Titanium surfaces immobilized with the major antimicrobial fragment FK-16 of human cathelicidin LL-37 are potent against multiple antibiotic-resistant bacteria.

PubMed

Mishra, Biswajit; Wang, Guangshun

2017-08-01

Infections on implanted medical devices are a challenging problem, especially when bacteria form difficult-to-treat biofilms. Antimicrobial peptides are considered to be a solution due to their potency against antibiotic-resistant superbugs. Previously, the authors' laboratory demonstrated the prevention of staphylococcal biofilm formation in an animal catheter model by injecting merecidin (formerly known as 17BIPHE2), a peptide engineered based on the only human cathelicidin. This study documents an alternative solution via covalent immobilization of FK-16, amino acid sequence FKRIVQRIKDFLRNLV-amide, which corresponds to the major antimicrobial region (residues 17-32) of LL-37. FK-16 is superior to the longer peptide LL-37 in terms of synthesis cost and the shorter peptide KR-12 in terms of activity spectrum. Indeed, the FK16-coated titanium surface showed a broad-spectrum activity against the ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. It also demonstrated anti-adhesion and biofilm inhibition capabilities against both S. aureus and E. coli.

The fabrication and in vitro properties of antibacterial polydopamine-LL-37-POPC coatings on micro-arc oxidized titanium.

PubMed

He, Ye; Zhang, Yangyang; Shen, Xinkun; Tao, Bailong; Liu, Ju; Yuan, Zhang; Cai, Kaiyong

2018-05-31

Bacterial infection commonly occurs in clinical settings when the procedure involves a medical implant. Thus, the fabrication of antimicrobial medical materials has attracted much attention in recent years. To improve the antibacterial properties of titanium (Ti)-based biomedical materials, surface microporous structures, with antimicrobial peptide coatings, were employed in this study. Native Ti substrates were endowed with a certain level of antibacterial activity after treatment with the micro-arc oxidation (MAO). A multilayer consisting of polydopamine, cationic antimicrobial peptides LL-37, and phospholipid (POPC) was coated onto MAO substrates, leading to antibacterial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. The combination of polydopamine-LL-37-POPC was found to alleviate the burst release of LL-37 in the initial phase. This multilayer coated onto microporous Ti substrates also showed favorable cytocompatibility to both mesenchymal stem cells (MSCs) and osteoblasts. These findings illustrate a novel strategy for the development of antibacterial Ti-based implants. Copyright © 2018 Elsevier B.V. All rights reserved.

LL-37-derived membrane-active FK-13 analogs possessing cell selectivity, anti-biofilm activity and synergy with chloramphenicol and anti-inflammatory activity.

PubMed

Rajasekaran, Ganesan; Kim, Eun Young; Shin, Song Yub

2017-05-01

Although the human-derived antimicrobial peptide (AMP) LL-37 has potent antimicrobial and anti-inflammatory activities, its therapeutic application is limited by its low cell selectivity and high production cost due to its large size. To overcome these problems, we tried to develop novel LL-37-derived short α-helical AMPs with improved cell selectivity and without a significant loss of anti-inflammatory activity relative to that of parental LL-37. Using amino acid substitution, we designed and synthesized a series of FK13 analogs based on the sequence of the 13-meric short FK13 peptide (residues 17-29 of LL-37) that has been identified as the region responsible for the antimicrobial activity of LL-37. Among the designed FK13 analogs, FK-13-a1 and FK-13-a7 showed high cell selectivity and retained the anti-inflammatory activity. The therapeutic index (a measure of cell selectivity) of FK-13-a1 and FK-13-a7 was 6.3- and 2.3-fold that of parental LL-37, respectively. Furthermore, FK-13-a1 and FK-13-a7 displayed more potent antimicrobial activity against antibiotic-resistant bacteria including MRSA, MDRPA, and VREF, than did LL-37. In addition, FK-13-a1 and FK-13-a7 exhibited greater synergistic effects with chloramphenicol against MRSA and MDRPA and were more effective anti-biofilm agents against MDRPA than LL-37 was. Moreover, FK-13-a1 and FK-13-a7 maintained their activities in the presence of physiological salts and human serum. SYTOX green uptake, membrane depolarization and killing kinetics revealed that FK13-a1 and FK13-a7 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Taken together, our results suggest that FK13-a1 and FK13-a7 can be developed as novel antimicrobial/anti-inflammatory agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37

PubMed Central

2017-01-01

Antimicrobial peptides (AMPs) are thought to kill bacterial cells by permeabilizing their membranes. However, some antimicrobial peptides inhibit E. coli growth more efficiently in aerobic than in anaerobic conditions. In the attack of the human cathelicidin LL-37 on E. coli, real-time, single-cell fluorescence imaging reveals the timing of membrane permeabilization and the onset of oxidative stress. For cells growing aerobically, a CellROX Green assay indicates that LL-37 induces rapid formation of oxidative species after entry into the periplasm, but before permeabilization of the cytoplasmic membrane (CM). A cytoplasmic Amplex Red assay signals a subsequent burst of oxidative species, most likely hydrogen peroxide, shortly after permeabilization of the CM. These signals are much stronger in the presence of oxygen, a functional electron transport chain, and a large proton motive force (PMF). They are much weaker in cells growing anaerobically, by either fermentation or anaerobic respiration. In aerobic growth, the oxidative signals are attenuated in a cytochrome oxidase–bd deletion mutant, but not in a –bo3 deletion mutant, suggesting a specific effect of LL-37 on the electron transport chain. The AMPs melittin and LL-37 induce strong oxidative signals and exhibit O2-sensitive MICs, while the AMPs indolicidin and cecropin A do not. These results suggest that AMP activity in different tissues may be tuned according to the local oxygen level. This may be significant for control of opportunistic pathogens while enabling growth of commensal bacteria. PMID:28665988

Antibacterial function of the human cathelicidin-18 peptide (LL-37) between theory and practice.

PubMed

Iacob, Simona A; Iacob, Diana G

2014-01-01

The human cathelicidin-18 is an antimicrobial, immunomodulatory and tissue repair peptide. The LL-37 fragment of this peptide which is in fact the active domain of the cathelicidin-18 is critical for the human antibacterial defense and epithelial integrity. It's activity against resistant pathogens, the potential of epithelial healing after microbial injury and the neutralization of bacterial endotoxin underlie the most important benefits of this peptide. However, there are still a number of questions that remain to be answered regarding the precise interactions of cathelicidin-18 within the immune system, the exact tissue concentrations or its possible pro-tumoral activity. In this respect, the therapeutic potential of cathelicidin-18 in various infections has been proved by in vitro experiments, but additional detailed clinical studies are still required to ascertain its antimicrobial role in vivo. We present a short review on the antibacterial activity of human cathelicidin-18 (LL-37) according to in vitro experiments while discussing its potential use in the clinical practice.

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria

PubMed Central

Puklo, M.; Guentsch, A.; Hiemstra, P. S.; Eick, S.; Potempa, J.

2008-01-01

Introduction During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. Methods GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and α-defensins (HNP1–3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. Results Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1–3 in samples from patients, compared to controls. The amount of HNP1–3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Conclusion The lack of a correlation between LL-37, HNP1–3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis. PMID:18582333

Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications.

PubMed

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K; Paranjape, Shruti M; Montelaro, Ronald C; Mietzner, Timothy A

2005-08-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg(2+) and Ca(2+) ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg(2+) and Ca(2+). WLBU2 is able to kill P. aeruginosa (10(6) CFU/ml) in human serum, with a minimum bactericidal concentration of <9 microM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 microM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis.

Activity of the De Novo Engineered Antimicrobial Peptide WLBU2 against Pseudomonas aeruginosa in Human Serum and Whole Blood: Implications for Systemic Applications

PubMed Central

Deslouches, Berthony; Islam, Kazi; Craigo, Jodi K.; Paranjape, Shruti M.; Montelaro, Ronald C.; Mietzner, Timothy A.

2005-01-01

Cationic amphipathic peptides have been extensively investigated as a potential source of new antimicrobials that can complement current antibiotic regimens in the face of emerging drug-resistant bacteria. However, the suppression of antimicrobial activity under certain biologically relevant conditions (e.g., serum and physiological salt concentrations) has hampered efforts to develop safe and effective antimicrobial peptides for clinical use. We have analyzed the activity and selectivity of the human peptide LL37 and the de novo engineered antimicrobial peptide WLBU2 in several biologically relevant conditions. The host-derived synthetic peptide LL37 displayed high activity against Pseudomonas aeruginosa but demonstrated staphylococcus-specific sensitivity to NaCl concentrations varying from 50 to 300 mM. Moreover, LL37 potency was variably suppressed in the presence of 1 to 6 mM Mg2+ and Ca2+ ions. In contrast, WLBU2 maintained its activity in NaCl and physiologic serum concentrations of Mg2+ and Ca2+. WLBU2 is able to kill P. aeruginosa (106 CFU/ml) in human serum, with a minimum bactericidal concentration of <9 μM. Conversely, LL37 is inactive in the presence of human serum. Bacterial killing kinetic assays in serum revealed that WLBU2 achieved complete bacterial killing in 20 min. Consistent with these results was the ability of WLBU2 (15 to 20 μM) to eradicate bacteria from ex vivo samples of whole blood. The selectivity of WLBU2 was further demonstrated by its ability to specifically eliminate P. aeruginosa in coculture with human monocytes or skin fibroblasts without detectable adverse effects to the host cells. Finally, WLBU2 displayed potent efficacy against P. aeruginosa in an intraperitoneal infection model using female Swiss Webster mice. These results establish a potential application of WLBU2 in the treatment of bacterial sepsis. PMID:16048927

LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

PubMed

van der Does, Anne M; Beekhuizen, Henry; Ravensbergen, Bep; Vos, Tim; Ottenhoff, Tom H M; van Dissel, Jaap T; Drijfhout, Jan W; Hiemstra, Pieter S; Nibbering, Peter H

2010-08-01

The human cathelicidin LL-37 has broad-spectrum antimicrobial activity. It also participates at the interface of innate and adaptive immunity by chemoattracting immune effector cells, modulating the production of a variety of inflammatory mediators by different cell types, and regulating the differentiation of monocytes into dendritic cells. In this study, we investigated the effects of LL-37 on the differentiation of human monocytes into anti-inflammatory macrophages (MPhi-2; driven by M-CSF) versus proinflammatory macrophages (MPhi-1; driven by GM-CSF) as well as on fully differentiated MPhi-1 and MPhi-2. Results revealed that monocytes cultured with M-CSF in the presence of LL-37 resulted in macrophages displaying a proinflammatory signature, namely, low expression of CD163 and little IL-10 and profound IL-12p40 production on LPS stimulation. The effects of LL-37 on M-CSF-driven macrophage differentiation were dose- and time-dependent with maximal effects observed at 10 microg/ml when the peptide was present from the start of the cultures. The peptide enhanced the GM-CSF-driven macrophage differentiation. Exposure of fully differentiated MPhi-2 to LL-37 for 6 d resulted in macrophages that produced less IL-10 and more IL-12p40 on LPS stimulation than control MPhi-2. In contrast, LL-37 had no effect on fully differentiated MPhi-1. Peptide mapping using a set of 16 overlapping 22-mer peptides covering the complete LL-37 sequence revealed that the C-terminal portion of LL-37 is responsible for directing macrophage differentiation. Our results furthermore indicate that the effects of LL-37 on macrophage differentiation required internalization of the peptide. Together, we conclude that LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis.

PubMed

Rivas-Santiago, Bruno; Rivas Santiago, Cesar E; Castañeda-Delgado, Julio E; León-Contreras, Juan C; Hancock, Robert E W; Hernandez-Pando, Rogelio

2013-02-01

Tuberculosis (TB) is a major worldwide health problem in part due to the lack of development of new treatments and the emergence of new strains such as multidrug-resistant (MDR) and extensively drug-resistant strains that are threatening and impairing the control of this disease. In this study, the efficacy of natural and synthetic cationic antimicrobial (host defence) peptides that have been shown often to possess broad-spectrum antimicrobial activity was tested. The natural antimicrobial peptides human LL-37 and mouse CRAMP as well as synthetic peptides E2, E6 and CP26 were tested for their activity against Mycobacterium tuberculosis both in in vitro and in vivo models. The peptides had moderate antimicrobial activities, with minimum inhibitory concentrations ranging from 2 μg/mL to 10 μg/mL. In a virulent model of M. tuberculosis lung infection, intratracheal therapeutic application of these peptides three times a week at doses of ca. 1mg/kg led to significant 3-10-fold reductions in lung bacilli after 28-30 days of treatment. The treatments worked both against the drug-sensitive H37Rv strain and a MDR strain. These results indicate that antimicrobial peptides might constitute a novel therapy against TB. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37.

PubMed

Nan, Yong Hai; Bang, Jeong-Kyu; Jacob, Binu; Park, Il-Seon; Shin, Song Yub

2012-06-01

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. Copyright © 2012. Published by Elsevier Inc.

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis.

PubMed

Hell, E; Giske, C G; Nelson, A; Römling, U; Marchini, G

2010-02-01

The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device-related infections. We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l(-1)) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l(-1) concentration of LL37, indicating a direct effect of the peptide on biofilm production. As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.

Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wada, Akihiro, E-mail: a-wada@nagasaki-u.ac.jp; Wong, Pooi-Fong; Hojo, Hironobu

Highlights: • Alarin inhibits the growth of E. coli but not S. aureus. • Alarin’s potency is comparable to LL-37 in inhibiting the growth of E. coli. • Alarin can cause bacterial membrane blebbing. • Alalin does not induce hemolysis on erythrocytes. -- Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radialmore » diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.« less

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

PubMed Central

Limoli, Dominique H.; Rockel, Andrea B.; Host, Kurtis M.; Jha, Anuvrat; Kopp, Benjamin T.; Hollis, Thomas; Wozniak, Daniel J.

2014-01-01

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections. PMID:24763694

PLGA nanoparticles loaded with host defense peptide LL37 promote wound healing.

PubMed

Chereddy, Kiran Kumar; Her, Charles-Henry; Comune, Michela; Moia, Claudia; Lopes, Alessandra; Porporato, Paolo E; Vanacker, Julie; Lam, Martin C; Steinstraesser, Lars; Sonveaux, Pierre; Zhu, Huijun; Ferreira, Lino S; Vandermeulen, Gaëlle; Préat, Véronique

2014-11-28

Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Poly (lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. LL37 is an endogenous human host defense peptide that modulates wound healing and angiogenesis and fights infection. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) promotes wound closure due to the sustained release of both LL37 and lactate. In full thickness excisional wounds, the treatment with PLGA-LL37 NP significantly accelerated wound healing compared to PLGA or LL37 administration alone. PLGA-LL37 NP-treated wounds displayed advanced granulation tissue formation by significant higher collagen deposition, re-epithelialized and neovascularized composition. PLGA-LL37 NP improved angiogenesis, significantly up-regulated IL-6 and VEGFa expression, and modulated the inflammatory wound response. In vitro, PLGA-LL37 NP induced enhanced cell migration but had no effect on the metabolism and proliferation of keratinocytes. It displayed antimicrobial activity on Escherichia coli. In conclusion, we developed a biodegradable drug delivery system that accelerated healing processes due to the combined effects of lactate and LL37 released from the nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37.

PubMed

Holásková, Edita; Galuszka, Petr; Mičúchová, Alžbeta; Šebela, Marek; Öz, Mehmet Tufan; Frébort, Ivo

2018-06-01

The peptide LL-37, a component of the human innate immune system, represents a promising drug candidate. In particular, the development of low-cost production platform technology is a critical bottleneck in its use in medicine. In the present study, a viable approach for the LL-37 production in transgenic barley is developed. First, comparative analyses of the effects of different fused peptide epitope tags applicable for accumulation and purification on LL-37 production yield are performed using transient expression in tobacco leaves. Following the selection of the most yielding fusion peptide strategies, eight different constructs for the expression of codon optimized chimeric LL-37 genes in transgenic barley plants are created. The expression of individual constructs is driven either by an endosperm-specific promoter of the barley B1 hordein gene or by the maize ubiquitin promoter. The transgenes are stably integrated into the barley genome and inherited in the subsequent generation. All transgenic lines show normal phenotypes and are fertile. LL-37 accumulated in the barley seeds up to 0.55 mg per 1 kg of grain. The fused epitope tags are cleaved off by the use of enterokinase. Furthermore, in planta produced LL-37 including the fused versions is biologically active. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells.

PubMed

Gambade, Audrey; Zreika, Sami; Guéguinou, Maxime; Chourpa, Igor; Fromont, Gaëlle; Bouchet, Ana Maria; Burlaud-Gaillard, Julien; Potier-Cartereau, Marie; Roger, Sébastien; Aucagne, Vincent; Chevalier, Stéphan; Vandier, Christophe; Goupille, Caroline; Weber, Günther

2016-04-26

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells

PubMed Central

Guéguinou, Maxime; Chourpa, Igor; Fromont, Gaëlle; Bouchet, Ana Maria; Burlaud-Gaillard, Julien; Potier-Cartereau, Marie; Roger, Sébastien; Aucagne, Vincent; Chevalier, Stéphan; Vandier, Christophe

2016-01-01

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects. PMID:26993604

Real-time Attack of LL-37 on Single Bacillus subtilis Cells

PubMed Central

Barns, Kenneth J.; Weisshaar, James C.

2013-01-01

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12 s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2 μM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4 μM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5 frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel “growth recovery” assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2 μM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4 μM LL-37 is not. PMID:23454084

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

PubMed Central

Hutchinson, Andrew T.; To, Joyce; Taylor, Nicole L.; Norton, Raymond S.; Perugini, Matthew A.

2011-01-01

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation. PMID:21589904

A QCM-D study of the concentration- and time-dependent interactions of human LL37 with model mammalian lipid bilayers.

