Synergistic actions of antibacterial neutrophil defensins and cathelicidins

OBJECTIVE: Activated neutrophils extracellularly release antibacterial defensins and cathelicidins from the granules. In this study, to elucidate the interactions between defensins and cathelicidins in the extracellular environment, we evaluated the individual and synergistic actions of defensins and cathelicidins in the presence of physiological concentration of NaCl (150 mM). MATERIALS AND METHODS: Antibacterial activities against Escherichia coli and Staphylococcus aureus were assessed using human and guinea pig defensins and cathelicidins. Furthermore, the effect of defensins and cathelicidins on membrane permeabilization was examined using E. coli ML-35p, as a target organism. RESULTS: In the absence of NaCl, human defensin (HNP-1) and guinea pig defensins (GNCPs) exhibited the antibacterial activities in a dose-dependent manner (0.1-10 microg/ml); however, their activities were completely lost in the presence of 150 mM NaCl. In contrast, the antibacterial activities of human cathelicidin (CAP18/LL-37) and guinea pig cathelicidin (CAP11) were resistant to NaCl. Interestingly, HNP-1 and GNCPs synergized with CAP18/LL-37 and CAP11 to enhance the antibacterial activities against E. coli and S. aureus in the presence of 150 mM NaCl (p<0.05). Similarly, HNP-1 and GNCPs were synergistic with CAP18/LL-37 and CAP11 to potentiate the outer and inner membrane permeabilization of E. coli ML-35p (p<0.05). CONCLUSIONS: Together these observations indicate that when extracellularly released from neutrophils, defensins cannot function as antibacterial molecules by themselves, but can synergistically work with cathelicidins to exert the antibacterial activity in the extracellular milieu by augmenting the membrane permeabilization of target cells.     

Augmentation of Innate Host Defense by Expression of a Cathelicidin Antimicrobial Peptide

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.     

Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content.

The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy. This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593. However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P. aeruginosa and S. aureus. The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity. Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.     

Bactericidal activity of synthetic peptides based on the structure of the 55-kilodalton bactericidal protein from human neutrophils.

Short (10- to 11-mer) hydrophilic peptides based on the structure of the 55-kDa bactericidal protein (BP55, B/PI, and CAP57) from human neutrophil granules were identified from the hydropathy plot of the 456-amino-acid sequence predicted from the nucleotide sequences of cDNA clones for BP55 and B/PI. Peptides corresponding to amino acid residues 90 to 99 (peptide #90-99), 86 to 99, or 90 to 102 of BP55 were bactericidal toward 5 x 10(6) Pseudomonas aeruginosa cells at 0.6 x 10(-5) to 1.5 x 10(-5) M and killed an Escherichia coli rough strain at 3 x 10(-5) M. The #90-99 peptide with a cysteine added at the amino terminus (C#90-99) was approximately 10 times more active than #90-99, killing P. aeruginosa at 1.5 x 10(-6) M. Peptides representing amino acid residues 27 to 37, 118 to 127, and 160 to 170 and the first 10 amino acids of the signal sequence for BP55 were not bactericidal. When coupled to either keyhole limpet hemocyanin or ovalbumin protein carriers through the thiol group, the C#90-99 peptide was not diminished on a molar basis in its capacity for killing of P. aeruginosa. Two other relatively hydrophilic peptides with an added amino-terminal cysteine, peptides C#227-236 and C#418-427, were not bactericidal at 1.2 x 10(-4) M or at 100 times the effective bactericidal concentration of C#90-99. The C#90-99 peptide killed E. coli at 1.5 x 10(-5) M, or at 10 times the concentration required to kill an equal number of P. aeruginosa cells. Although Pseudomonas cepacia and Staphylococcus aureus were resistent to killing by the parent BP55 molecule, they were susceptible to the C#90-99 and #90-99 peptides in the same concentration range as was E. coli. When all peptides were compared for the ability to neutralize E. coli O55:B5 endotoxin in a Limulus amoebocyte lysate assay, the C#227-236, C#418-427, and #160-170 peptides completely inhibited gelation at a 10(-4) M concentration. All other synthetic peptides, including bactericidal peptide #90-99 and its congeners, lacked endotoxin-neutralizing activity at the highest concentration tested (4.5 x 10(-4) M). A hybrid of the C#227-236 and #90-99 peptides (CHybrid) was identical to the C#227-236 peptide component in effectiveness for carrying out endotoxin neutralization and was fivefold better than the #90-99 peptide in its capacity for killing P. aeruginosa.     

Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide

Cathelicidins, a group of cationic peptides found in leukocytes and epithelial cells, play a central role in the early innate immune defense against infection. Although these host defense peptides have been reported in several mammalian species, including primates, no cathelicidins have been identified in carnivores. Here we report the cloning, tissue expression and biological activity of a novel canine cathelicidin (K9CATH). The full-length cDNA sequence of K9CATH encodes a predicted 172 amino acid pre-propeptide that is 60–70% similar to other mammalian cathelicidins. Mass spectrometry analysis confirmed that the 38 aa mature K9CATH peptide was present in neutrophil granule contents. Synthetic K9CATH displayed broad antimicrobial activity against Gram-positive bacteria (Listeria monocytogenes, and Staphylococcus aureus; MICs (minimal inhibitory concentrations) 0.5 and 50 μM, respectively), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Pseudomonas aeruginosa, Proteus mirabilis; MICs 1.25 μM, Salmonella serotype Enteritidis; MIC 0.5 μM, and Neisseria gonorrhoeae; MIC 0.06 μM), and yeast (Candida albicans; MIC 12.5–50 μM). K9CATH demonstrated high antimicrobial activity against Ureaplasma canigenitalium, and lower activity against Ureaplasma urealyticum (MIC 0.06 and 50 μM, respectively). Similar to its ovine congener SMAP-29, K9CATH possesses salt-independent antimicrobial activity and LPS binding capacity. K9CATH displayed minimal hemolytic activity against human, dog and chicken erythrocytes. The potency and broad antimicrobial activity of K9CATH suggest that this peptide may act as a fundamental contributor to the innate immune responses in this carnivore species     

Staphylococcus aureus susceptibility to innate antimicrobial peptides,beta-defensins and CAP18, expressed by human keratinocytes

The antimicrobial peptides human beta-defensin-1 (hBD1), hBD2, hBD3, and CAP18 expressed by keratinocytes have been implicated in mediation of the innate defense against bacterial infection. To gain insight into Staphylococcus aureus infection, the susceptibility of S. aureus, including methicillin-resistant S. aureus (MRSA), to these antimicrobial peptides was examined. Based on quantitative PCR, expression of hBD2 mRNA by human keratinocytes was significantly induced by contact with S. aureus, and expression of hBD3 and CAP18 mRNA was slightly induced, while hBD1 mRNA was constitutively expressed irrespective of the presence of S. aureus. Ten clinical S. aureus isolates, including five MRSA isolates, induced various levels of expression of hBD2, hBD3, and CAP18 mRNA by human kertinocytes. The activities of hBD3 and CAP18 against S. aureus were found to be greater than those of hBD1 and hBD2. A total of 44 S. aureus clinical isolates, including 22 MRSA strains, were tested for susceptibility to hBD3 and CAP18. Twelve (55%) and 13 (59%) of the MRSA strains exhibited more than 20% survival in the presence of hBD3 (1 microg/ml) and CAP18 (0.5 microg/ml), respectively. However, only three (13%) and two (9%) of the methicillin-sensitive S. aureus isolates exhibited more than 20% survival with hBD3 and CAP18, respectively, suggesting that MRSA is more resistant to these peptides. A synergistic antimicrobial effect between suboptimal doses of methicillin and either hBD3 or CAP18 was observed with 10 MRSA strains. Furthermore, of several genes associated with methicillin resistance, inactivation of the fmtC gene in MRSA strain COL increased susceptibility to the antimicrobial effect mediated by hBD3 or CAP18.     

Synthetic peptides of human lysosomal cathepsin G with potent antipseudomonal activity.

