S. cerevisiae. It also was determined found that the lysosomes have antimicrobial activity against seven different microorganisms, including E. coli, and starvation-induced lysosomes showed enhanced antimicrobial activity compared to those from normal lysosomes. These results suggest the possibility that lysosomal alterations during starvation may induce conditions that activate lysosomes for future development of efficient antimicrobial agents.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

Purification and characterization of antimicrobial peptides from the skin secretion of Rana dybowskii

Six antimicrobial peptides designated dybowskins were isolated from the skin secretion of Rana dybowskii, an edible frog in Korea. Dybowskin-1 (FLIGMTHGLICLISRKC) and dybowskin-2 (FLIGMTQGLICLITRKC) were isoforms differing in only two amino acid residues at the 7th and 14th positions from the N-terminus, and they showed amino acid sequence similarities with ranalexin peptides. Dybowskin-3 (GLFDVVKGVLKGVGKNVAGSLLEQLKCKLSGGC), dybowskin-4 (VWPLGLVICKALKIC), dybowskin-5 (GLFSVVTGVLKAVGKNVAKNVGGSLLEQLKCKISGGC), and dybowskin-6 (FLPLLLAGLPLKLCFLFKKC) differed in both size and sequence, and they were, in terms of amino acid sequence similarities, related to brevinin-2, japonicin-2, esculentin-2, and brevinin-1 peptides, respectively. All the peptides presented in this paper contained Rana-box, the cyclic heptapeptide domain, which is conserved in other antimicrobial peptides derived from the genus Rana. All the dybowskin peptides showed a broad spectrum of antimicrobial activity against the Gram-positive and Gram-negative bacteria (minimum inhibition concentrations (MIC), 12.5 to >100 microg/ml) and against Candida albicans (MIC, 25 to >100 microg/ml). Especially, dybowskin-4 with valine at its N-terminus was the most abundant and showed the strongest antimicrobial activity among all the dybowskin peptides. This result indicates that the dybowskin peptides from R. dybowskii, whose main habitats are mountains or forests, have evolved differently from antimicrobial peptides isolated from other Korean frogs, whose habitats are plain fields.     

Antimicrobial activity of Lactobacillus reuteri DPC16 supernatants against selected food borne pathogens

Lactobacillus reuteri DPC16 is a human-isolated strain recently patented in New Zealand. The antimicrobial activity of cell-free supernatants from different fermentation processes, with or without glycerol supplementation was studied. When grown in just MRS broth, the cultural supernatant significantly inhibited the growth of selected food-borne pathogens, possibly due to acidic effect as this activity was pH-dependent. The cell-free supernatants from secondary fermentation of DPC16 resting cells in glycerol-supplemented media have shown very different antimicrobial activities. A very potent antimicrobial activity gradually developed during the fermentation process which was observed only when growing in MRS-glycerol broth (such supernatant is denoted MRSg). This strong antimicrobial activity was pH-independent, dose-dependant and affected both Gram-negative and Gram-positive pathogens. Reuterin detected in MRSg is believed to be responsible for these activities. The susceptibility of the selected pathogens (grown to stationary phase) to MRSg was tested and found that exposure to MRSg for 180 min led to a significant reduction in cell viability in all pathogens. These results suggest that this is a reuterin-producing strain, which has potent antimicrobial activity against both Gram-negative and Gram-positive pathogens. These findings have indicated a clear potential of this novel strain in industrial applications.     

Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.