PubMed

Lozeau, Lindsay D; Rolle, Marsha W; Camesano, Terri A

2018-07-01

The human antimicrobial peptide LL37 is promising as an alternative to antibiotics due to its biophysical interactions with charged bacterial lipids. However, its clinical potential is limited due to its interactions with zwitterionic mammalian lipids leading to cytotoxicity. Mechanistic insight into the LL37 interactions with mammalian lipids may enable rational design of less toxic LL37-based therapeutics. To this end, we studied concentration- and time-dependent interactions of LL37 with zwitterionic model phosphatidylcholine (PC) bilayers with quartz crystal microbalance with dissipation (QCM-D). LL37 mass adsorption and PC bilayer viscoelasticity changes were monitored by measuring changes in frequency (Δf) and dissipation (ΔD), respectively. The Voigt-Kelvin viscoelastic model was applied to Δf and ΔD to study changes in bilayer thickness and density with LL37 concentration. At low concentrations (0.10-1.00 μM), LL37 adsorbed onto bilayers in a concentration-dependent manner. Further analyses of Δf, ΔD and thickness revealed that peptide saturation on the bilayers was a threshold for interactions observed above 2.00 μM, interactions that were rapid, multi-step, and reached equilibrium in a concentration- and time-dependent manner. Based on these data, we proposed a model of stable transmembrane pore formation at 2.00-10.0 μM, or transition from a primarily lipid to a primarily protein film with a transmembrane pore formation intermediate state at concentrations of LL37 > 10 μM. The concentration-dependent interactions between LL37 and PC bilayers correlated with the observed concentration-dependent biological activities of LL37 (antimicrobial, immunomodulatory and non-cytotoxic at 0.1-1.0 μM, hemolytic and some cytotoxicity at 2.0-13 μM and cytotoxic at >13 μM). Copyright © 2018 Elsevier B.V. All rights reserved.

Potential Use of Antimicrobial Peptides as Vaginal Spermicides/Microbicides

PubMed Central

Tanphaichitr, Nongnuj; Srakaew, Nopparat; Alonzi, Rhea; Kiattiburut, Wongsakorn; Kongmanas, Kessiri; Zhi, Ruina; Li, Weihua; Baker, Mark; Wang, Guanshun; Hickling, Duane

2016-01-01

The concurrent increases in global population and sexually transmitted infection (STI) demand a search for agents with dual spermicidal and microbicidal properties for topical vaginal application. Previous attempts to develop the surfactant spermicide, nonoxynol-9 (N-9), into a vaginal microbicide were unsuccessful largely due to its inefficiency to kill microbes. Furthermore, N-9 causes damage to the vaginal epithelium, thus accelerating microbes to enter the women’s body. For this reason, antimicrobial peptides (AMPs), naturally secreted by all forms of life as part of innate immunity, deserve evaluation for their potential spermicidal effects. To date, twelve spermicidal AMPs have been described including LL-37, magainin 2 and nisin A. Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use for the following reasons. First, it is a human AMP naturally produced in the vagina after intercourse. Second, LL-37 exerts microbicidal effects to numerous microbes including those that cause STI. Third, its cytotoxicity is selective to sperm and not to the female reproductive tract. Furthermore, the spermicidal effects of LL-37 have been demonstrated in vivo in mice. Therefore, the availability of LL-37 as a vaginal spermicide/microbicide will empower women for self-protection against unwanted pregnancies and STI. PMID:26978373

LL-37-derived short antimicrobial peptide KR-12-a5 and its d-amino acid substituted analogs with cell selectivity, anti-biofilm activity, synergistic effect with conventional antibiotics, and anti-inflammatory activity.

PubMed

Kim, Eun Young; Rajasekaran, Ganesan; Shin, Song Yub

2017-08-18

KR-12-a5 is a 12-meric α-helical antimicrobial peptide (AMP) with dual antimicrobial and anti-inflammatory activities designed from human cathelicidin LL-37. We designed and synthesized a series of d-amino acid-substituted analogs of KR-12-a5 with the aim of developing novel α-helical AMPs that possess higher cell selectivity than KR-12-a5, while maintaining the anti-inflammatory activity. d-amino acid incorporation into KR-12-a5 induced a significant improvement in the cell selectivity by 2.6- to 13.6-fold as compared to KR-12-a5, while maintaining the anti-inflammatory activity. Among the three analogs, KR-12-a5 (6- D L) with d-amino acid in the polar-nonpolar interface (Leu 6 ) showed the highest cell selectivity (therapeutic index: 61.2). Similar to LL-37, KR-12-a5 and its analogs significantly inhibited the expression and secretion of NO, TNF-α, IL-6 and MCP-1 from LPS-stimulated RAW264.7 cells. KR-12-a5 and its analogs showed a more potent antimicrobial activity against antibiotic-resistant bacteria, including clinically isolated MRSA, MDRPA, and VREF than LL-37 and melittin. Furthermore, compared to LL-37, KR-12-a5 and its analogs showed greater synergistic effects with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin against MDRPA; KR-12-a5 and its analogs had a FICI range between 0.25 and 0.5, and LL-37 had a range between 0.75 and 1.5. KR-12-a5 and its analogs were found to be more effective anti-biofilm agents against MDRPA than LL-37. In addition, KR-12-a5 and its analogs maintained antimicrobial activity in physiological salts and human serum. SYTOX Green uptake and membrane depolarization studies revealed that KR-12-a5 and its analogs kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Taken together, our results suggest that KR-12-a5 and its analogs can be developed further as novel antimicrobial/anti-inflammatory agents to treat antibiotic-resistant infections. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

LL-37 modulates human neutrophil responses to influenza A virus

PubMed Central

Tripathi, Shweta; Verma, Anamika; Kim, Eun-Jeong; White, Mitchell R.; Hartshorn, Kevan L.

2014-01-01

Recent studies have shown that the human cathelicidin, LL-37, has antiviral activity against IAV in vitro and in vivo. Neutrophils are important cellular components of the initial innate response to IAV infection. In addition to its direct antimicrobial activities, LL-37 has important immunomodulatory effects. In this study, we explore how LL-37 affects interactions of IAV with human neutrophils. LL-37 did not alter neutrophil uptake of IAV but significantly increased neutrophil H2O2 responses to the virus. IAV stimulated production of NETs in vitro, and this response was increased by preincubating the virus with LL-37. NADPH-oxidase blockade did not reduce IAV-induced NET formation or the increased NET response stimulated by LL-37 + IAV. The increased respiratory burst and NET responses were, however, inhibited by preincubating cells with a formyl peptide receptor blocker, indicating that LL-37 engages these receptors when complexed with IAV. Responses to IAV alone were not inhibited by formyl peptide receptor blockade. It has been reported that LL-37 reduces proinflammatory cytokine responses during IAV infection in vivo. We now show that IAV alone potentiated release of IL-8 from neutrophils, and preincubation with LL-37 reduced IAV-stimulated IL-8 release. These results confirm that LL-37 modulates human neutrophil responses to IAV in a distinctive manner and could have important bearing on the protective effects of LL-37 during IAV infection in vivo. PMID:25082153

Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells.

PubMed

Rivas-Santiago, Bruno; Hernandez-Pando, Rogelio; Carranza, Claudia; Juarez, Esmeralda; Contreras, Juan Leon; Aguilar-Leon, Diana; Torres, Martha; Sada, Eduardo

2008-03-01

The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis.

Expression of Cathelicidin LL-37 during Mycobacterium tuberculosis Infection in Human Alveolar Macrophages, Monocytes, Neutrophils, and Epithelial Cells▿

PubMed Central

Rivas-Santiago, Bruno; Hernandez-Pando, Rogelio; Carranza, Claudia; Juarez, Esmeralda; Contreras, Juan Leon; Aguilar-Leon, Diana; Torres, Martha; Sada, Eduardo

2008-01-01

The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis. PMID:18160480

Transformation of Human Cathelicidin LL-37 into Selective, Stable, and Potent Antimicrobial Compounds

PubMed Central

2015-01-01

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens. PMID:25061850

Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

PubMed

Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

2016-02-01

Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections.

Vitamin D status and antimicrobial peptide cathelicidin (LL-37) concentrations in patients with active pulmonary tuberculosis.

PubMed

Yamshchikov, Alexandra V; Kurbatova, Ekaterina V; Kumari, Meena; Blumberg, Henry M; Ziegler, Thomas R; Ray, Susan M; Tangpricha, Vin

2010-09-01

Vitamin D insufficiency is common in industrialized and developing nations. Recent studies have shown that vitamin D insufficiency is associated with a higher risk of active tuberculosis. Laboratory studies provided a mechanism for this link on the basis of findings that vitamin D metabolites regulate the expression of cathelicidin (LL-37), which is an endogenous antimicrobial peptide with activity against Mycobacterium tuberculosis. Little information is available on the clinical relation between vitamin D, LL-37 concentrations, and disease severity in patients with tuberculosis. The primary objective of the study was to evaluate the relation between vitamin D nutriture, serum LL-37 concentrations, and tuberculosis by using samples stored in the Tuberculosis Trials Consortium serum repository. We measured 25-hydroxyvitamin D [25(OH)D] and LL-37 concentrations in 95 serum specimens from patients with culture-confirmed pulmonary tuberculosis and correlated these concentrations to clinical and demographic variables. The prevalence of vitamin D insufficiency [serum 25(OH)D concentration lt 30 ng/mL] in patients with active tuberculosis was 86% (n = 95) with a mean baseline serum 25(OH)D concentration of 20.4 ng/mL. Factors associated with vitamin D insufficiency were black race and indoor lifestyle. The mean ( plusmn SD) baseline LL-37 concentration was 49.5 plusmn 23.8 ng/mL. Higher LL-37 concentrations correlated with acid fast bacilli sputum smear positivity and weight gt 10% below ideal body weight. Serum vitamin D status of the study subjects did not correlate with serum LL-37 concentrations. More prospectively designed studies are needed to evaluate the clinical implications of vitamin D insufficiency in patients with tuberculosis and the utility of circulating LL-37 as a potential biomarker in patients with active tuberculosis disease. The parent trial was registered at clinicaltrials.gov as NCT00023335.

LL-37-Derived Peptides Eradicate Multidrug-Resistant Staphylococcus aureus from Thermally Wounded Human Skin Equivalents

PubMed Central

de Breij, Anna; Chan, Heelam; van Dissel, Jaap T.; Drijfhout, Jan W.; Hiemstra, Pieter S.; El Ghalbzouri, Abdoelwaheb; Nibbering, Peter H.

2014-01-01

Burn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus (MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria. PMID:24841266

Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages

PubMed Central

Rekha, Rokeya Sultana; Rao Muvva, SSV Jagadeeswara; Wan, Min; Raqib, Rubhana; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta

2015-01-01

LL-37 is a human antimicrobial peptide (AMP) of the cathelicidin family with multiple activities including a mediator of vitamin D-induced autophagy in human macrophages, resulting in intracellular killing of Mycobacterium tuberculosis (Mtb). In a previous trial in healthy volunteers, we have shown that LL-37 expression and subsequent Mtb-killing can be further enhanced by 4-phenylbutyrate (PBA), also an inducer of LL-37 expression. Here, we explore a potential mechanism(s) behind PBA and LL-37-induced autophagy and intracellular killing of Mtb. Mtb infection of macrophages downregulated the expression of both the CAMP transcript and LL-37 peptide as well as certain autophagy-related genes (BECN1 and ATG5) at both the mRNA and protein levels. In addition, activation of LC3-II in primary macrophages and THP-1 cells was not detected. PBA and the active form of vitamin D3 (1,25[OH]2D3), separately or particularly in combination, were able to overcome Mtb-induced suppression of LL-37 expression. Notably, reactivation of autophagy occurred by stimulation of macrophages with PBA and promoted colocalization of LL-37 and LC3-II in autophagosomes. Importantly, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells, when the expression of LL-37 was silenced. However, PBA-induced autophagy was restored when the LL-37 knockdown cells were supplemented with synthetic LL-37. Interestingly, we have found that LL-37-induced autophagy was mediated via P2RX7 receptor followed by enhanced cytosolic free Ca2+, and activation of AMPK and PtdIns3K pathways. Altogether, these results suggest a novel activity for PBA as an inducer of autophagy, which is LL-37-dependent and promotes intracellular killing of Mtb in human macrophages. PMID:26218841

Induction of group A Streptococcus virulence by a human antimicrobial peptide.

PubMed

Gryllos, Ioannis; Tran-Winkler, Hien J; Cheng, Ming-Fang; Chung, Hachung; Bolcome, Robert; Lu, Wuyuan; Lehrer, Robert I; Wessels, Michael R

2008-10-28

Group A streptococci (Streptococcus pyogenes or GAS) freshly isolated from individuals with streptococcal sore throat or invasive ("flesh-eating") infection often grow as mucoid colonies on primary culture but lose this colony appearance after laboratory passage. The mucoid phenotype is due to abundant production of the hyaluronic acid capsular polysaccharide, a key virulence determinant associated with severe GAS infections. These observations suggest that signal(s) from the human host trigger increased production of capsule and perhaps other virulence factors during infection. Here we show that subinhibitory concentrations of the human antimicrobial cathelicidin peptide LL-37 stimulate expression of the GAS capsule synthesis operon (hasABC). Up-regulation is mediated by the CsrRS 2-component regulatory system: it requires a functional CsrS sensor protein and can be antagonized by increased extracellular Mg(2+), the other identified environmental signal for CsrS. Up-regulation was also evident for other CsrRS-regulated virulence genes, including the IL-8 protease PrtS/ScpC and the integrin-like/IgG protease Mac/IdeS, findings that suggest a coordinated GAS virulence response elicited by this antimicrobial immune effector peptide. LL-37 signaling through CsrRS led to a marked increase in GAS resistance to opsonophagocytic killing by human leukocytes, an in vitro measure of enhanced GAS virulence, consistent with increased expression of the antiphagocytic capsular polysaccharide and Mac/IdeS. We propose that the human cathelicidin LL-37 has the paradoxical effect of stimulating CsrRS-regulated virulence gene expression, thereby enhancing GAS pathogenicity during infection. The ability of GAS to sense and respond to LL-37 may explain, at least in part, the unique susceptibility of the human species to streptococcal infection.

Antimicrobial and immunomodulatory activity of host defense peptides, clavanins and LL-37, in vitro: An endodontic perspective.

PubMed

Lima, Stella Maris F; Freire, Mirna S; Gomes, Ana Luisa O; Cantuária, Ana Paula C; Dutra, Flávia Rodrigues P; Magalhães, Beatriz S; Sousa, Maurício Gonçalves C; Migliolo, Ludovico; Almeida, Jeeser A; Franco, Octávio L; Rezende, Taia Maria B

2017-09-01

Endodontic treatment is mainly based on root canal disinfection and its failure may be motivated by microbial resistance. Endodontic therapy can be benefitted by host defense peptides (HDPs), which are multifunctional molecules that act against persistent infection and inflammation. This study aimed to evaluate the antimicrobial, cytotoxic and immunomodulatory activity of several HDPs, namely clavanin A, clavanin A modified (MO) and LL-37, compared to intracanal medication Ca(OH) 2 . HDPs and Ca(OH) 2 were evaluated by: (1) antimicrobial assays against Candida albicans and Enterococcus faecalis, (2) cytotoxicity assays and (3) cytokine tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1α, IL-6, IL-10 and IL-12 and nitric oxide (NO) production by RAW 264.7 cells incubated with or without heat-killed (HK) C. albicans or E. faecalis combined or not with interferon-γ. The minimum inhibitory concentration (MIC) was established only for E. faecalis (LL-37, 57μM). Considering cytotoxicity, clavanin MO was able to reduce cell viability in many groups and demonstrated lowest LC 50 . The Ca(OH) 2 up-regulated the production of MCP-1, TNF-α, IL-12 and IL-6 and down-regulated IL-1α, IL-10 and NO. Clavanins up-regulated the TNF-α and NO and down-regulated IL-10 production. LL-37 demonstrated up-regulation of IL-6 and TNF-α production and down-regulation in IL-10 and NO production. In conclusion, LL-37 demonstrated better antibacterial potential. In addition, Ca(OH) 2 demonstrated a proinflammatory response, while the HDPs modulated the inflammatory response from non-interference with the active cytokines in the osteoclastogenesis process, probably promoting the health of periradicular tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Vitamin D3 analog maxacalcitol (OCT) induces hCAP-18/LL-37 production in human oral epithelial cells.

PubMed

Tada, Hiroyuki; Shimizu, Takamitsu; Nagaoka, Isao; Takada, Haruhiko

2016-01-01

Maxacalcitol (22-oxacalcitriol: OCT) is a synthetic vitamin D3 analog with a limited calcemic effect. In this study, we investigated whether OCT increases the production of LL-37/CAP-18, a human cathelicidin antimicrobial peptide, in human gingival/oral epithelial cells. A human gingival epithelial cell line (Ca9-22) and human oral epithelial cell lines (HSC-2, HSC-3, and HSC-4) exhibited the enhanced expression of LL-37 mRNA upon stimulation with OCT as well as active metabolites of vitamins D3 and D2. Among the human epithelial cell lines, Ca9-22 exhibited the strongest response to these vitamin D-related compounds. OCT induced the higher production of CAP-18 (ng/mL order) until 6 days time-dependently in Ca9-22 cells in culture. The periodontal pathogen Porphyromonas gingivalis was killed by treatment with the LL-37 peptide. These findings suggest that OCT induces the production of hCAP-18/LL-37 in a manner similar to that induced by the active metabolite of vitamin D3.

Effects of conditioned medium from LL-37 treated adipose stem cells on human fibroblast migration.

PubMed

Yang, Eun-Jung; Bang, Sa-Ik

2017-07-01

Adipose stem cell-conditioned medium may promote human dermal fibroblast (HDF) proliferation and migration by activating paracrine peptides during the re-epithelization phase of wound healing. Human antimicrobial peptide LL-37 is upregulated in the skin epithelium as part of the normal response to injury. The effects of conditioned medium (CM) from LL-37 treated adipose stem cells (ASCs) on cutaneous wound healing, including the mediation of fibroblast migration, remain to be elucidated, therefore the aim of the present study was to determine how ASCs would react to an LL-37-rich microenvironment and if CM from LL-37 treated ASCs may influence the migration of HDFs. The present study conducted migration assays with HDFs treated with CM from LL-37 treated ASCs. Expression of CXC chemokine receptor 4 (CXCR4), which controls the recruitment of HDFs, was analyzed at the mRNA and protein levels. To further characterize the stimulatory effects of LL-37 on ASCs, the expression of stromal cell-derived factor-1α (SDF-1α), a CXC chemokine, was investigated. CM from LL-37-treated ASCs induced migration of HDFs in a time- and dose-dependent manner, with a maximum difference in migration observed 24 h following stimulation with LL-37 at a concentration of 10 µg/ml. The HDF migration and the expression of CXCR4 in fibroblasts was markedly increased upon treatment with CM from LL-37-treated ASCs compared with CM from untreated ASCs. SDF-1α expression was markedly increased in CM from LL-37 treated ASCs. It was additionally observed that SDF-1α blockade significantly reduced HDF migration. These findings suggest the feasibility of CM from LL-37-treated ASCs as a potential therapeutic for human dermal fibroblast migration.