Enzymatically active and inactive (diisopropylfluorophosphate-treated) cathepsin G exerted antibacterial action in vitro against Staphylococcus aureus, whereas only enzymatically active cathepsin G displayed bactericidal action against Pseudomonas aeruginosa. In order to further test the requirement for protease activity for the antipseudomonal action of cathepsin G, synthetic peptides spanning the full-length mature protein were prepared and examined for antibacterial action. Surprisingly, three structurally distinct peptides that correspond to residues 61 to 80, 117 to 136, and 198 to 223 within the full-length protein were found to exert potent antipseudomonal action (> 4.5 logs of killing at 500 micrograms/ml) against P. aeruginosa ATCC 27853 and four mucoid clinical isolates. Only the peptide (CG117-136) corresponding to residues 117 to 136 (117-RPGTLCTVAGWGRVSMRRGT-136) within cathepsin G exerted antibacterial action against the gram-positive pathogen S. aureus. The antipseudomonal action of CG117-136 was rapid and could be inhibited either by increasing concentrations of NaCl or by 0.5 mM MgCl2 plus 0.5 mM CaCl2, and these conditions appeared to reduce binding of the peptide to whole bacteria. Variants of peptide CG117-136 lacking either a hydrophobic N-terminal domain or a positively charged C-terminal domain were found to have significantly less antipseudomonal action than CG117-136. The antibacterial capacity of the all-D-enantiomeric form of peptide CG117-136 was found to be identical to that of the all-L-peptide, suggesting that the mechanism of killing does not require the recognition of a target site possessing a chiral center.     

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.     

Human lactoferrin and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant bacteria

Since human lactoferrin (hLF) binds to bacterial products through its highly positively charged N terminus, we investigated which of the two cationic domains is involved in its bactericidal activity. The results revealed that hLF lacking the first three residues (hLF(-3N)) was less efficient than hLF in killing of antibiotic-resistant Staphylococcus aureus, Listeria monocytogenes, and Klebsiella pneumoniae. Both hLF preparations failed to kill Escherichia coli O54. In addition, hLF(-3N) was less effective than hLF in reducing the number of viable bacteria in mice infected with antibiotic-resistant S. aureus and K. pneumoniae. Studies with synthetic peptides corresponding to the first 11 N-terminal amino acids, designated hLF(1-11), and fragments thereof demonstrated that peptides lacking the first three N-terminal residues are less effective than hLF(1-11) in killing of bacteria. Furthermore, a peptide corresponding to residues 21 to 31, which comprises the second cationic domain, was less effective than hLF(1-11) in killing of bacteria in vitro and in mice having an infection with antibiotic-resistant S. aureus or K. pneumoniae. Using fluorescent probes, we found that bactericidal hLF peptides, but not nonbactericidal peptides, caused an increase of the membrane permeability. In addition, hLF killed the various bacteria, most probably by inducing intracellular changes in these bacteria without affecting the membrane permeability. Together, hLF and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant S. aureus and K. pneumoniae, and the first two arginines play an essential role in this activity.     

Anti-microbial activity of human CAP18 peptides

Background: CAP18 derived from rabbit leukocytes is a 142-amino acid protein recently demonstrated to have Lipopolysaccharide (LPS) binding and anti-microbial activity. The C-terminal 37 amino acids of rabbit CAP18 (CAP18_106–142) comprise the LPS-binding and anti-microbial domain. The homologous domain of human CAP18 (huCAP18_104–140) was identified from the recently cloned human CAP18 cDNA. Objectives: To evaluate the antimicrobial activity of C-terminal peptides derived from human CAP18. Study design: Prepare synthetic human CAP18_104–140 and study anti-microbial activity versus various gram-negative and gram-positive bacteria. Results: Synthetic human CAP18_104–140 has broad anti-microbial activity versus both gram-positive (IC₅₀ = 2.5 μg/ml) and gram-negative bacteria (IC₅₀ = 0.5–5 μg/ml). Susceptible strains include Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. A 32-amino acid peptide lacking five amino acids from the C-terminus of CAP18_104–140 has higher activity. Unlike previously characterized anti-microbial peptides derived from granulocyte proteins, CAP18_104–140 is active in serum. Conclusion: Human CAP18_104–140 or a derivative peptide may have therapeutic potential for bacterial sepsis.     