Decoding the Functional Roles of Cationic Side Chains of the Major Antimicrobial Region of Human Cathelicidin LL-37

PubMed Central

Epand, Raquel F.; Mishra, Biswajit; Lushnikova, Tamara; Thomas, Vinai Chittezham; Bayles, Kenneth W.; Epand, Richard M.

2012-01-01

Human cathelicidin LL-37 is a critical cationic antimicrobial peptide for host defense against infection, immune modulation, and wound healing. This article elucidates the functional roles of the cationic side chains of the major antimicrobial region of LL-37, corresponding to residues 17 to 32 (designated GF-17). Antimicrobial assays, killing kinetics studies, and vesicle leakage experiments all indicate that a conversion of lysines to arginines affected the ability of the peptide to kill the Gram-positive Staphylococcus aureus strain USA300. Alanine scanning experiments show that S. aureus is less sensitive than Escherichia coli to a single cationic residue mutation of GF-17. Among the five cationic residues, R23 appears to be somewhat important in killing S. aureus. However, R23 and K25 of GF-17 are of prime importance in killing the Gram-negative organism E. coli. In particular, R23 is essential for (i) rapid recognition, (ii) permeation of the E. coli outer membrane, (iii) clustering of anionic lipids in a membrane system mimicking the E. coli inner membrane, and (iv) membrane disruption. Bacterial aggregation (i.e., rapid recognition via charge neutralization) is the first step of the peptide action. Structurally, R23 is located in the interface (i.e., the first action layer), a situation ideal for the interactions listed above. In contrast, residues K18, R19, and R29 are on the hydrophilic surface of the amphipathic helix and play only a secondary role. Mapping of the functional spectrum of cationic residues of GF-17 provides a solid basis for engineering bacterium-specific antimicrobials using this highly potent template. PMID:22083479

Activity of innate antimicrobial peptides and ivacaftor against clinical cystic fibrosis respiratory pathogens.

PubMed

Payne, Joanna E; Dubois, Alice V; Ingram, Rebecca J; Weldon, Sinead; Taggart, Clifford C; Elborn, J Stuart; Tunney, Michael M

2017-09-01

There is a clear need for new antimicrobials to improve current treatment of chronic lung infection in people with cystic fibrosis (CF). This study determined the activities of antimicrobial peptides (AMPs) and ivacaftor, a novel CF transmembrane conductance regulator potentiator, for CF treatment. Antimicrobial activities of AMPs [LL37, human β-defensins (HβD) 1-4 and SLPI] and ivacaftor against clinical respiratory isolates (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus spp., Achromobacter spp. and Stenotrophomonas maltophilia) were determined using radial diffusion and time-kill assays, respectively. Synergy of LL37 and ivacaftor with tobramycin was determined by time-kill, with in vivo activity of ivacaftor and tobramycin compared using a murine infection model. LL37 and HβD3 were the most active AMPs tested, with MICs ranging from 3.2- ≥ 200 mg/L and 4.8- ≥ 200 mg/L, respectively, except for Achromobacter that was resistant. HβD1 and SLPI demonstrated no antimicrobial activity. LL37 demonstrated synergy with tobramycin against 4/5 S. aureus and 2/5 Streptococcus spp. isolates. Ivacaftor demonstrated bactericidal activity against Streptococcus spp. (mean log 10 decrease 3.31 CFU/mL) and bacteriostatic activity against S. aureus (mean log 10 change 0.13 CFU/mL), but no activity against other genera. Moreover, ivacaftor demonstrated synergy with tobramycin, with mean log 10 decreases of 5.72 CFU/mL and 5.53 CFU/mL at 24 h for S. aureus and Streptococcus spp., respectively. Ivacaftor demonstrated immunomodulatory but no antimicrobial activity in a P. aeruginosa in vivo murine infection model. Following further modulation to enhance activity, AMPs and ivacaftor offer real potential as therapeutics to augment antibiotic therapy of respiratory infection in CF. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

A Murine Model of Inflammatory Bladder Disease: Cathelicidin Peptide Induced Bladder Inflammation and Treatment With Sulfated Polysaccharides

PubMed Central

Oottamasathien, Siam; Jia, Wanjian; McCoard, Lindsi; Slack, Sean; Zhang, Jianxing; Skardal, Aleksander; Job, Kathleen; Kennedy, Thomas P.; Dull, Randal O.; Prestwich, Glenn D.

2013-01-01

Purpose Studies show that LL-37 is a naturally occurring urinary defensin peptide that is up-regulated during urinary tract infections. Although normal urinary LL-37 levels are antimicrobial, we propose that increased LL-37 may trigger bladder inflammation. We further suggest that anti-inflammatory sulfated polysaccharides known as semi-synthetic glycosaminoglycan ether compounds can treat/prevent LL-37 mediated bladder inflammation. Materials and Methods C57BL/6 mice were catheterized/instilled with LL-37 (320 μM at 150 μl) for 45 minutes. Animals were sacrificed at 12 and 24 hours, and tissues were examined using hematoxylin and eosin. Separate experiments were performed for myeloperoxidase to quantify inflammation. GM-1111 semi-synthetic glycosaminoglycan ether treatments involved instillation of 10 mg/ml for 45 minutes directly before or after LL-37. Tissues were harvested at 24 hours. To compare semi-synthetic glycosaminoglycan ether efficacy experiments were performed using 10 mg/ml heparin. Finally, tissue localization of semi-synthetic glycosaminoglycan ether was examined using a fluorescent GM-1111-Alexa Fluor® 633 conjugate. Results Profound bladder inflammation developed after LL-37. Greater tissue inflammation occurred after 24 hours compared to that at 12 hours. Myeloperoxidase assays revealed a 21 and 61-fold increase at 12 and 24 hours, respectively. Semi-synthetic glycosaminoglycan ether treatment after LL-37 showed mild attenuation of inflammation with myeloperoxidase 2.5-fold below that of untreated bladders. Semi-synthetic glycosaminoglycan ether treatment before LL-37 demonstrated almost complete attenuation of inflammation. Myeloperoxidase results mirrored those in controls. In heparin treated bladders minimal attenuation of inflammation occurred. Finally, instillation of GM-1111-Alexa Fluor 633 revealed urothelial coating, significant tissue penetration and binding to endovasculature. Conclusions We developed what is to our knowledge a new model of inflammatory bladder disease by challenge with the naturally occurring urinary peptide LL-37. We also noted that a new class of anti-inflammatory sulfated polysaccharides prevents and mitigates bladder inflammation. PMID:21855919

Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression

PubMed Central

Grether-Beck, Susanne; Felsner, Ingo; Brenden, Heidi; Kohne, Zippora; Majora, Marc; Marini, Alessandra; Jaenicke, Thomas; Rodriguez-Martin, Marina; Trullas, Carles; Hupe, Melanie; Elias, Peter M.; Krutmann, Jean

2012-01-01

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin. PMID:22418868

Modulation of Neutrophil Apoptosis by Antimicrobial Peptides

PubMed Central

Nagaoka, Isao; Suzuki, Kaori; Niyonsaba, François; Tamura, Hiroshi; Hirata, Michimasa

2012-01-01

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Human antimicrobial peptides, α-defensins (human neutrophil peptides, HNPs), human β-defensins (hBDs), and cathelicidin (LL-37) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also function as immunomodulatory molecules by inducing cytokine and chemokine production, and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, we provided the evidence that HNP-1, hBD-3, and LL-37 cannot only destroy bacteria but also potently modulate (suppress) neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/-2, the downregulation of tBid (an proapoptotic protein) and upregulation of Bcl-xL (an antiapoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity, possibly via the actions on the distinct receptors, the P2Y6 nucleotide receptor, the chemokine receptor CCR6, and the low-affinity formyl-peptide receptor FPRL1/the nucleotide receptor P2X7, respectively. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion. PMID:23724322

Identification of Human Cathelicidin Peptide LL-37 as a Ligand for Macrophage Integrin αMβ2 (Mac-1, CD11b/CD18) that Promotes Phagocytosis by Opsonizing Bacteria

PubMed Central

Lishko, Valeryi K.; Moreno, Benjamin; Podolnikova, Nataly P.; Ugarova, Tatiana P.

2016-01-01

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor(s) mediating the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the αMI-domain of integrin αMβ2 (Mac-1), a major receptor on the surface of myeloid cells, and induces a migratory response in Mac-1-expressing monocyte/macrophages as well as activation of Mac-1 on neutrophils. Here, we show that LL-37 and its C-terminal derivative supported strong adhesion of various Mac-1-expressing cells, including HEK293 cells stably transfected with Mac-1, human U937 monocytic cells and murine IC-21 macrophages. The cell adhesion to LL-37 was partially inhibited by specific Mac-1 antagonists, including mAb against the αM integrin subunit and neutrophil inhibitory factor, and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface heparan sulfate proteoglycans act cooperatively with integrin Mac-1. Coating both Gram-negative and Gram-positive bacteria with LL-37 significantly potentiated their phagocytosis by macrophages, and this process was blocked by a combination of anti-Mac-1 mAb and heparin. Furthermore, phagocytosis by wild-type murine peritoneal macrophages of LL-37-coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. By contrast, LL-37 failed to augment phagocytosis of beads by Mac-1-deficient macrophages. These results identify LL-37 as a novel ligand for integrin Mac-1 and demonstrate that the interaction between Mac-1 on macrophages and bacteria-bound LL-37 promotes phagocytosis. PMID:27990411

Induced resistance to the antimicrobial peptide lactoferricin B in Staphylococcus aureus.

PubMed

Samuelsen, Orjan; Haukland, Hanne H; Jenssen, Håvard; Krämer, Manuela; Sandvik, Kjersti; Ulvatne, Hilde; Vorland, Lars H

2005-06-20

This study was designed to investigate inducible intrinsic resistance against lactoferricin B in Staphylococcus aureus. Serial passage of seven S. aureus strains in medium with increasing concentrations of peptide resulted in an induced resistance at various levels in all strains. The induced resistance was unstable and decreased relatively rapidly during passages in peptide free medium but the minimum inhibitory concentration remained elevated after thirty passages. Cross-resistance to penicillin G and low-level cross-resistance to the antimicrobial peptides indolicidin and Ala(8,13,18)-magainin-II amide [corrected] was observed. No cross-resistance was observed to the human cathelicidin LL-37. In conclusion, this study shows that S. aureus has intrinsic resistance mechanisms against antimicrobial peptides that can be induced upon exposure, and that this may confer low-level cross-resistance to other antimicrobial peptides.

Antimicrobial peptides in saliva of children with severe early childhood caries.

PubMed

Colombo, Natália H; Ribas, Laís F F; Pereira, Jesse A; Kreling, Paula F; Kressirer, Christine A; Tanner, Anne C R; Duque, Cristiane

2016-09-01

Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n=29), ECC group (n=25) and S-ECC group (n=29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human β-defensin 2 (hBD-2), human β-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

Recombinant production of antimicrobial peptides in Escherichia coli: a review.

PubMed

Li, Yifeng

2011-12-01

Antimicrobial peptides are of great interest due to their potential application as novel antibiotics. Large quantities of highly purified peptides are required to meet the needs of basic research and clinical trials. Compared with isolation from natural sources and chemical synthesis, recombinant approach offers the most cost-effective means for large-scale peptide manufacture. Among the systems available for heterologous protein production, Escherichia coli has been the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, a strategy necessary to mask these peptides' lethal effect towards the host and protect them from proteolytic degradation. The present article reviews commonly used fusion partners (e.g., solubility-enhancing, aggregation-promoting and self-cleavable carriers, etc.), cleavage methods and optimization options for antimicrobial peptides production in E. coli. In addition, the various approaches developed to generate recombinant human antimicrobial peptide LL-37, which offer excellent examples demonstrating effective production strategies, were briefly discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Pyrazole derived ultra-short antimicrobial peptidomimetics with potent anti-biofilm activity.

PubMed

Ahn, Mija; Gunasekaran, Pethaiah; Rajasekaran, Ganesan; Kim, Eun Young; Lee, Soo-Jae; Bang, Geul; Cho, Kun; Hyun, Jae-Kyung; Lee, Hyun-Ju; Jeon, Young Ho; Kim, Nam-Hyung; Ryu, Eun Kyoung; Shin, Song Yub; Bang, Jeong Kyu

2017-01-05

In this study, we report on the first chemical synthesis of ultra-short pyrazole-arginine based antimicrobial peptidomimetics derived from the newly synthesized N-alkyl/aryl pyrazole amino acids. Through the systematic tuning of hydrophobicity, charge, and peptide length, we identified the shortest peptide Py11 with the most potent antimicrobial activity. Py11 displayed greater antimicrobial activity against antibiotic-resistant bacteria, including MRSA, MDRPA, and VREF, which was approximately 2-4 times higher than that of melittin. Besides its higher selectivity (therapeutic index) toward bacterial cells than LL-37, Py11 showed highly increased proteolytic stability against trypsin digestion and maintained its antimicrobial activity in the presence of physiological salts. Interestingly, Py11 exhibited higher anti-biofilm activity against MDRPA compared to LL-37. The results from fluorescence spectroscopy and transmission electron microscopy (TEM) suggested that Py11 kills bacterial cells possibly by integrity disruption damaging the cell membrane, leading to the cytosol leakage and eventual cell lysis. Furthermore, Py11 displayed significant anti-inflammatory (endotoxin-neutralizing) activity by inhibiting LPS-induced production of nitric oxide (NO) and TNF-α. Collectively, our results suggest that Py11 may serve as a model compound for the design of antimicrobial and antisepsis agents. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Primate cathelicidin orthologues display different structures and membrane interactions.

PubMed

Morgera, Francesca; Vaccari, Lisa; Antcheva, Nikolinka; Scaini, Denis; Pacor, Sabrina; Tossi, Alessandro

2009-02-01

The human cathelicidin LL-37 displays both direct antibacterial activities and the capacity to modulate host-cell activities. These depend on structural characteristics that are subject to positive selection for variation, as observed in a previous analysis of the CAMP gene (encoding LL-37) in primates. The altered balance between cationic and anionic residues in different primate orthologues affects intramolecular salt-bridging and influences the stability of the helical conformation and tendency to aggregate in solution of the peptide. In the present study, we have analysed the effects of these structural variations on membrane interactions for human LL-37, rhesus RL-37 and orang-utan LL-37, using several complementary biophysical and biochemical methods. CD and ATR (attenuated total reflection)-FTIR (Fourier-transform IR) spectroscopy on model membranes indicate that RL-37, which is monomeric and unstructured in bulk solution [F-form (free form)], and human LL-37, which is partly structured and probably aggregated [A-form (aggregated form)], bind biological membranes in different manners. RL-37 may insert more deeply into the lipid bilayer than LL-37, which remains aggregated. AFM (atomic force microscopy) performed on the same supported bilayer as used for ATR-FTIR measurements suggests a carpet-like mode of permeabilization for RL37 and formation of more defined worm-holes for LL-37. Comparison of data from the biological activity on bacterial cells with permeabilization of model membranes indicates that the structure/aggregation state also affects the trajectory of the peptides from bulk solution through the outer cell-wall layers to the membrane. The results of the present study suggest that F-form cathelicidin orthologues may have evolved to have primarily a direct antimicrobial defensive capacity, whereas the A-forms have somewhat sacrificed this to gain host-cell modulating functions.

Antimicrobial Peptides in 2014

PubMed Central

Wang, Guangshun; Mishra, Biswajit; Lau, Kyle; Lushnikova, Tamara; Golla, Radha; Wang, Xiuqing

2015-01-01

This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms. PMID:25806720

Anaerobic bacteria growth in the presence of cathelicidin LL-37 and selected ceragenins delivered as magnetic nanoparticles cargo.

PubMed

Durnaś, Bonita; Piktel, Ewelina; Wątek, Marzena; Wollny, Tomasz; Góźdź, Stanisław; Smok-Kalwat, Jolanta; Niemirowicz, Katarzyna; Savage, Paul B; Bucki, Robert

2017-07-26

Cationic antibacterial peptides (CAPs) and synthetic molecules mimicking the amphiphilic structure of CAPs, such as ceragenins, are promising compounds for the development of new antimicrobials. We tested the in vitro activity of ceragenins CSA-13 and CSA-131 against several anaerobic bacteria including Bacteroides spp. and Clostridium difficile. We compared results to the activity of cathelicidin LL-37, metronidazole and nanosystems developed by attachment of CSA-13 and CSA-131 to magnetic nanoparticles (MNPs). The antibacterial effect was tested using killing assay and modified CLSI broth microdilution assay. Ceragenins CSA-13 and CSA-131 displayed stronger bactericidal activity than LL-37 or metronidazole against all of the tested bacterial strains. Additionally CSA-131 revealed an enhanced ability to prevent the formation of Bacteroides fragilis and Propionibacterium acnes biofilms. These data confirmed that ceragenins display antimicrobial activity against a broad range of microorganisms including anaerobic bacteria and deserve further investigations as compounds serving to develop new treatment against anaerobic and mixed infections.

Novel Synthetic, Host-defense Peptide Protects Against Organ Injury/Dysfunction in a Rat Model of Severe Hemorrhagic Shock.

PubMed

Yamada, Noriaki; Martin, Lukas B; Zechendorf, Elisabeth; Purvis, Gareth S D; Chiazza, Fausto; Varrone, Barbara; Collino, Massimo; Shepherd, Joanna; Heinbockel, Lena; Gutsmann, Thomas; Correa, Wilmar; Brandenburg, Klaus; Marx, Gernot; Schuerholz, Tobias; Brohi, Karim; Thiemermann, Christoph

2017-03-10

To evaluate (1) levels of the host-defense/antimicrobial peptide LL-37 in patients with trauma and hemorrhagic shock (HS) and (2) the effects of a synthetic host-defense peptide; Pep19-4LF on multiple organ failure (MOF) associated with HS. HS is a common cause of death in severely injured patients. There is no specific therapy that reduces HS-associated MOF. (1) LL-37 was measured in 47 trauma/HS patients admitted to an urban major trauma center. (2) Male Wistar rats were submitted to HS (90 min, target mean arterial pressure: 27-32 mm Hg) or sham operation. Rats were treated with Pep19-4LF [66 (n = 8) or 333 μg/kg · h (n = 8)] or vehicle (n = 12) for 4 hours following resuscitation. Plasma LL-37 was 12-fold higher in patients with trauma/HS compared to healthy volunteers. HS rats treated with Pep19-4LF (high dose) had a higher mean arterial pressure at the end of the 4-hour resuscitation period (79 ± 4 vs 54 ± 5 mm Hg) and less renal dysfunction, liver injury, and lung inflammation than HS rats treated with vehicle. Pep19-4LF enhanced (kidney/liver) the phosphorylation of (1) protein kinase B and (2) endothelial nitric oxide synthase. Pep19-4LF attenuated the HS-induced (1) translocation of p65 from cytosol to nucleus, (2) phosphorylation of IκB kinase on Ser, and (3) phosphorylation of IκBα on Ser resulting in inhibition of nuclear factor kappa B and formation of proinflammatory cytokines. Pep19-4LF prevented the release of tumor necrosis factor alpha caused by heparan sulfate in human mononuclear cells by binding to this damage-associated molecular pattern. Trauma-associated HS results in release of LL-37. The synthetic host-defense/antimicrobial peptide Pep19-4LF attenuates the organ injury/dysfunction associated with HS.

[Prokaryotic expression and purification of antimicrobial peptide LL-37 and the inhibiting effect against Candida albicans].

PubMed

Huo, Y; Wang, F; Sun, B; Yin, L R; Zhang, P P; Zhang, Y J; Zhang, B M

2016-02-01

To study the inhibitory effect of antimicrobial peptide LL-37 on Candida albicans through its ability to promote the secretion of immune factors by vaginal epithelial cells. (1) LL-37 prokaryotic expression vector pET-Duet/LL-37 was constructed and its expression was induced in Escherichia coli M15. The expressed LL-37 fusion protein was purified and identified by western blot. Antifungal activity of the purified protein was initially identified by Kirby-Bauer (K-B) method. (2) Purified LL-37 protein was added to human vaginal epithelial cells co-cultured with Candida, and inhibitory effect on Candida growth was determined by the glucose consumption method. Interferon γ (IFN-γ), interleukin 10 (IL-10) concentration and IFN-γ/IL-10 ratio were measured by ELISA at different time points. (1) LL-37 fusion protein was purified to 96% purity at a concentration of 433.92 μg/ml, and was shown to possess anti-fungal activity confirmed by the K-B method. (2) A Candida-vaginal epithelial cells co-culture system was successfully constructed. LL-37 recombinant protein inhibited the growth of Candida with absorbance values significantly higher in the treatment group compared to the control group at all measured time points (12-hour: 3.008±0.003 versus 2.967±0.003, 24-hour: 2.941±0.003 versus 2.601±0.003, 48-hour: 2.893 ± 0.004 versus 2.409 ± 0.003; all P<0.01). Furthermore, the rate of decrease was also much slower compared to the control group. In both control and experimental groups, IFN-γ and IL-10 secretion levels were observed to rise at first peaking at 24 hours and subsequently decrease. For each time period, IFN-γ concentration in the experimental group was significantly higher at 24 hours compared to the control group [(104.00 ± 1.07) versus (85.17 ± 0.28) pg/ml,P<0.01]. In contrast, IL-10 concentrations were significantly lower than the control group at all time points (P<0.01). IFN-γ/IL-10 ratio was also observed to be significantly higher than the control group at all measured time points (P<0.01). (1) Recombinant protein LL-37 could significantly inhibit the growth of Candida. (2) By influencing the secretion of immune factors such as IFN-γ, IL-10, etc, recombinant protein LL-37 is able to adjust vaginal epithelial cells local immunity, and enhance resistance to Candida infection.

LL-37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection.

PubMed

Sun, Wenyan; Zheng, Yan; Lu, Zhuoyang; Wang, Hui; Feng, Zhihui; Wang, Juan; Xiao, Shengxiang; Liu, Feng; Liu, Jiankang

2014-09-01

The human cationic antimicrobial protein LL-37 is a multifunctional host defense peptide with a wide range of immunomodulatory activities. Previous work has shown that LL-37 exerts both pro- and anti-inflammatory effects. The role of mitochondria in the skin inflammatory effects of LL-37 has not been well studied. Therefore, our aim was to investigate the immunomodulatory effect of LL-37 in HaCaT cells and to delineate the underlying mechanisms related to mitochondrial function. Immunohistochemistry results from tissue microarrays showed strong cytoplasmic LL-37 staining in inflammatory cells in chronic dermatic inflammation. Using exogenous LL-37 stimulation and LL-37 knockdown and overexpression, LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL-1α and tumor necrosis factor-α (TNF-α), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 are dependent upon its ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. Furthermore, we observed that LL-37 enhances the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin can neutralize the protective effects of LL-37 on mitochondria. In conclusion, these results suggest that high LL-37 expression levels correlate with chronic skin inflammation; mitochondrial dysfunction occurs in HaCaT cells during inflammation; and LL-37 attenuates inflammatory impairment by stimulating mitochondrial biogenesis and protecting mitochondrial function, which are dependent upon mTOR signaling. These findings provide new insights into targeting mitochondria with LL-37 to prevent skin inflammatory reactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

LL-37 immunomodulatory activity during Mycobacterium tuberculosis infection in macrophages.

PubMed

Torres-Juarez, Flor; Cardenas-Vargas, Albertina; Montoya-Rosales, Alejandra; González-Curiel, Irma; Garcia-Hernandez, Mariana H; Enciso-Moreno, Jose A; Hancock, Robert E W; Rivas-Santiago, Bruno

2015-12-01

Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Alzheimer's Disease-Associated Amyloid β-Protein Is an Antimicrobial Peptide

PubMed Central

Soscia, Stephanie J.; Kirby, James E.; Washicosky, Kevin J.; Tucker, Stephanie M.; Ingelsson, Martin; Hyman, Bradley; Burton, Mark A.; Goldstein, Lee E.; Duong, Scott; Tanzi, Rudolph E.; Moir, Robert D.

2010-01-01

Background The amyloid β-protein (Aβ) is believed to be the key mediator of Alzheimer's disease (AD) pathology. Aβ is most often characterized as an incidental catabolic byproduct that lacks a normal physiological role. However, Aβ has been shown to be a specific ligand for a number of different receptors and other molecules, transported by complex trafficking pathways, modulated in response to a variety of environmental stressors, and able to induce pro-inflammatory activities. Methodology/Principal Findings Here, we provide data supporting an in vivo function for Aβ as an antimicrobial peptide (AMP). Experiments used established in vitro assays to compare antimicrobial activities of Aβ and LL-37, an archetypical human AMP. Findings reveal that Aβ exerts antimicrobial activity against eight common and clinically relevant microorganisms with a potency equivalent to, and in some cases greater than, LL-37. Furthermore, we show that AD whole brain homogenates have significantly higher antimicrobial activity than aged matched non-AD samples and that AMP action correlates with tissue Aβ levels. Consistent with Aβ-mediated activity, the increased antimicrobial action was ablated by immunodepletion of AD brain homogenates with anti-Aβ antibodies. Conclusions/Significance Our findings suggest Aβ is a hitherto unrecognized AMP that may normally function in the innate immune system. This finding stands in stark contrast to current models of Aβ-mediated pathology and has important implications for ongoing and future AD treatment strategies. PMID:20209079

Children with cystic fibrosis demonstrate no respiratory immunological, infective or physiological, consequences of vitamin D deficiency.

PubMed

Thursfield, Rebecca M; Naderi, Khayam; Leaver, Neil; Rosenthal, Mark; Alton, Eric W F W; Bush, Andrew; Davies, Jane C

2018-04-06

Vitamin D has health benefits in many respiratory diseases but the evidence in CF is unclear. Induction of the antimicrobial peptides cathelicidin (LL37) and human-beta-defensin-2 (HBD-2) may be the mechanism of any benefit. We hypothesised that antimicrobial peptide levels would be decreased, and airway infection and inflammation greater, in CF children with vitamin D deficiency. The objective of the study was to explore relationships between vitamin D, LL37 and HBD-2, and airway infection, inflammation and physiology in children with CF. Bronchoalveolar lavage (BALF) and blood were obtained from children undergoing fibreoptic bronchoscopy. Serum vitamin D, BALF HBD-2 and LL37, cultured bacteria and inflammatory markers were measured. Clinical parameters were recorded. 113 patients with CF, 23 with non-CF chronic suppurative lung disease (CSLD) and 6 healthy controls were included. We found no relationship between serum vitamin D and BALF HBD-2 or LL-37. There were no differences in infective or inflammatory markers between vitamin D sufficient and deficient groups. Vitamin D deficient patients (<50 nmol/L) did not have a worse FEV 1 (CF: 66 (58-71)% vs. 71.5 (61-76)%, ns; non-CF CSLD: 69 (36-88)% vs. 70 (62-95)%, ns). In the first bronchoscopic study exploring this question, we demonstrate that vitamin D deficiency is not associated with immunological, infective or clinical markers of disease severity in patients with CF or CSLD. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

[Effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes in the murine model of vaginal candidiasis].

PubMed

Wang, F; Huo, Y; Yin, L R; Sun, B; Zhang, P P

2016-07-25

To study the effects of antimicrobial peptide LL-37 expressed and purified from prokaryotes on candida albicans growth. (1)Thirty female Kunming mice were treated with estrogen and white candida yeast suspension were poured into vagina to establish a vulvovaginal candidiasis(VVC)murine model. After successful establishing the VVC mouse model, mice were randomly sorted into test group(n=15)and control group(n=15). Suspension(30 μl, 100 μg/ml)of recombinant peptide LL-37 expressed and purified in Prokaryotes was given by intravaginal administration to the test group for 5 days, while the same amount of phosphate buffered saline(PBS)was given to the control group.(2)Tweenty-four hours after treatment, the fungal burden and colony-forming unit(CFU)of vaginal fluids were evaluated. All mice were subsequently sacrificed and vaginal tissues were harvested for tissue homogenate preparation. ELISA was used to determine the levels of nterleukin-10(IL-10)and interferon-γ(IFN-γ)in the isolated vaginal tissues. (1)VVC mouse model was established successfully in this study. Vaginal mucosa congestion, edema, vaginal plica disappearing were obviously observed in the control group. After treatment with recombinant protein LL-37 vaginal mucosa has no obvious change in the test group.(2)Fungal burden and CFU of vaginal fluids were significantly lower in the test group [(4.8±1.0)×10(4) CFU/ml]than that in the control group [(8.5±2.1)×10(4) CFU/ml, P=0.017]. IFN-γ level of the test group was increased [(257±11)vs(197±4)pg/ml, P=0.000], while the level of IL-10 was reduced [(287 ± 15)vs(379 ± 17)pg/ml P=0.000] resulting in a the ratio of IFN-γ/IL-10 was in significantly higher in test group(0.892±0.008 vs 0.496±0.013, P=0.000). Recombinant protein LL-37 expressed and purified from prokaryotes inhibits the growth candida albicans and improves vaginal immunity by adjusting IFN-γ and IL-10 secretion in the VVC mouse model, highlighting the therapeutic potential of LL-37 for VVC.

Prevention of ESKAPE pathogen biofilm formation by antimicrobial peptides WLBU2 and LL37.

PubMed

Lin, Qiao; Deslouches, Berthony; Montelaro, Ronald C; Di, Y Peter

2018-05-09

Bacterial biofilm-dependent infections (e.g., cystic fibrosis, surgical sites, and medical implants) are associated with enhanced drug-resistance and thus difficult to eradicate. The goal of this study was to systematically compare three distinct classes of antimicrobial peptides (AMPs) that include the clinically used antibiotic colistin, the natural AMP LL37, the engineered cationic-AMP WLBU2, and four commonly used antibiotics with different bactericidal mechanisms (tobramycin, ciprofloxacin, ceftazidime and vancomycin) for biofilm prevention properties. Using biofilm-prevention assays, we detected bacterial biomass post-attachment in subinhibitory concentrations (1/3 of the MIC) for each AMP, by the crystal violet method, to distinguish the commonly known bactericidal from potentially distinct mechanisms of biofilm prevention. Biofilm regulatory gene expression was assessed using RT-qPCR for correlation with biofilm growth inhibition. Commonly used antibiotics at 1x MIC showed modest ESKAPE biofilm prevention while 1/3 MIC of AMPs demonstrated up to 90% of biofilm prevention. WLBU2 was generally more effective in preventing bacterial attachment than colistin and LL37. Changes in expression of bacterial genes known to affect biofilm regulation were consistent with biofilm prevention. The data warrant further exploration of AMPs with optimized structures to fill a knowledge gap on the potential application of AMPs to difficult-to-cure bacterial biofilm-related infections. Copyright © 2018. Published by Elsevier B.V.

Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells*

PubMed Central

Adase, Christopher A.; Borkowski, Andrew W.; Zhang, Ling-juan; Williams, Michael R.; Sato, Emi; Sanford, James A.

2016-01-01

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment. PMID:27048655

Expression of Antimicrobial Peptides by Uveal and Cutaneous Melanoma Cells and Investigation of Their Role in Tumor Cell Migration and Vasculogenic Mimicry.

PubMed

Manarang, Joseph C; Otteson, Deborah C; McDermott, Alison M

2017-11-01

Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy. mRNA expression of the AMPs α defensin (HNP-1); cathelicidin (LL-37); and β defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy. Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process. Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.

Plasma LL-37 correlates with vitamin D and is reduced in human immunodeficiency virus-1 infected individuals not receiving antiretroviral therapy

PubMed Central

Connick, Elizabeth; MaWhinney, Samantha; Chan, Edward D.; Flores, Sonia C.

2014-01-01

Low levels of the vitamin D-regulated antimicrobial peptide cathelicidin (LL-37) may negatively impact the immune status of human immunodeficiency virus-1 (HIV-1) infected individuals (HIV+). We compared plasma LL-37 levels in healthy controls (HIV−) and HIV+ individuals on or off antiretroviral therapies (ARTs) (ART+ and ART−, respectively), and evaluated the relationship between vitamin D and LL-37 levels. In this cross-sectional study, levels of LL-37, 25-hydroxycholecalciferol [25(OH)D3] and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] were measured from an initial cohort of 18 healthy controls and 10 HIV+/ART− individuals. Because this cohort lacked HIV+/ART+ subjects, LL-37 was also quantified from a second cohort of 10 HIV+/ART− and 13 HIV+/ART+ individuals. LL-37 levels were significantly lower in the HIV+/ART− group compared to the healthy controls (P = 0.01). A direct relationship was observed between LL-37 and both 25(OH)D3 and 1,25(OH)2D3. The level of 25(OH)D3 was predictive of higher LL-37 (P = 0.04) and for any given level of 25(OH)D3, HIV+/ART− subjects averaged 20 % lower LL-37 compared to the healthy controls (P = 0.045). For any given level of 1,25(OH)2D3, HIV+/ART− subjects averaged 25 % lower LL-37 compared to the healthy controls (P = 0.018), although 1,25(OH)2D3 was not predictive of higher LL-37 (P = 0.28). Finally, LL-37 levels were significantly lower in the HIV+/ART− group compared to the HIV+/ART+ group from the second cohort (P = 0.045). Untreated HIV infection may contribute to lower LL-37 levels, independent of vitamin D levels. ART treatment may potentially mitigate this decrease in LL-37 levels. PMID:24821067

Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37.

PubMed

Seib, K L; Serruto, D; Oriente, F; Delany, I; Adu-Bobie, J; Veggi, D; Aricò, B; Rappuoli, R; Pizza, M

2009-01-01

Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log(10) CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log(10) CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

Differential expression pattern of antimicrobial peptides in nasal mucosa and secretion.

PubMed

Laudien, Martin; Dressel, Stefanie; Harder, Jürgen; Gläser, Regine

2011-03-01

The intact nasal barrier is a prerequisite for a functioning defense of the upper airway system, in particular the permanent threat by inhaled potentially harmful microorganisms. Antimicrobial peptides (AMP) play an important role in maintaining barrier function. There is few data about AMP in respect of nasal mucosa. This study is addressed to gain further insight into the differential AMP expression and secretion pattern according to defined anatomical regions of the vestibulum nasi and turbinates. ELISA was applied to quantify concentrations of AMP RNase-7, psoriasin, hBD-2, hBD-3 and LL-37 in nasal secretions of 20 healthy volunteers. Immunohistochemistry was used to detect the local cellular sources of AMP in the vestibulum nasi (squamous epithelium) and compared to the mucosa of the turbinates (pseudostratified epithelium) in 10 healthy volunteers. Expression of RNase 7 and psoriasin was detected in all nasal secretion specimens, whereas LL-37 was detected in 16, hBD-2 in 5 and hBD-3 in 6 specimens. In the vestibulum nasi, luminal cell layers were demonstrated as local cellular sources for hBD-3 and RNase 7, whereas psoriasin was found in all layers of the stratified squamous epithelium. LL-37 was detected in 1 stroma cells sample, whereas hBD-2 was not detected at all. In turbinate biopsie,s hBD-3 and LL-37 were detectable in the epithelium, stroma cells and submucosal glands. RNase 7 was only present in submucosal glands. HBD-2 and psoriasin were not detected. These data demonstrate that the nasal epithelium contains a chemical defense shield through the expression and secretion of various AMP.

Circulating cathelicidin levels correlate with mucosal disease activity in ulcerative colitis, risk of intestinal stricture in Crohn's disease, and clinical prognosis in inflammatory bowel disease.

PubMed

Tran, Diana Hoang-Ngoc; Wang, Jiani; Ha, Christina; Ho, Wendy; Mattai, S Anjani; Oikonomopoulos, Angelos; Weiss, Guy; Lacey, Precious; Cheng, Michelle; Shieh, Christine; Mussatto, Caroline C; Ho, Samantha; Hommes, Daniel; Koon, Hon Wai

2017-05-12

Cathelicidin (LL-37) is an antimicrobial peptide known to be associated with various autoimmune diseases. We attempt to determine if cathelicidin can accurately reflect IBD disease activity. We hypothesize that serum cathelicidin correlates with mucosal disease activity, stricture, and clinical prognosis of IBD patients. Serum samples were collected from two separate cohorts of patients at the University of California, Los Angeles. Cohort 1 consisted of 50 control, 23 UC, and 28 CD patients. Cohort 2 consisted of 20 control, 57 UC, and 67 CD patients. LL-37 levels were determined by ELISA. Data from both cohorts were combined for calculation of accuracies in indicating mucosal disease activity, relative risks of stricture, and odds ratios of predicting disease development. Serum cathelicidin levels were inversely correlated with Partial Mayo Scores of UC patients and Harvey-Bradshaw Indices of CD patients. Among IBD patients with moderate or severe initial disease activity, the patients with high initial LL-37 levels had significantly better recovery than the patients with low initial LL-37 levels after 6-18 months, suggesting that high LL-37 levels correlate with good prognosis. Co-evaluation of LL-37 and CRP levels was more accurate than CRP alone or LL-37 alone in the correlation with Mayo Endoscopic Score of UC patients. Low LL-37 levels indicated a significantly elevated risk of intestinal stricture in CD patients. Co-evaluation of LL-37 and CRP can indicate mucosal disease activity in UC patients. LL-37 can predict future clinical activity in IBD patients and indicate risk of intestinal stricture in CD patients.