Late intraphagosomal hydrogen ion concentration favors the in vitro antimicrobial capacity of a 37-kilodalton cationic granule protein of human neutrophil granulocytes.

We described previously (W.M. Shafer, L.E. Martin, and J.K. Spitznagel, Infect. Immun. 45:29-35, 1984) the presence of a 37-kilodalton cationic antimicrobial protein (37K CAP) in extracts of granules prepared from human polymorphonuclear granulocytes (PMN). In this investigation, we prepared 37K CAP from PMN granule extracts by sequential ion-exchange and molecular-sieve chromatography and examined its antimicrobial activity against a number of gram-negative and gram-positive bacteria. At concentrations of 5 micrograms/ml or lower, 37K CAP exerted selective antimicrobial activity against gram-negative bacteria. These bacteria included Acinetobacter lwoffii, Escherichia coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Pseudomonas cepacia, Salmonella typhi, Salmonella typhimurium, and Shigella sonnei. However, at 5 micrograms of 37K CAP per ml, Proteus mirabilis, Proteus vulgaris, and Serratia marcescens resisted this antimicrobial activity. The bactericidal activity of 37K CAP was greatest in acidic (pH 5.5) as opposed to alkaline (pH 7.5) media. The level of S. typhimurium resistance to 37K CAP correlated with the presence of O antigen in the lipopolysaccharide. In the absence of O antigen repeat units, resistance was proportional to the length of the core oligosaccharide. These results suggest that 37K CAP may contribute significantly to the ability of PMN to kill gram-negative bacteria by nonoxidative means, particularly as the maturing phagolysosome becomes acidified.     

Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions

Objective: Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading micro-organisms. Among these, human cathelicidin CAP18/LL-37 (L¹-S³⁷) possesses potent antibacterial activities against Gram-positive and Gram-negative bacteria. In this study, to develop peptide derivatives with improved bactericidal actions, we utilized the amphipathic 18-mer peptide (K¹⁵–V³²) of LL-37 as a template, and evaluated the activities of modified peptides.Methods: Antibacterial activities of the peptides (0.022 ~ 4.4 M corresponding to 0.1 ~ 10 g/ml) were assessed by alamarBlue^TM assay using Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa as target organisms. Furthermore, the membrane-permeabilization activities of the peptides were examined by using E. coli ML-35p as a target.Results: By substituting E¹⁶ and K²⁵ with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further substituting Q²², D²⁶ and N³⁰ with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK exhibited the most potent antibacterial actions against S. aureus (methicillin-resistant and -sensitive), S. pneumoniae, S. pyogenes, E. coli and P. aeruginosa, and possessed the most powerful membrane-permeabilizing activities against E. coli ML-35p at the effective concentrations (p < 0.05, 18-mer LLKKK vs. 18-mer LL, 18-mer K¹⁵-V³² and LL-37).Conclusions: Bactericidal activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and 18-mer LLKKK is the most potent among peptide derivatives with therapeutic potential for Gram-positive and Gram-negative bacterial infections.Received 1 September 2004; returned for revision 1 October 2004; accepted by M. Katori 23 October 2004     

Antimicrobial peptides from human platelets

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin beta-4 (Tbeta-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tbeta-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.     

Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L¹ to S³⁷) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K¹⁵ to V³²) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14⁺ murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E¹⁶ and K²⁵ with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q²², D²⁶, and N³⁰ with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14⁺ cells and attenuating production of tumor necrosis factor alpha (TNF-α) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-α production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.     

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.     