Database-Guided Discovery of Potent Peptides to Combat HIV-1 or Superbugs

PubMed Central

Wang, Guangshun

2013-01-01

Antimicrobial peptides (AMPs), small host defense proteins, are indispensable for the protection of multicellular organisms such as plants and animals from infection. The number of AMPs discovered per year increased steadily since the 1980s. Over 2,000 natural AMPs from bacteria, protozoa, fungi, plants, and animals have been registered into the antimicrobial peptide database (APD). The majority of these AMPs (>86%) possess 11–50 amino acids with a net charge from 0 to +7 and hydrophobic percentages between 31–70%. This article summarizes peptide discovery on the basis of the APD. The major methods are the linguistic model, database screening, de novo design, and template-based design. Using these methods, we identified various potent peptides against human immunodeficiency virus type 1 (HIV-1) or methicillin-resistant Staphylococcus aureus (MRSA). While the stepwise designed anti-HIV peptide is disulfide-linked and rich in arginines, the ab initio designed anti-MRSA peptide is linear and rich in leucines. Thus, there are different requirements for antiviral and antibacterial peptides, which could kill pathogens via different molecular targets. The biased amino acid composition in the database-designed peptides, or natural peptides such as θ-defensins, requires the use of the improved two-dimensional NMR method for structural determination to avoid the publication of misleading structure and dynamics. In the case of human cathelicidin LL-37, structural determination requires 3D NMR techniques. The high-quality structure of LL-37 provides a solid basis for understanding its interactions with membranes of bacteria and other pathogens. In conclusion, the APD database is a comprehensive platform for storing, classifying, searching, predicting, and designing potent peptides against pathogenic bacteria, viruses, fungi, parasites, and cancer cells. PMID:24276259

Bladder pain in an LL-37 interstitial cystitis and painful bladder syndrome model.

PubMed

Jia, Wanjian; Schults, Austin J; Jensen, Mark Martin; Ye, Xiangyang; Alt, Jeremiah A; Prestwich, Glenn D; Oottamasathien, Siam

2017-01-01

Our goal was to evaluate the pain response in an LL-37 induced murine model for interstitial cystitis/painful bladder syndrome (IC/PBS). In particular, we sought to characterize the dose dependence, time-course, and relationship of LL-37 induced bladder inflammation and pain. The IC/PBS model was induced in C57Bl/6 mice by instilling 50 μL of LL-37, an immunomodulatory human cathelicidin (anti-microbial peptide), in the bladder for 1 hr. Pain responses were measured using von Frey filaments (0.04 gm to 4.0 gm) before and after LL-37 instillation. Inflammation was evaluated using tissue myeloperoxidase (MPO) assay, gross inspection, and microscopic histologic examination. The dose response experiment demonstrated a graded pain response, with higher concentrations of LL-37 challenge yielding higher pain responses across all stimuli tested. Statistical significance was seen when comparing 1.0 gm von Frey filament results at 320 μM (68 ± 8% response) vs. 0 μM (38 ± 6% response). Interestingly, pain responses did not attenuate across time but increased significantly after 5 (p=0.0012) and 7 days (p=0.0096). Comparison with MPO data suggested that pain responses could be independent of inflammation. We demonstrated within our LL-37 induced IC/PBS model pain occurs in a dose-dependent fashion, pain responses persist beyond the initial point of insult, and our dose response and time course experiments demonstrated that pain was independent of inflammation.

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens.

PubMed

Herasimenka, Yury; Benincasa, Monica; Mattiuzzo, Maura; Cescutti, Paola; Gennaro, Renato; Rizzo, Roberto

2005-07-01

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.

The impact of cathelicidin, the human antimicrobial peptide LL-37 in urinary tract infections.

PubMed

Babikir, Ibrahim H; Abugroun, Elsir A; Bilal, Naser Eldin; Alghasham, Abdullah Ali; Abdalla, Elmuataz Elmansi; Adam, Ishag

2018-01-08

The defense mechanisms of the urinary tract are attributed mainly to the innate immune system and the urinary tract urothelium which represent the first line of defense against invading pathogens and maintaining sterility of the urinary tract. There are only a few publications regarding cathelicidin (LL-37) and a urinary tract infection (UTI). This study was done to investigate the plasma and urine levels of human LL-37 in patients with UTI. A case-control study was conducted at Omdurman Hospital, Sudan during the period from August 2014 to May 2017. The cases were patients with confirmed UTI and the controls were healthy volunteers without UTI. Sociodemographic and clinical data were obtained from each participant using questionnaires. Urine cultures and antimicrobial susceptibility were tested. Plasma and urine levels of LL-37 were determined using an enzyme-linked immunosorbent assay (ELISA) kit. SPSS (version 16.0) was used for analyses. Cases and controls (87 in each arm) were matched according to their basic characteristics. Compared with controls, the median (inter-quartile) LL-37 level in plasma [2.100 (1.700-2.700) vs. 1.800 (1.000-2.200) ng/ml, P = 0.002] and in urine [0.900 (0.300-1.600) vs. 0.000 (0.000-1.000) ng/mg creatinine, P < 0.001] was significantly higher in cases. There was no significant difference in the median plasma [2.1 (1.7-2.9) vs. 2.000 (1.700-2.400) ng/ml, P = 0.561] and urine [0.850 (0.275-2.025) vs. 0.900 (0.250-1.350) ng/mg creatinine, P = 0.124]. The uropathogenic Escherichia coli (UPEC) was the predominant isolate, n = 38 (43.7%). LL-37 levels between the E. coli isolates and the other isolated organisms. There was no significant correlation between plasma and urine LL-37 levels (r = 0.221), even when the data of the cases were analyzed separately. LL-37 is notably increased among patients with UTI compared with normal control subjects. Severity of UTI increases the levels of LL-37. The increased level was not only in the patient's urine, but has also been observed in the patient's plasma. Detection of increased levels of LL-37 could help to differentiate subjects with suspected UTI. Accordingly, LL-37 could act as a good marker for diagnosing UTIs.

1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

PubMed Central

Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A.; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU. PMID:25337708

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

PubMed

Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Cellular Basis of Secondary Infections and Impaired Desquamation in Certain Inherited Ichthyoses

PubMed Central

Chan, Aegean; Godoy-Gijon, Elena; Nuno-Gonzalez, Almudena; Crumrine, Debra; Hupe, Melanie; Choi, Eung-Ho; Gruber, Robert; Williams, Mary L.; Choate, Keith; Fleckman, Philip H.; Elias, Peter M.

2015-01-01

IMPORTANCE Secondary infections and impaired desquamation complicate certain inherited ichthyoses, but their cellular basis remains unknown. In healthy human epidermis, the antimicrobial peptides cathelicidin (LL-37) and human β-defensin 2 (HBD2), as well as the desquamatory protease kallikrein-related peptidase 7 (KLK7), are delivered to the stratum corneum (SC) interstices by lamellar body (LB) exocytosis. OBJECTIVE To assess whether abnormalities in the LB secretory system could account for increased risk of infections and impaired desquamation in inherited ichthyoses with known abnormalities in LB assembly (Harlequin ichthyosis [HI]), secretion (epidermolytic ichthyosis [EI]), or postsecretory proteolysis (Netherton syndrome [NS]). DESIGN, SETTING, AND PARTICIPANTS Samples from library material were taken from patients with HI, EI, NS, and other ichthyoses, but with a normal LB secretory system, and in healthy controls and were evaluated by electron microscopy and immunohistochemical analysis from July 1, 2010, through March 31, 2013. MAIN OUTCOME AND MEASURES Changes in LB secretion and in the fate of LB-derived enzymes and antimicrobial peptides in ichthyotic patients vs healthy controls. RESULTS In healthy controls and patients with X-linked ichthyosis, neutral lipid storage disease with ichthyosis, and Gaucher disease, LB secretion is normal, and delivery of LB-derived proteins and LL-37 immunostaining persists high into the SC. In contrast, proteins loaded into nascent LBs and their delivery to the SC interstices decrease markedly in patients with HI, paralleled by reduced immunostaining for LL-37, HBD2, and KLK7 in the SC. In patients with EI, the cytoskeletal abnormality impairs the exocytosis of LB contents and thus results in decreased LL-37, HBD2, and KLK7 secretion, causing substantial entombment of these proteins within the corneocyte cytosol. Finally, in patients with NS, although abundant enzyme proteins loaded in parallel with accelerated LB production, LL-37 disappears, whereas KLK7 levels increase markedly in the SC. CONCLUSIONS AND RELEVANCE Together, these results suggest that diverse abnormalities in the LB secretory system account for the increased risk of secondary infections and impaired desquamation in patients with HI, EI, and NS. PMID:25565224

Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence.

PubMed

Pence, Morgan A; Haste, Nina M; Meharena, Hiruy S; Olson, Joshua; Gallo, Richard L; Nizet, Victor; Kristian, Sascha A

2015-01-01

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing.

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

PubMed

Trombley, Michael P; Post, Deborah M B; Rinker, Sherri D; Reinders, Lorri M; Fortney, Kate R; Zwickl, Beth W; Janowicz, Diane M; Baye, Fitsum M; Katz, Barry P; Spinola, Stanley M; Bauer, Margaret E

2015-01-01

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Amaranthus caudatus extract inhibits the invasion of E. coli into uroepithelial cells.

PubMed

Mohanty, Soumitra; Zambrana, Silvia; Dieulouard, Soizic; Kamolvit, Witchuda; Nilsén, Vera; Gonzales, Eduardo; Östenson, Claes-Göran; Brauner, Annelie

2018-06-28

Amaranthus caudatus is traditionally used to treat infections. Based on its traditional usage, we investigated the effect of A. caudatus on the bladder epithelial cells in the protection of E. coli infection. The direct antimicrobial effects of A. caudatus on uropathogenic bacteria were investigated using minimum inhibitory concentration (MIC) assay. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli strain #12 were used to investigate the effect of A. caudatus. Bacterial adhesion and invasion into bladder cells treated with A. caudatus was analyzed. Expression of uroplakin-1a (UPK1A), β1 integrin (ITGB1), caveolin-1 (CAV1) and the antimicrobial peptides human β defensin-2 (DEFB4A) and LL-37 (CAMP) was evaluated using RT-PCR. No direct antibacterial effect on E. coli or any of the tested uropathogenic strains was observed by A. caudatus. However, we demonstrated reduced mRNA expression of uroplakin-1a and caveolin-1, but not β1 integrin after treatment of uroepithelial cells, mirrored by the decreased adhesion and invasion of E. coli. A. caudatus treatment did not induce increased gene expression of the antimicrobial peptides, LL-37 and human β-defensin-2. Our results showed that A. caudatus has a protective role on bladder epithelial cells against uropathogenic E. coli infection by decreasing the bacterial adhesion and invasion, thereby preventing infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials

PubMed Central

Malhotra, Sankalp; Limoli, Dominique H.; English, Anthony E.; Parsek, Matthew R.

2018-01-01

ABSTRACT Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo. Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H2O2). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H2O2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung. PMID:29588399

Defensins and Other Antimicrobial Peptides at the Ocular Surface

PubMed Central

McDermott, Alison M.

2006-01-01

Although constantly exposed to the environment and “foreign bodies” such as contact lenses and unwashed fingertips, the ocular surface succumbs to infection relatively infrequently. This is, in large part, due to a very active and robust innate immune response mounted at the ocular surface. Studies over the past 20 years have revealed that small peptides with antimicrobial activity are a major component of the human innate immune response system. The ocular surface is no exception, with peptides of the defensin and cathelicidin families being detected in the tear film and secreted by corneal and conjunctival epithelial cells. There is also much evidence to suggest that the role of some antimicrobial peptides is not restricted to direct killing of pathogens, but, rather, that they function in various aspects of the immune response, including recruitment of immune cells, and through actions on dendritic cells provide a link to adaptive immunity. A role in wound healing is also supported. In this article, the properties, mechanisms of actions and functional roles of antimicrobial peptides are reviewed, with particular emphasis on the potential multifunctional roles of defensins and LL-37 (the only known human cathelicidin) at the ocular surface. PMID:17216098

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens.

PubMed

Caiaffa, Karina Sampaio; Massunari, Loiane; Danelon, Marcelle; Abuna, Gabriel Flores; Bedran, Telma Blanca Lombardo; Santos-Filho, Norival Alves; Spolidorio, Denise Madalena Palomari; Vizoto, Natalia Leal; Cilli, Eduardo Maffud; Duque, Cristiane

2017-11-01

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3-1C V and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 μg ml -1 , respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.

Engineered Cationic Antimicrobial Peptides To Overcome Multidrug Resistance by ESKAPE Pathogens

PubMed Central

Deslouches, Berthony; Steckbeck, Jonathan D.; Craigo, Jodi K.; Doi, Yohei; Burns, Jane L.

2014-01-01

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings. PMID:25421473

Cathelicidin (LL-37) and its correlation with pro-oxidant, antioxidant balance and disease activity in systemic lupus erythematosus: a cross-sectional human study.

PubMed

Sahebari, M; Roshandel, G; Saadati, N; Saghafi, M; Abdolahi, N; Rezaieyazdi, Z

2017-08-01

Background Cathelicidin (LL-37), an endogenous antimicrobial peptide, has recently been involved in the pathogenesis of autoimmune diseases. To assess whether LL-37 reflects disease activity, we measured serum levels of it in systemic lupus erythematosus (SLE) patients with active and inactive disease compared to healthy controls. LL-37 was also compared between new and old cases. Moreover, the correlation of LL-37 and pro-oxidant, antioxidant balance (PAB) was measured. Methods The study population consisted of 50 SLE patients and 28 healthy controls. Of those, 39 patients had active and 11 patients had inactive disease. Serum levels of LL-37 were measured by ELISA and PAB values by a special method. Results There was no difference in levels of LL-37 between patients and healthy controls (50.9 ± 20.8 vs. 67.7 ± 43.3 ng/ml, P = 0.31). LL-37 did not correlate with SLEDAI and its items in total patients. LL-37 had a positive correlation with SLEDAI in active patients ( P = 0.01, r = 0.4). In active patients (78% of patients), multivariate regression analysis showed significant negative correlation between LL-37 and C3 ( P = 0.01, standardized beta -0.50). No difference was found in levels of PAB between patients and controls (90.4 ± 34.1 vs. 86.9 ± 25.6 HK, P = 0.4).There was no difference in the levels of PAB between patients with active and inactive disease (93.2 ± 34.1 vs. 80.2 ± 33.7 HK, P = 0.27). No correlation was found between levels of PAB and SLEDAI items and total score. However, a positive correlation between the levels of LL-37 and PAB in SLE patients was found ( r = 0.3, P < 0.01). Conclusion Based on this study, serum LL-37 and PAB did not increase in lupus compared with healthy individuals. LL-37 serum values rose in parallel with SLEDAI in active disease. Positive correlation between serum PAB and LL-37 could be a great achievement of this study that may suggest the role of antioxidants in controlling NETosis.

Vesicular LL-37 Contributes to Inflammation of the Lesional Skin of Palmoplantar Pustulosis

PubMed Central

Murakami, Masamoto; Kaneko, Takaaki; Nakatsuji, Teruaki; Kameda, Kenji; Okazaki, Hidenori; Dai, Xiuju; Hanakawa, Yasushi; Tohyama, Mikiko; Ishida-Yamamoto, Akemi; Sayama, Koji

2014-01-01

“Pustulosis palmaris et plantaris”, or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin. PMID:25330301

Mycobacterial r32-kDa antigen-specific T-cell responses correlate with successful treatment and a heightened anti-microbial response in human leprosy patients.

PubMed

Neela, Venkata Sanjeev Kumar; Devalraju, Kamakshi Prudhula; Pydi, Satya Sudheer; Sunder, Sharada Ramaseri; Adiraju, Kameswara Rao; Singh, Surya Satyanarayana; Anandaraj, M P J S; Valluri, Vijaya Lakshmi

2016-09-01

Immunological characterization of mycobacterial peptides may help not only in the preparation of a vaccine for leprosy but also in developing in vitro T-cell assays that could perhaps be used as an in vitro correlate for treatment outcome. The main goal of this study was to evaluate the use of Mycobacterium bovis recombinant 32-kDa protein (r32-kDa) antigen-stimulated T-cell assay as a surrogate marker for treatment outcome and monitor vitamin D receptor (VDR)-mediated anti-microbial responses during multidrug therapy (MDT) in leprosy. Newly diagnosed tuberculoid and lepromatous leprosy patients were enrolled and followed up during their course of MDT at 6 and 12 months. IFN-γ, IL-10, IL-17 and IL-23 levels in culture supernatants and expression of VDR, TLR2, LL37 and DEFB in r32-kDa-stimulated PBMCs were measured. Controls comprised household contacts (HHCs) and healthy endemic subjects (HCs). Significant differences were observed in the levels of IFN-γ, IL-17, IL-23, VDR and anti-microbial peptides LL37 and DEFB after treatment and when compared with that of HHCs and HCs, respectively. These findings suggest that responses to r32-kDa antigen reflect an improved immunological and anti-microbial response in leprosy patients during therapy, thereby indicating its potential use as an immune correlate in the treatment of leprosy patients. © The Japanese Society for Immunology. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport

PubMed Central

Kumaraswamy, Monika; Kousha, Armin; Nizet, Victor

2017-01-01

ABSTRACT This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo. PMID:29242830

Activity of Genital Tract Secretions and Synthetic Antimicrobial Peptides against Group B Streptococcus.

PubMed

Agarwal, Nidhi; Buckley, Niall; Nakra, Natasha; Gialanella, Philip; Yuan, Weirong; Ghartey, Jeny P

2015-12-01

Genital tract secretions inhibit Escherichia coli (E. coli) through antimicrobial peptides (AMP) secreted by the host and vaginal microbiota. However, there are limited data against group B Streptococcus (GBS). Group B Streptococcus were incubated with cervico-vaginal lavage (CVL) samples from healthy non-pregnant women (n = 12) or synthetic AMP and monitored for bacterial growth using a turbidimetric approach. E. coli inhibitory activity was determined by a colony-forming unit assay. None of the CVL samples inhibited GBS. The human neutrophil peptide-1 and human defensin 5 inhibited GBS growth by ≥80% at concentrations ≥20 μg/mL and ≥50 μg/mL, respectively, while human beta-defensin 2 and LL-37 did not inhibit at highest concentration tested (100 μg/mL). In contrast, all AMP inhibited E. coli. Antimicrobial peptides may protect against E. coli colonization but have more limited activity against GBS. Future studies will focus on augmenting host defense with specific AMP to prevent genitourinary infection with these pathogenic organisms. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens.

PubMed

Deslouches, Berthony; Steckbeck, Jonathan D; Craigo, Jodi K; Doi, Yohei; Burns, Jane L; Montelaro, Ronald C

2015-02-01

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

PubMed

Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

2015-08-01

Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

Expression of an additional cathelicidin antimicrobial peptide protects against bacterial skin infection.

PubMed

Lee, Phillip H A; Ohtake, Takaaki; Zaiou, Mohamed; Murakami, Masamoto; Rudisill, Jennifer A; Lin, Kenneth H; Gallo, Richard L

2005-03-08

Cathelicidin antimicrobial peptides are effectors of innate immune defense in mammals. Humans and mice have only one cathelicidin gene, whereas domesticated mammals such as the pig, cow, and horse have multiple cathelicidin genes. We hypothesized that the evolution of multiple cathelicidin genes provides these animals with enhanced resistance to infection. To test this, we investigated the effects of the addition of cathelicidins by combining synthetic cathelicidin peptides in vitro, by producing human keratinocytes that overexpress cathelicidins in culture, or by producing transgenic mice that constitutively overexpress cathelicidins in vivo. The porcine cathelicidin peptide PR-39 acted additively with human cathelicidin LL-37 to kill group A Streptococcus (GAS). Lentiviral delivery of PR-39 enhanced killing of GAS by human keratinocytes. Finally, transgenic mice expressing PR-39 under the influence of a K14 promoter showed increased resistance to GAS skin infection (50% smaller necrotic ulcers and 60% fewer surviving bacteria). Similarly constructed transgenic mice designed to overexpress their native cathelicidin did not show increased resistance. These findings demonstrate that targeted gene transfer of a xenobiotic cathelicidin confers resistance against infection and suggests the benefit of duplication and divergence in the evolution of antimicrobial peptides.

Selectivity in the potentiation of antibacterial activity of α-peptide/β-peptoid peptidomimetics and antimicrobial peptides by human blood plasma.

PubMed

Hein-Kristensen, Line; Knapp, Kolja M; Franzyk, Henrik; Gram, Lone

2013-11-01

Antimicrobial peptides (AMPs) are promising leads for novel antibiotics; however, their activity is often compromised under physiological conditions. The purpose of this study was to determine the activity of α-peptide/β-peptoid peptidomimetics and AMPs against Escherichia coli and Staphylococcus aureus in the presence of human blood-derived matrices and immune effectors. The minimum inhibitory concentration (MIC) of two peptidomimetics against E. coli decreased by up to one order of magnitude when determined in 50% blood plasma as compared to MHB media. The MIC of a membrane-active AMP, LL-I/3, also decreased, whereas two intracellularly acting AMPs were not potentiated by plasma. Blood serum had no effect on activity against E. coli and neither matrix had an effect on activity against S. aureus. Unexpectedly, physiological concentrations of human serum albumin did not influence activity. Plasma potentiation was not mediated by an LL-37 analogue, lysozyme or hydrogen peroxide; however, plasma potentiation of activity was abolished when the complement system was heat-inactivated. Time-course experiments indicated that potentiation was due to plasma-mediated effects on bacterial cells prior to activities of peptidomimetics. The unexpected enhancement of antibacterial activity of peptidomimetics and AMPs under physiological conditions significantly increases the therapeutic potential of these compounds. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Identifying the Critical Domain of LL-37 Involved in Mediating Neutrophil Activation in the Presence of Influenza Virus: Functional and Structural Analysis

PubMed Central

Tripathi, Shweta; Wang, Guangshun; White, Mitchell; Rynkiewicz, Michael; Seaton, Barbara; Hartshorn, Kevan

2015-01-01

The human cathelicidin LL-37 has been shown to play a role in host defense against influenza A viruses (IAV) through direct antiviral effects and through modulating inflammatory responses to infection. We recently showed that LL-37 increases neutrophil respiratory burst and neutrophil extracellular trap (NET) responses to IAV through engaging formyl peptide receptor 2 (FPR-2). In this paper we show that a fragment of LL-37, GI-20, which is composed of the central helical segment of the peptide, has similar effects as LL-37 on neutrophil activation. In addition to increasing respiratory burst and NET responses of the cells to IAV through an FPR-2 dependent mechanism, it reduces neutrophil IL-8 production to IAV (also like LL-37). The N-terminal fragment, LL-23, did not have similar effects. Both GI-20 and LL-37 increase neutrophil intracellular calcium levels and their ability to increase neutrophil activation responses was calcium dependent and partially inhibited by pertussis toxin. These studies show that the central helix of LL-37 retains the ability of LL-37 to modulate neutrophil responses through FPR-2. Based on our findings we developed a homology model of FPR-2 and performed docking experiments of LL-37 and GI-20 with the receptor. PMID:26308522

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

PubMed Central

Szafranski-Schneider, Eva; Swidergall, Marc; Cottier, Fabien; Tielker, Denis; Román, Elvira; Pla, Jesus; Ernst, Joachim F.

2012-01-01

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance. PMID:22319443

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

PubMed

Chionis, Kostas; Krikorian, Dimitrios; Koukkou, Anna-Irini; Sakarellos-Daitsiotis, Maria; Panou-Pomonis, Eugenia

2016-11-01

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G 1 LLKR 5 IKT 8 LL-NH 2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly 1 , Arg 5 , and Thr 8 and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF 5 IKK 8 LL-NH 2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions.

PubMed

Kreling, Paula Fernanda; Aida, Kelly Limi; Massunari, Loiane; Caiaffa, Karina Sampaio; Percinoto, Célio; Bedran, Telma Blanca Lombardo; Spolidorio, Denise Madalena Palomari; Abuna, Gabriel Flores; Cilli, Eduardo Maffud; Duque, Cristiane

2016-10-01

This study evaluated the cytotoxicity and effect of fragments derived from three oral cationic peptides (CP): LL-37, D6-17 and D1-23 against cariogenic bacteria under planktonic and biofilm conditions. For cytotoxicity analysis, two epithelial cell lines were used. The minimum inhibitory concentration and the minimal bactericidal concentration were determined for the CP fragments and the control (chlorhexidine-CHX) against cariogenic bacteria. The fractional inhibitory concentration was obtained for the combinations of CP fragments on Streptococcus mutans. Biofilm assays were conducted with the best antimicrobial CP fragment against S. mutans. The results indicated that D6-17 was not cytotoxic. D1-23, LL-37 and CHX were not cytotoxic in low concentrations. D1-23 presented the best bactericidal activity against S. mutans, S. mitis and S. salivarius. Combinations of CP fragments did not show a synergic effect. D1-23 presented a higher activity against S. mutans biofilm than CHX. It was concluded that D1-23 showed a substantial effect against cariogenic bacteria and low cytotoxicity.

Prostate cancer-derived cathelicidin-related antimicrobial peptide facilitates macrophage differentiation and polarization of immature myeloid progenitors to protumorigenic macrophages.

PubMed

Cha, Ha-Ram; Lee, Joo Hyoung; Hensel, Jonathan A; Sawant, Anandi B; Davis, Brittney H; Lee, Carnellia M; Deshane, Jessy S; Ponnazhagan, Selvarangan

2016-05-01

A growing body of evidence indicates a positive correlation between expression of human antimicrobial peptide leucin leucin 37 (LL-37) and progression of epithelial cancers, including prostate cancer (PCa). Although the molecular mechanisms for this correlation has not yet been elucidated, the primary function of LL-37 as a chemotactic molecule for innate immune effector cells suggests its possible association in coordinating protumorigenic mechanisms, mediated by tumor-infiltrating immune cells. To investigate protumorigenic role(s) of cathelicidin-related antimicrobial peptide (CRAMP), a murine orthologue of LL-37, the present study compared tumor growth kinetics between mouse PCa cell lines with and without CRAMP expression (TRAMP-C1 and TRAMP-C1(CRAMP-sh) , respectively) in immunocompetent mice. CRAMP-mediated chemotaxis of different innate immune cell types to the tumor microenvironment (TME) was observed in vivo and confirmed by in vitro chemotaxis assay. The role of CRAMP in differentiation and polarization of immature myeloid progenitors (IMPs) to protumorigenic type 2 macrophages (M2) in TME was determined by adoptive transfer of IMPs into mice bearing CRAMP(+) and CRAMP(-) tumors. To differentiate protumorigenic events mediated by tumor-derived CRAMP from host immune cell-derived CRAMP, tumor challenge study was performed in CRAMP-deficient mice. To identify mechanisms of CRAMP function, macrophage colony stimulating factor (M-CSF) and monocyte chemoattractant protein 1 (MCP-1) gene expression was analyzed by QRT-PCR and STAT3 signaling was determined by immunoblotting. Significantly delayed tumor growth was observed in wild-type (WT) mice implanted with TRAMP-C1(CRAMP-sh) cells compared to mice implanted with TRAMP-C1 cells. CRAMP(+) TME induced increased number of IMP differentiation into protumorigenic M2 macrophages compared to CRAMP(-) TME, indicating tumor-derived CRAMP facilitates differentiation and polarization of IMPs toward M2. Tumor challenge study in CRAMP deficient mice showed comparable tumor growth kinetics with WT mice, suggesting tumor-derived CRAMP plays a crucial role in PCa progression. In vitro study demonstrated that overexpressed M-CSF and MCP-1 in TRAMP-C1 cells through CRAMP-mediated autocrine signaling, involving p65, regulates IMP-to-M2 differentiation/polarization through STAT3 activation. Altogether, the present study suggests that overexpressed CRAMP in prostate tumor initially chemoattracts IMPs to TME and mediates differentiation and polarization of early myeloid progenitors into protumorigenic M2 macrophages during PCa progression. Thus, selective downregulation of CRAMP in tumor cells in situ may benefit overcoming immunosuppressive mechanisms in PCa. © 2016 Wiley Periodicals, Inc.

The Cutaneous Microbiome and Aspects of Skin Antimicrobial Defense System Resist Acute Treatment with Topical Skin Cleansers.

PubMed

Two, Aimee M; Nakatsuji, Teruaki; Kotol, Paul F; Arvanitidou, Evangelia; Du-Thumm, Laurence; Hata, Tissa R; Gallo, Richard L

2016-10-01

The human skin microbiome has been suggested to play an essential role in maintaining health by contributing to innate defense of the skin. These observations have inspired speculation that the use of common skin washing techniques may be detrimental to the epidermal antibacterial defense system by altering the microbiome. In this study, several common skin cleansers were used to wash human forearms and the short-term effect on the abundance of the antimicrobial peptide LL-37 and the abundance and diversity of bacterial DNA was measured. Despite small but significant decreases in the amount of LL-37 on the skin surface shortly after washing, no significant change in the bacterial community was detected. Furthermore, Group A Streptococcus did not survive better on the skin after washing. In contrast, the addition of antimicrobial compounds such as benzalkonium chloride or triclocarban to soap before washing decreased the growth of Group A Streptococcus applied after rinse. These results support prior studies that hand washing techniques in the health care setting are beneficial and should be continued. Additional research is necessary to better understand the effects of chronic washing and the potential impact of skin care products on the development of dysbiosis in some individuals. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic.

PubMed

Chang, Hao-Teng; Tsai, Pei-Wen; Huang, Hsin-Hui; Liu, Yu-Shu; Chien, Tzu-Shan; Lan, Chung-Yu

2012-02-01

The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human β-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall β-1,3-exoglucanase Xog1p, which is involved in cell-wall β-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the β-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3 μM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated β-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.

Green tea polyphenol epigallocatechin-3-gallate and cranberry proanthocyanidins act in synergy with cathelicidin (LL-37) to reduce the LPS-induced inflammatory response in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts.

PubMed

Lombardo Bedran, Telma Blanca; Palomari Spolidorio, Denise; Grenier, Daniel

2015-06-01

The human antimicrobial peptide cathelicidin (LL-37) possesses anti-inflammatory properties that may contribute to attenuating the inflammatory process associated with chronic periodontitis. Plant polyphenols, including those from cranberry and green tea, have been reported to reduce inflammatory cytokine secretion by host cells. In the present study, we hypothesized that A-type cranberry proanthocyanidins (AC-PACs) and green tea epigallocatechin-3-gallate (EGCG) act in synergy with LL-37 to reduce the secretion of inflammatory mediators by oral mucosal cells. A three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts treated with non-cytotoxic concentrations of AC-PACs (25 and 50 μg/ml), EGCG (1 and 5 μg/ml), and LL-37 (0.1 and 0.2 μM) individually and in combination (AC-PACs+LL-37 and EGCG+LL-37) were stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). Multiplex ELISA assays were used to quantify the secretion of 54 host factors, including chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). LL-37, AC-PACs, and EGCG, individually or in combination, had no effect on the regulation of MMP and TIMP secretion but inhibited the secretion of several cytokines. AC-PACs and LL-37 acted in synergy to reduce the secretion of CXC-chemokine ligand 1 (GRO-α), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6), and had an additive effect on reducing the secretion of interleukin-8 (IL-8), interferon-γ inducible protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) in response to LPS stimulation. EGCG and LL-37 acted in synergy to reduce the secretion of GRO-α, G-CSF, IL-6, IL-8, and IP-10, and had an additive effect on MCP-1 secretion. The combination of LL-37 and natural polyphenols from cranberry and green tea acted in synergy to reduce the secretion of several cytokines by an LPS-stimulated 3D co-culture model of oral mucosal cells. Such combinations show promising results as potential adjunctive therapies for treating inflammatory periodontitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes.

PubMed

Wang, Xiuqing; Junior, José Carlos Bozelli; Mishra, Biswajit; Lushnikova, Tamara; Epand, Richard M; Wang, Guangshun

2017-08-01

Antimicrobial peptides are essential components of the innate immune system of multicellular organisms. Although cationic and hydrophobic amino acids are known determinants of these amphipathic molecules for bacterial killing, it is not clear how lysine-arginine (K-R) positional swaps influence peptide structure and activity. This study addresses this question by investigating two groups of peptides (GF-17 and 17BIPHE2) derived from human cathelicidin LL-37. K-R positional swap showed little effect on minimal inhibitory concentrations of the peptides. However, there are clear differences in bacterial killing kinetics. The membrane permeation patterns vary with peptide and bacterial types, but not changes in fluorescent dyes, salts or pH. In general, the original peptide is more efficient in bacterial killing, but less toxic to human cells, than the K-R swapped peptides, revealing the evolutionary significance of the native sequence for host defense. The characteristic membrane permeation patterns for different bacteria suggest a possible application of these K-R positional-swapped peptides as molecular probes for the type of bacteria. Such differences are related to bacterial membrane compositions: minimal for Gram-positive Staphylococcus aureus with essentially all anionic lipids (cardiolipin and phosphatidylglycerol), but evident for Gram-negative Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli with a mixture of phosphatidylethanolamine and phosphatidylglycerol. Biophysical characterization found similar structures and binding affinities for these peptides in vesicle systems mimicking E. coli and S. aureus. It seems that interfacial arginines of GF-17 are preferred over lysines in bacterial membrane permeation. Our study sheds new light on the design of cationic amphipathic peptides. Copyright © 2017 Elsevier B.V. All rights reserved.

C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

PubMed

Papareddy, Praveen; Kalle, Martina; Kasetty, Gopinath; Mörgelin, Matthias; Rydengård, Victoria; Albiger, Barbara; Lundqvist, Katarina; Malmsten, Martin; Schmidtchen, Artur

2010-09-03

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.

Cathelicidin (LL-37) level in the scalp hair of patients with tinea capitis.

PubMed

Abdelaal, Nagwa H; Rashed, Laila A; Ibrahim, Sahar Y; Abd El Halim, Mona H; Ghoneim, Noha; Saleh, Noha A; Saleh, Marwah A

2017-10-01

Antimicrobial peptides (AMPs) are considered an important first line of defense against pathogens. Cathelicidin LL-37 was upregulated in response to fungal infection. In this work we aimed to evaluate cathelicidin LL-37 in the hair of tinea capitis and compare it to normal controls. Hair samples were collected from 30 children and 30 controls aged from 2 to10 years old, and the level of cathelicidin LL-37 in the hair was detected by quantitative real-time PCR. The 30 patients were further subdivided into three subgroups according to their clinical type. Ten patients were scaly type, 10 patients were black dots type, and 10 patients were kerion type. Cathelicidin level in patients ranged from 6.0 to 17.5 with mean ± SD (11.3 ± 2.3) and in control ranged from 1.02 to 6.2, with mean ± SD (2.8 ± 1.5). There was a significant difference between the patients and controls regarding the cathelicidin level; P value was 0. The mean cathelicidin level was lowest in the kerion type10.73 ± 2.6 and highest in the black dot type 12.05 ± 2.76. However, there was no significant difference between the cathelicidin level of the different clinical types of tinea capitis; P value was 0.58. In conclusion, the level of cathelicidin LL-37 in hair specimens of human tinea capitis was significantly higher than controls. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

De novo design and synthesis of ultra-short peptidomimetic antibiotics having dual antimicrobial and anti-inflammatory activities.

PubMed

Murugan, Ravichandran N; Jacob, Binu; Ahn, Mija; Hwang, Eunha; Sohn, Hoik; Park, Hyo-Nam; Lee, Eunjung; Seo, Ji-Hyung; Cheong, Chaejoon; Nam, Ky-Youb; Hyun, Jae-Kyung; Jeong, Ki-Woong; Kim, Yangmee; Shin, Song Yub; Bang, Jeong Kyu

2013-01-01

Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability. In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.

Cost-effective Expression and Purification of Antimicrobial and Host Defense Peptides in Escherichia coli

PubMed Central

Bommarius, B.; Jenssen, H.; Elliott, M.; Kindrachuk, J.; Pasupuleti, Mukesh; Gieren, H; Jaeger, K.-E.; Hancock, R.E. W.