Hedistin: A novel antimicrobial peptide containing bromotryptophan constitutively expressed in the NK cells-like of the marine annelid, Nereis diversicolor

A novel antimicrobial peptide, named hedistin was identified from the coelomocytes of Nereis diversicolor. Hedistin shows no obvious similarities with other known peptides and constitutes the first antimicrobial peptide containing bromotryptophans demonstrated in annelids. cDNA and mass spectrometry analysis revealed that, upon bacteria challenge, this peptide is secreted following processing of a precursor containing a signal peptide and prosequences. Hedistin was shown to possess an activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The gene was demonstrated to be constitutively and exclusively expressed in circulating NK cells like known to play an important role in the immunity of the sand worm. These data contrast with those observed in another annelid, the leech, in which genes coding for antimicrobial peptides are upregulated in a specific tissue and peptides are rapidly released into the hemolymph after septic injury.     

Insights into the membrane interaction mechanism and antibacterial properties of chensinin-1b

Antimicrobial peptides (AMPs) with non-specific membrane disrupting activities are thought to exert their antimicrobial activity as a result of their cationicity, hydrophobicity and α-helical or β-sheet structures. Chensinin-1, a native peptide from skin secretions of Rana chensinensis, fails to manifest its desired biological properties because its low hydrophobic nature and an adopted random coil structure in a membrane-mimetic environment. In this study, chensinin-1b was designed by rearranging the amino acid sequence of its hydrophilic/polar residues on one face and its hydrophobic/nonpolar residues on the opposite face according to its helical diagram, and by replacing three Gly residues with three Trp residues. Introduction of Trp residues significantly promoted the binding of the peptide to the bacterial outer membrane and exerted bactericidal activity through cytoplasmic membrane damage. Chensinin-1b demonstrates higher antimicrobial activity and greater cell selectivity than its parent peptide, chensinin-1. The electrostatic interactions between chensinin-1b and lipopolysaccharide (LPS) may have facilitated the uptake of the peptide into Gram-negative cells and be also helpful to disrupt the bacterial cytoplasmic membrane, as evidenced by depolarisation of the membrane potential and leakage of calceins from the liposomes of Escherichia coli and Staphylococcus aureus. Chensinin-1b was also found to penetrate mouse skin and was also effective in vivo, as measured by hydroxyproline levels in a wound infection mouse model, and could therefore act as an anti-infective agent for wound healing.     

Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3.     

Antimicrobial peptides derived from growth factors

Growth factors, comprising diverse protein and peptide families, are involved in a multitude of developmental processes, including embryogenesis, angiogenesis, and wound healing. Here we show that peptides derived from HB-EGF, amphiregulin, hepatocyte growth factor, PDGF-A and PDGF-B, as well as various FGFs are antimicrobial, demonstrating a previously unknown activity of growth factor-derived peptides. The peptides killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis, as well as the fungus Candida albicans. Several peptides were also active against the Gram-positive S. aureus. Electron microscopy analysis of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. Furthermore, HB-EGF was antibacterial per se, and its epitope GKRKKKGKGLGKKRDPCLRKYK retained its activity in presence of physiological salt and plasma. No discernible hemolysis was noted for the growth factor-derived peptides. Besides providing novel templates for design of peptide-based antimicrobials, our findings demonstrate a previously undisclosed link between the family of growth factors and antimicrobial peptides, both of which are induced during tissue remodelling and repair.     

In vitro antibacterial activity of bovine dialyzable leukocyte extract

The rapidly developing resistance of many infectious pathogenic organisms to modern drugs has spurred scientists to search for new sources of antibacterial compounds. One potential candidate, bDLE (dialysis at 10 to 12 kDa cut-off) and its fractions ("S" and "L" by 3.5 kDa cut-off and I, II, III, and IV by molecular exclusion chromatography), was evaluated for antibacterial activity against pathogenic bacterial strains (Staphylococcus aureus, Streptococcus pyogenes, Lysteria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi) using standard antimicrobial assays. A minimum inhibitory concentration (MIC) of bDLE and its fractions was determined by agar and broth dilutions methods. Only bDLE and its "S" fraction had an effect upon all bacteria evaluated (MIC ranging from 0.29 to 0.62 U/ml), and the bactericidal and bacteriostatic effects (evaluated by MTT assay) were bacterial species-dependent. These results showed a remarkable in vitro antibacterial property of bDLE against several pathogenic bacteria.