2010-01-01

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity; and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10 L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans. PMID:20713107

Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials.

PubMed

Malhotra, Sankalp; Limoli, Dominique H; English, Anthony E; Parsek, Matthew R; Wozniak, Daniel J

2018-03-27

Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H 2 O 2 ). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H 2 O 2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys , encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung. IMPORTANCE P. aeruginosa mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of P. aeruginosa demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified P. aeruginosa communities in vivo , as these variants can differentially shield one another from components of the host response. Copyright © 2018 Malhotra et al.

DrsG from Streptococcus dysgalactiae subsp. equisimilis Inhibits the Antimicrobial Peptide LL-37

PubMed Central

Smyth, Danielle; Cameron, Ainslie; Davies, Mark R.; McNeilly, Celia; Hafner, Louise; Sriprakash, Kadaba S.

2014-01-01

SIC and DRS are related proteins present in only 4 of the >200 Streptococcus pyogenes emm types. These proteins inhibit complement-mediated lysis and/or the activity of certain antimicrobial peptides (AMPs). A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp. equisimilis. Here we show that geographically dispersed isolates representing 14 of 50 emm types examined possess variants of drsG. However, not all isolates within the drsG-positive emm types possess the gene. Sequence comparisons also revealed a high degree of conservation in different S. dysgalactiae subsp. equisimilis emm types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatants. Unlike SIC, but similar to DRS, DrsG does not inhibit complement-mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelicidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. Conservation of prolines in the C-terminal region also suggests that these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP-inhibitory protein in S. dysgalactiae subsp. equisimilis and suggests that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of the complement-inhibitory activity of SIC may reflect its continuing evolution. PMID:24664506

Cathelin-related antimicrobial peptide differentially regulates T- and B-cell function

PubMed Central

Kin, Nicholas W.; Chen, Yao; Stefanov, Emily K.; Gallo, Richard L.; Kearney, John F.

2011-01-01

Mammalian antimicrobial peptides (AMPs) play an important role in host defense via direct antimicrobial activity as well as immune regulation. The mouse cathelin-related antimicrobial peptide (mCRAMP), produced from the mouse gene Camp, is the only mouse cathelicidin identified and the ortholog of the human gene encoding the peptide LL-37. This study tested the hypothesis that mouse B and T cells produce and respond to mCRAMP. We show that all mature mouse B-cell subsets, including follicular (FO), marginal zone (MZ), B1a, and B1b cells, as well as CD4+ and CD8+ T cells produce Camp mRNA and mCRAMP protein. Camp−/− B cells produced equivalent levels of IgM, IgG3, and IgG2c but less IgG1 and IgE, while Camp−/− CD4+ T cells cultured in Th2-inducing conditions produced more IL-4-expressing cells when compared with WT cells, effects that were reversed upon addition of mCRAMP. In vivo, Camp−/− mice immunized with TNP-OVA absorbed in alum produced an enhanced TNP-specific IgG1 response when compared with WT mice. ELISpot analysis revealed increased numbers of TNP-specific IgG1-secreting splenic B cells and FACS analysis revealed increased CD4+ T-cell IL-4 expression. Our results suggest that mCRAMP differentially regulates B- and T-cell function and implicate mCRAMP in the regulation of adaptive immune responses. PMID:21773974

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging.

PubMed

Ordonez, Soledad R; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

2014-01-01

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37.

Diagnostic value of anti-microbial peptide, cathelicidin in congenital pneumonia.

PubMed

Gad, Ghada I; Abushady, Nancy M; Fathi, Marwa S; Elsaadany, Wafaa

2015-01-01

To evaluate the diagnostic value of anti-microbial peptide (cathelicidin), LL-37, in congenital pneumonia and its relation to 25 hydroxycholecalciferol [(25 OH)D] status. The study included 30 neonates with congenital pneumonia and culture proven sepsis admitted to neonatal intensive care unit of Ain Shams University and 30 healthy neonates as control group. All neonates were subjected to history taking, clinical examination and measurement of serum 25(OH)D and cathelicidin. Neonates with congenital pneumonia had significantly higher serum cathelicidin and lower serum 25(OH)D compared to controls. Serum cathelicidin was negatively correlated with Apgar score at 1 and 5 min and positively correlated with length of stay among patient group. Cut-off value of cathelicidin to diagnose congenital pneumonia was 17 pg/mmol with 93% sensitivity and 86% specificity. Neonates with congenital pneumonia had significantly high cathelicidin and low 25(OH)D suggesting a possible role of fetal 25(OH)D deficiency as predisposing factor for congenital pneumonia.

Host defence peptides in human burns.

PubMed

Kaus, Aljoscha; Jacobsen, Frank; Sorkin, Michael; Rittig, Andrea; Voss, Bruno; Daigeler, Adrien; Sudhoff, Holger; Steinau, Hans-Ulrich; Steinstraesser, Lars

2008-02-01

The goal of this study was to analyse expression profiles of human epithelial host defence peptides in burned and unburned skin tissue, samples of which were obtained during debridements and snap-frozen in liquid nitrogen. Total RNA was isolated, and cDNA of epithelial host defence peptides and proteins (hCAP-18/LL-37, hBD1-hBD4, dermcidin, S100A7/psoriasin and RNAse7) was quantified by qRT-PCR. In situ hybridisation and immunohistochemical staining localised gene expression of hCAP-18/LL-37, hBD2 and hBD3 in histological sections. Most of the analysed host defence peptides and proteins showed higher mRNA levels in partial-thickness burns than in unburned tissue. In situ hybridisation revealed expression of hCAP-18/LL-37, hBD2 and hBD3 at the surface of burns that was independent of burn depth. However, the finding of higher host defence peptide gene expression rates does not correlate with the incidence of wound infection in burns. We hypothesise that the epithelial innate immune response in burns is complex.

De Novo Design and Synthesis of Ultra-Short Peptidomimetic Antibiotics Having Dual Antimicrobial and Anti-Inflammatory Activities

PubMed Central

Ahn, Mija; Hwang, Eunha; Sohn, Hoik; Park, Hyo-Nam; Lee, Eunjung; Seo, Ji-Hyung; Cheong, Chaejoon; Nam, Ky-Youb; Hyun, Jae-Kyung; Jeong, Ki-Woong; Kim, Yangmee; Shin, Song Yub; Bang, Jeong Kyu

2013-01-01

Background Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability. Methodology/Principal Findings In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. Conclusion/Significance The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics. PMID:24302996

Antimicrobial and Biophysical Properties of Surfactant Supplemented with an Antimicrobial Peptide for Treatment of Bacterial Pneumonia

PubMed Central

Veldhuizen, Edwin J. A.; Keating, Eleonora; Haagsman, Henk P.; Zuo, Yi Y.; Yamashita, Cory M.; Veldhuizen, Ruud A. W.

2015-01-01

Antibiotic-resistant bacterial infections represent an emerging health concern in clinical settings, and a lack of novel developments in the pharmaceutical pipeline is creating a “perfect storm” for multidrug-resistant bacterial infections. Antimicrobial peptides (AMPs) have been suggested as future therapeutics for these drug-resistant bacteria, since they have potent broad-spectrum activity, with little development of resistance. Due to the unique structure of the lung, bacterial pneumonia has the additional problem of delivering antimicrobials to the site of infection. One potential solution is coadministration of AMPs with exogenous surfactant, allowing for distribution of the peptides to distal airways and opening of collapsed lung regions. The objective of this study was to test various surfactant-AMP mixtures with regard to maintaining pulmonary surfactant biophysical properties and bactericidal functions. We compared the properties of four AMPs (CATH-1, CATH-2, CRAMP, and LL-37) suspended in bovine lipid-extract surfactant (BLES) by assessing surfactant-AMP mixture biophysical and antimicrobial functions. Antimicrobial activity was tested against methillicin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. All AMP/surfactant mixtures exhibited an increase of spreading compared to a BLES control. BLES+CATH-2 mixtures had no significantly different minimum surface tension versus the BLES control. Compared to the other cathelicidins, CATH-2 retained the most bactericidal activity in the presence of BLES. The BLES+CATH-2 mixture appears to be an optimal surfactant-AMP mixture based on in vitro assays. Future directions involve investigating the potential of this mixture in animal models of bacterial pneumonia. PMID:25753641

Antimicrobial and biophysical properties of surfactant supplemented with an antimicrobial peptide for treatment of bacterial pneumonia.

PubMed

Banaschewski, Brandon J H; Veldhuizen, Edwin J A; Keating, Eleonora; Haagsman, Henk P; Zuo, Yi Y; Yamashita, Cory M; Veldhuizen, Ruud A W

2015-01-01

Antibiotic-resistant bacterial infections represent an emerging health concern in clinical settings, and a lack of novel developments in the pharmaceutical pipeline is creating a "perfect storm" for multidrug-resistant bacterial infections. Antimicrobial peptides (AMPs) have been suggested as future therapeutics for these drug-resistant bacteria, since they have potent broad-spectrum activity, with little development of resistance. Due to the unique structure of the lung, bacterial pneumonia has the additional problem of delivering antimicrobials to the site of infection. One potential solution is coadministration of AMPs with exogenous surfactant, allowing for distribution of the peptides to distal airways and opening of collapsed lung regions. The objective of this study was to test various surfactant-AMP mixtures with regard to maintaining pulmonary surfactant biophysical properties and bactericidal functions. We compared the properties of four AMPs (CATH-1, CATH-2, CRAMP, and LL-37) suspended in bovine lipid-extract surfactant (BLES) by assessing surfactant-AMP mixture biophysical and antimicrobial functions. Antimicrobial activity was tested against methillicin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. All AMP/surfactant mixtures exhibited an increase of spreading compared to a BLES control. BLES+CATH-2 mixtures had no significantly different minimum surface tension versus the BLES control. Compared to the other cathelicidins, CATH-2 retained the most bactericidal activity in the presence of BLES. The BLES+CATH-2 mixture appears to be an optimal surfactant-AMP mixture based on in vitro assays. Future directions involve investigating the potential of this mixture in animal models of bacterial pneumonia. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

LL-37 boosts immunosuppressive function of placenta-derived mesenchymal stromal cells.

PubMed

Oliveira-Bravo, Martha; Sangiorgi, Bruno Braga; Schiavinato, Josiane Lilian Dos Santos; Carvalho, Juliana Lott; Covas, Dimas Tadeu; Panepucci, Rodrigo Alexandre; Neves, Francisco de Assis Rocha; Franco, Octávio Luiz; Pereira, Rinaldo Wellerson; Saldanha-Araujo, Felipe

2016-12-30

Although promising for graft-versus-host disease (GvHD) treatment, MSC therapy still faces important challenges. For instance, increasing MSC migratory capacity as well as potentializing immune response suppression are of interest. For GvHD management, preventing opportunistic infections is also a valuable strategy, since immunocompromised patients are easy targets for infections. LL-37 is a host defense peptide (HDP) that has been deeply investigated due to its immunomodulatory function. In this scenario, the combination of MSC and LL-37 may result in a robust combination to be clinically used. In the present study, the effects of LL-37 upon the proliferation and migratory capacity of human placenta-derived MSCs (pMSCs) were assessed by MTT and wound scratch assays. The influence of LL-37 over the immunosuppressive function of pMSCs was then investigated using CFSE cell division kit. Flow cytometry and real-time PCR were used to investigate the molecular mechanisms involved in the effects observed. LL-37 had no detrimental effects over MSC proliferation and viability, as assessed by MTT assay. Moreover, the peptide promoted increased migratory behavior of pMSCs and enhanced their immunomodulatory function over activated human PBMCs. Strikingly, our data shows that LL-37 treatment leads to increased TLR3 levels, as shown by flow cytometry, and to an increased expression of factors classically related to immunosuppression, namely IDO, IL-10, TGF-β, IL-6, and IL-1β. Taken together, our observations may serve as groundwork for the development of new therapeutic strategies based on the combined use of LL-37 and MSCs, which may provide patients not only with an enhanced immunosuppression regime, but also with an agent to prevent opportunistic infections.

Designing effective anticancer-radiopeptides. A Molecular Dynamics study of their interaction with model tumor and healthy cell membranes.

PubMed

Capozzi, E; Aureli, S; Minicozzi, V; Rossi, G C; Stellato, F; Morante, S

2018-06-06

One of the greatest merit of the use of radiopeptides in oncology is their selectivity which, however, brings about the drawback that each radiopeptide is specific for a given tumor type. To overcome this problem the direction currently taken in drug design is that of radiolabelling peptide hormones (or their analogues), relying on their intrinsic ability to bind to specific receptors in precise areas of the human body, at the cost, however, of a poor selectivity against healthy cells. We present here an extensive Molecular Dynamics study of a promising alternative inspired by the mechanism through which antimicrobial peptides interact with the negatively charged bacterial membranes. Appropriately modifying the human antimicrobial peptide, LL-37, we designed a functionalized radionuclide carrier capable of binding more strongly to the negatively charged (model) tumor membranes than to the neutral healthy ones. The mechanism behind this behaviour relies on the fact that at the slight acidic pH surrounding tumor tissues the histidines belonging to the peptide get protonated thus making it positively charged. We have investigated by an extended numerical study the way in which this artificial peptide interacts with models of tumor and healthy cell membranes, proving by Potential Mean Force calculations that the affinity of the peptide to model tumor membranes is significantly larger than to healthy ones. These features (high affinity and generic tumor selectivity) recommend antimicrobial derived customized carriers as promising theranostic constructs in cancer diagnostic and therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4.

PubMed

Schiemann, Florian; Brandt, Ernst; Gross, Roland; Lindner, Buko; Mittelstädt, Jessica; Sommerhoff, Christian P; Schulmistrat, Jan; Petersen, Frank

2009-08-15

The cathelicidin LL-37 represents a potent antimicrobial and cell-stimulating agent, most abundantly expressed in peripheral organs such as lung and skin during inflammation. Because mast cells (MC) overtake prominent immunomodulatory roles in these organs, we wondered whether interactions exist between MC and LL-37. In this study, we show for the first time to our knowledge that physiological concentrations of LL-37 induce degranulation in purified human lung MC. Intriguingly, as a consequence LL-37 rapidly undergoes limited cleavage by a released protease. The enzyme was identified as beta-tryptase by inhibitor studies and by comparison to the recombinant protease. Examining the resulting LL-37 fragments for their functional activity, we found that none of the typical capacities of intact LL-37, i.e., MC degranulation, bactericidal activity, and neutralization of LPS, were retained. Conversely, we found that another inflammatory protein, the platelet-derived chemokine CXCL4, protects LL-37 from cleavage by beta-tryptase. Interestingly, CXCL4 did not act as a direct enzyme inhibitor, but destabilized active tetrameric beta-tryptase by antagonizing the heparin component required for the integrity of the tetramer. Altogether our results suggest that interaction of LL-37 and MC initiates an effective feedback loop to limit cathelicidin activity during inflammation, whereas CXCL4 may represent a physiological counter-regulator of beta-tryptase activity.

Enhanced antitumor effects by docetaxel/LL37-loaded thermosensitive hydrogel nanoparticles in peritoneal carcinomatosis of colorectal cancer

PubMed Central

Fan, Rangrang; Tong, Aiping; Li, Xiaoling; Gao, Xiang; Mei, Lan; Zhou, Liangxue; Zhang, Xiaoning; You, Chao; Guo, Gang

2015-01-01

Intraperitoneal chemotherapy was explored in clinical trials as a promising strategy to improve the therapeutic effects of chemotherapy. In this work, we developed a biodegradable and injectable drug-delivery system by coencapsulation of docetaxel (Doc) and LL37 peptide polymeric nanoparticles (Doc+LL37 NPs) in a thermosensitive hydrogel system for colorectal peritoneal carcinoma therapy. Firstly, polylactic acid (PLA)-Pluronic L35-PLA (PLA-L35-PLA) was explored to prepare the biodegradable Doc+LL37 NPs using a water-in-oil-in-water double-emulsion solvent-evaporation method. Then, biodegradable and injectable thermosensitive PLA-L64-PLA hydrogel with lower sol–gel transition temperature at around body temperature was also prepared. Transmission electron microscopy revealed that the Doc+LL37 NPs formed with the PLA-L35-PLA copolymer were spherical. Fourier-transform infrared spectra certified that Doc and LL37 were encapsulated successfully. X-ray diffraction diagrams indicated that Doc was encapsulated amorphously. Intraperitoneal administration of Doc+LL37 NPs–hydrogel significantly suppressed the growth of HCT116 peritoneal carcinomatosis in vivo and prolonged the survival of tumor-bearing mice. Our results suggested that Doc+LL37 NPs–hydrogel may have potential clinical applications. PMID:26664119

Effects of Vancomycin Versus Nafcillin in Enhancing Killing of Methicillin-Susceptible Staphylococcus aureus Causing Bacteremia by Human Cathelicidin LL37

PubMed Central

Le, Jennifer; Dam, Quang; Schweizer, Marin; Thienphrapa, Wdee; Nizet, Victor; Sakoulas, George

2016-01-01

Recent studies have demonstrated that anti-staphylococcal beta-lactam antibiotics, like nafcillin, render methicillin-resistant Staphylococcus aureus (MRSA) more susceptible to killing by innate host defense peptides (HDPs), such as cathelicidin LL-37. We compared the effects of growth in 1/4 minimum inhibitory concentration (MIC) of nafcillin or vancomycin on LL-37 killing of 92 methicillin-susceptible S. aureus (MSSA) isolates. For three randomly selected strains among these, we examined the effects of nafcillin, vancomycin, daptomycin, or linezolid on LL-37 killing and autolysis. Growth in the presence of sub-inhibitory nafcillin significantly enhanced LL-37 killing of MSSA compared to vancomycin and antibiotic-free controls. Nafcillin also reduced MSSA production of the golden staphylococcal pigment staphyloxanthin in 39% of pigmented strains vs. 14% for vancomycin. Among antibiotics tested, only nafcillin resulted in significantly increased MSSA autolysis. These studies point to additional mechanisms of anti-staphylococcal activity of nafcillin beyond direct bactericidal activity, properties that vancomycin and other antibiotic classes do not exhibit. The ability of nafcillin to enhance sensitivity to innate host defense peptides may contribute to its superior effectiveness against MSSA as suggested by studies comparing clinical outcomes to vancomycin treatment. PMID:27234592

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.

PubMed

Choi, Heejun; Yang, Zhilin; Weisshaar, James C

2015-01-20

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1-7 of cecropin A (from moth) with residues 2-9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2 (-), H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency.

Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process.

PubMed

van der Weerden, Nicole L; Hancock, Robert E W; Anderson, Marilyn A

2010-11-26

The antifungal activity of the plant defensin NaD1 involves specific interaction with the fungal cell wall, followed by permeabilization of the plasma membrane and entry of NaD1 into the cytoplasm. Prior to this study, the role of membrane permeabilization in the activity of NaD1, as well as the relevance of cell wall binding, had not been investigated. To address this, the permeabilization of Fusarium oxysporum f. sp. vasinfectum hyphae by NaD1 was investigated and compared with that by other antimicrobial peptides, including the cecropin-melittin hybrid peptide CP-29, the bovine peptide BMAP-28, and the human peptide LL-37, which are believed to act largely through membrane disruption. NaD1 appeared to permeabilize cells via a novel mechanism that required the presence of the fungal cell wall. NaD1 and Bac2A, a linear variant of the bovine peptide bactenecin, were able to enter the cytoplasm of treated hyphae, indicating that cell death is accelerated by interaction with intracellular targets.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

PubMed

Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

2013-04-11

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

PubMed Central

2013-01-01

HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

Suppression of Antimicrobial Peptide Expression by Ureaplasma Species

PubMed Central

Crabb, Donna M.; Dai, Yuling; Chen, Yuying; Waites, Ken B.; Atkinson, T. Prescott

2014-01-01

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization. PMID:24491573

Suppression of antimicrobial peptide expression by ureaplasma species.

PubMed

Xiao, Li; Crabb, Donna M; Dai, Yuling; Chen, Yuying; Waites, Ken B; Atkinson, T Prescott

2014-04-01

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.

The innate defense antimicrobial peptides hBD3 and RNase7 are induced in human umbilical vein endothelial cells by classical inflammatory cytokines but not Th17 cytokines.

PubMed

Burgey, Christine; Kern, Winfried V; Römer, Winfried; Sakinc, Türkan; Rieg, Siegbert

2015-05-01

Antimicrobial peptides are multifunctional effector molecules of innate immunity. In this study we investigated whether endothelial cells actively contribute to innate defense mechanisms by expression of antimicrobial peptides. We therefore stimulated human umbilical vein endothelial cells (HUVEC) with inflammatory cytokines, Th17 cytokines, heat-inactivated bacteria, bacterial conditioned medium (BCM) of Staphylococcus aureus and Streptococcus sanguinis, and lipoteichoic acid (LTA). Stimulation with single cytokines induced discrete expression of human β-defensin 3 (hBD3) by IFN-γ or IL-1β and of ribonuclease 7 (RNase7) by TNF-α without any effects on LL-37 gene expression. Stronger hBD3 and RNase7 induction was observed after combined stimulation with IL-1β, TNF-α and IFN-γ and was confirmed by high hBD3 and RNase7 peptide levels in cell culture supernatants. In contrast, Th17 cytokines or stimulation with LTA did not result in AMP production. Moreover, only BCM of an invasive S. aureus bacteremia isolate induced hBD3 in HUVEC. We conclude that endothelial cells actively contribute to prevent dissemination of pathogens at the blood-tissue-barrier by production of AMPs that exhibit microbicidal and immunomodulatory functions. Further investigations should focus on tissue-specific AMP induction in different endothelial cell types, on pathogen-specific induction patterns and potentially involved pattern-recognition receptors of endothelial cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

What is really in control of skin immunity: lymphocytes, dendritic cells, or keratinocytes? facts and controversies.

PubMed

Rupec, Rudolf A; Boneberger, Susanne; Ruzicka, Thomas

2010-01-01

The pathophysiology of atopic dermatitis is still under discussion. Although it is widely accepted that environmental factors and a genetic predisposition are essential, the role of the innate and adaptive immune system and the functional cascade of the cells involved is still unclear. A concept that integrates all immune cells as equally essential has allure. In addition, barrier abnormalities due to mutations of the gene coding for filaggrin and down-regulation of antimicrobial peptides, such as LL-37 and beta-defensins 2 and 3, were very recently found to be relevant for the pathogenesis of atopic dermatitis. Copyright 2010 Elsevier Inc. All rights reserved.

Antimicrobials from human skin commensal bacteria protect against Staphylococcus aureus and are deficient in atopic dermatitis

PubMed Central

Nakatsuji, Teruaki; Chen, Tiffany H.; Narala, Saisindhu; Chun, Kimberly A.; Two, Aimee M.; Yun, Tong; Shafiq, Faiza; Kotol, Paul F.; Bouslimani, Amina; Melnik, Alexey V.; Latif, Haythem; Kim, Ji-Nu; Lockhart, Alexandre; Artis, Keli; David, Gloria; Taylor, Patricia; Streib, Joanne; Dorrestein, Pieter C.; Grier, Alex; Gill, Steven R.; Zengler, Karsten; Hata, Tissa R.; Leung, Donald Y. M.; Gallo, Richard L.

2017-01-01

The microbiome can promote or disrupt human health by influencing both adaptive and innate immune functions. We tested whether bacteria that normally reside on human skin participate in host defense by killing Staphylococcus aureus, a pathogen commonly found in patients with atopic dermatitis (AD) and an important factor that exacerbates this disease. High-throughput screening for antimicrobial activity against S.aureus was performed on isolates of coagulase-negative Staphylococcus (CoNS) collected from the skin of healthy and AD subjects. CoNS strains with antimicrobial activity were common on the normal population but rare on AD subjects. A low frequency of strains with antimicrobial activity correlated with colonization by S.aureus. The antimicrobial activity was identified as previously unknown antimicrobial peptides (AMPs) produced by CoNS species including Staphylococcus epidermidis and Staphylococcus hominis. These AMPs were strain-specific, highly potent, selectively killed S.aureus, and synergized with the human AMP LL-37. Application of these CoNS strains to mice confirmed their defense function in vivo relative to application of nonactive strains. Strikingly, reintroduction of antimicrobial CoNS strains to human subjects with AD decreased colonization by S.aureus. These findings show how commensal skin bacteria protect against pathogens and demonstrate how dysbiosis of the skin microbiome can lead to disease. PMID:28228596

Myoinhibiting Peptides are the Ancestral Ligands of the Promiscuous Drosophila Sex Peptide Receptor

DTIC Science & Technology

2010-01-01

decreases the willingness to re-mate, induces egg production and egg laying, stimulates food intake, enhances antimicrobial peptide synthesis and reduces...polypropylene tubes, centrifuged to remove cell debris, and the supernatants dried. Each sample was dissolved in 250 ll of assay buffer (0.05 M Tris, 4...variations during the cDNA synthesis step, all RNA samples were reverse transcribed simultaneously. Furthermore, several negative control reactions, i.e

Peptides: β-cyclodextrin inclusion compounds as highly effective antimicrobial and anti-epithelial proliferation agents.

PubMed

Consuegra, Jessika; de Lima, Maria Elena; Santos, Daniel; Sinisterra, Rubén Dario; Cortés, Maria Esperanza

2013-12-01

The use of antimicrobial peptides (AMPs) as therapeutic agents for periodontal infections has great advantages, such as broad spectrum of action, low toxicity, and limited bacterial resistance. However, their practical use is limited because of the large amount of peptide required to exercise the microbicidal function. LyeTxI, LL37f, and KR12 cationic peptides were prepared with β-cyclodextrin (βCD) at 1:1 molar ratios. The susceptibility of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum were assessed in anaerobic conditions. Cytotoxicity assays were performed using osteoblast and Caco-2 epithelial cells, and hemolytic activity was assessed on rabbit erythrocytes at an absorbance of 414 nm. Parameters of surface roughness and electrical charge were established by atomic force microscopy and zeta (ζ) potential, respectively. AMP/βCDs drastically decreased the peptide concentration required for activity against the bacteria tested. Moreover, AMPs associated with βCD were able to modify cell-surface parameters, such as roughness and ζ potential. On the other hand, AMP/βCD did not alter the degree of hemolysis induced by the pure AMPs. The effective concentration at half-maximum values of the peptides and compounds on osteoblasts were greater than the concentrations required to achieve inhibition of bacterial growth in all the species tested. AMP/βCDs inhibited the proliferation of Caco-2 epithelial cells in a more efficient manner than AMPs alone. AMP/βCD compounds more effectively inhibit periodontopathogenic bacteria than AMPs alone, with the additional ability of inhibiting the proliferation of epithelial cells at concentrations that are non-cytotoxic for osteoblasts and erythrocytes.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

PubMed Central

Yang, Zhilin; Weisshaar, James C.

2015-01-01

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1–7 of cecropin A (from moth) with residues 2–9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O2−, H2O2, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency. PMID:25561551

Heightened circulating levels of antimicrobial peptides in tuberculosis—Diabetes co-morbidity and reversal upon treatment

PubMed Central

Kumar, Nathella Pavan; Moideen, Kadar; Viswanathan, Vijay; Sivakumar, Shanmugam; Menon, Pradeep A.; Kornfeld, Hardy

2017-01-01

Background The association of antimicrobial peptides (AMPs) with tuberculosis—diabetes comorbidity (PTB-DM) is not well understood. Methods To study the association of AMPs with PTB-DM, we examined the systemic levels of cathelicidin (LL37), human beta defensin– 2 (HBD2), human neutrophil peptides 1–3, (HNP1-3) and granulysin in individuals with either PTB-DM, PTB, latent TB (LTB) or no TB infection (NTB). Results Circulating levels of cathelicidin and HBD2 were significantly higher and granulysin levels were significantly lower in PTB-DM compared to PTB, LTB or NTB, while the levels of HNP1-3 were significantly higher in PTB-DM compared to LTB or NTB individuals. Moreover, the levels of cathelicidin and/or HBD2 were significantly higher in PTB-DM or PTB individuals with bilateral and cavitary disease and also exhibited a significant positive relationship with bacterial burden. Cathelidin, HBD2 and HNP1-3 levels exhibited a positive relationship with HbA1c and/or fasting blood glucose levels. Finally, anti-tuberculosis therapy resulted in significantly diminished levels of cathelicidin, HBD2, granulysin and significantly enhanced levels of HNP1-3 and granulysin in PTB-DM and/or PTB individuals. Conclusion Therefore, our data demonstrate that PTB-DM is associated with markedly enhanced levels of AMPs and diminished levels of granulysin. PMID:28910369

Nafcillin enhances innate immune-mediated killing of methicillin-resistant Staphylococcus aureus.

PubMed

Sakoulas, George; Okumura, Cheryl Y; Thienphrapa, Wdee; Olson, Joshua; Nonejuie, Poochit; Dam, Quang; Dhand, Abhay; Pogliano, Joseph; Yeaman, Michael R; Hensler, Mary E; Bayer, Arnold S; Nizet, Victor

2014-02-01

Based on in vitro synergy studies, the addition of nafcillin to daptomycin was used to treat refractory methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Daptomycin is a de facto cationic antimicrobial peptide in vivo, with antistaphylococcal mechanisms reminiscent of innate host defense peptides (HDPs). In this study, the effects of nafcillin on HDP activity against MRSA were examined in vitro and in vivo. Exposures to β-lactam antimicrobials in general, and nafcillin in particular, significantly increased killing of S. aureus by selected HDPs from keratinocytes, neutrophils, and platelets. This finding correlated with enhanced killing of MRSA by whole blood, neutrophils, and keratinocytes after growth in nafcillin. Finally, nafcillin pretreatment ex vivo reduced MRSA virulence in a murine subcutaneous infection model. Despite the lack of direct activity against MRSA, these studies show potent, consistent, and generalized nafcillin-mediated "sensitization" to increased killing of MRSA by various components of the innate host response. The use of nafcillin as adjunctive therapy in MRSA bacteremia merits further study and should be considered in cases refractory to standard therapy. Nafcillin has been used as adjunctive therapy to clear persistent MRSA bacteremia. Nafcillin enhances killing of MRSA by a cadre of innate host defense peptides. Nafcillin increases binding of human cathelicidin LL-37 to the MRSA membrane. Nafcillin enhances killing of MRSA by neutrophils. Nafcillin reduces virulence of MRSA in a murine subcutaneous infection model.

NOD2 enhances the innate response of alveolar macrophages to Mycobacterium tuberculosis in humans.

PubMed

Juárez, Esmeralda; Carranza, Claudia; Hernández-Sánchez, Fernando; León-Contreras, Juan C; Hernández-Pando, Rogelio; Escobedo, Dante; Torres, Martha; Sada, Eduardo

2012-04-01

A role for the nucleotide-binding oligomerization domain 2 (NOD2) receptor in pulmonary innate immune responses has recently been explored. In the present study, we investigated the role that NOD2 plays in human alveolar macrophage innate responses and determined its involvement in the response to infection with virulent Mycobacterium tuberculosis. Our results showed that NOD2 was expressed in human alveolar macrophages, and significant amounts of IL-1β, IL-6, and TNF-α were produced upon ligand recognition with muramyldipeptide (MDP). NOD2 ligation induced the transcription and protein expression of the antimicrobial peptide LL37 and the autophagy enzyme IRGM in alveolar macrophages, demonstrating a novel function for this receptor in these cells. MDP treatment of alveolar macrophages improved the intracellular growth control of virulent M. tuberculosis; this was associated with a significant release of TNF-α and IL-6 and overexpression of bactericidal LL37. In addition, the autophagy proteins IRGM, LC3 and ATG16L1 were recruited to the bacteria-containing autophagosome after treatment with MDP. In conclusion, our results suggest that NOD2 can modulate the innate immune response of alveolar macrophages and play a role in the initial control of respiratory M. tuberculosis infections. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activity of antimicrobial peptides in the presence of polysaccharides produced by pulmonary pathogens.

PubMed

Benincasa, M; Mattiuzzo, M; Herasimenka, Y; Cescutti, P; Rizzo, R; Gennaro, R

2009-09-01

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and members of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the beta-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors. Copyright 2009 European Peptide Society and John Wiley & Sons, Ltd.

Expression profiles of antimicrobial peptides in the genital tract of women using progesterone intrauterine devices versus combined oral contraceptives.

PubMed

Introini, Andrea; Kaldensjö, Tove; Hirbod, Taha; Röhl, Maria; Tjernlund, Annelie; Andersson, Sonia; Broliden, Kristina

2014-11-01

Sex hormones can influence the immune defenses of the female genital tract (FGT) and its susceptibility to infections. Here we investigated the effect of different hormonal contraceptives on the production of antimicrobial peptides (AMPs) in different compartments of the female genital mucosa (FGM), secretions and tissue. Cervicovaginal secretions (CVS) and ectocervical tissue samples obtained from women using progesterone intrauterine devices (pIUD) (n = 23) and combined oral contraceptives (COC) (n = 23) were analyzed for the expression and in situ localization of HNP1-3, BD-2, LL-37, SLPI and trappin-2 by ELISA, real-time PCR and immunohistochemistry. Women using COC had significantly lower mRNA levels of BD-2 and trappin-2 in ectocervical tissue than pIUD users. The two groups showed no differences in CVS concentration, as well as similar in situ expression patterns in ectocervical tissue, of all five AMPs. The use of hormonal contraceptives influences AMP expression differently in genital secretions compared to ectocervical tissue. This suggests that the impact of sex hormones on local immune defenses varies in different compartments of the FGM, and likely in different locations across the FGT. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Candidacidal activity of synthetic peptides based on the antimicrobial domain of the neutrophil-derived protein, CAP37

PubMed Central

Pereira, H. Anne; Tsyshevskaya-Hoover, Irina; Hinsley, Heather; Logan, Sreemathi; Nguyen, Melissa; Nguyen, Thuy-Trang; Pohl, Jan; Wozniak, Karen; Fidel, Paul L.

2009-01-01

The primary bactericidal domain of CAP37, a cationic antimicrobial protein with potent activity against Gram-negative organisms was previously shown to reside between amino acids 20 through 44 (NQGRHFCGGALIHARFVMTAASCFQ) of the native protein. In this study, we explored the efficacy of four synthetic CAP37 peptide analogs, based on this sequence, against various Candida species including fluconazole-sensitive and -resistant isolates of C. albicans. Three of the peptides demonstrated strong antifungal activity for C. albicans, including fluconazole-resistant isolates of C. albicans and were active against C. guilliermondii, C. tropicalis, C. pseudotropicalis, C. parapsilosis, and C. dubliniensis. The peptides were ineffective against C. glabrata, C. krusei, and Saccharomyces cerevisiae. For C. albicans isolates, the peptides had relatively greater activity against blastoconidia than hyphal forms, although strong antifungal activity was observed with pseudohyphal forms of the various Candida species tested. Kinetic studies demonstrated fungicidal rather than fungistatic activity. These findings indicate that synthetic peptides based on the antimicrobial domain of CAP37 also have activity against eukaryotic organisms suggesting a broader range of activity than originally demonstrated and show for the first time their potent fungicidal activity. PMID:19626550

Vitamin D, serum 25(OH)D, LL-37 and polymorphisms in a Canadian First Nation population with endemic tuberculosis

PubMed Central

Larcombe, Linda; Mookherjee, Neeloffer; Slater, Joyce; Slivinski, Caroline; Dantouze, Joe; Singer, Matthew; Whaley, Chris; Denechezhe, Lizette; Matyas, Sara; Decter, Kate; Turner-Brannen, Emily; Ramsey, Clare; Nickerson, Peter; Orr, Pamela

2015-01-01

Background Canadian First Nation populations have experienced endemic and epidemic tuberculosis (TB) for decades. Vitamin D–mediated induction of the host defence peptide LL-37 is known to enhance control of pathogens such as Mycobacterium tuberculosis. Objective Evaluate associations between serum levels of 25-hydroxy vitamin D (25(OH)D) and LL-37, in adult Dene First Nation participants (N = 34) and assess correlations with single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). Design Venous blood was collected from all participants at baseline (winter and summer) and in conjunction with taking vitamin D supplements (1,000 IU/day) (winter and summer). Samples were analysed using ELISA for concentrations of vitamin D and LL-37, and SNPs in the VDR and VDBP regions were genotyped. Results Circulating levels of 25(OH)D were not altered by vitamin D supplementation, but LL-37 levels were significantly decreased. VDBP and VDR SNPs did not correlate with serum concentrations of 25(OH)D, but LL-37 levels significantly decreased in individuals with VDBP D432E T/G and T/T, and with VDR SNP Bsm1 T/T genotypes. Conclusions Our findings suggest that vitamin D supplementation may not be beneficial as an intervention to boost innate immune resistance to M. tuberculosis in the Dene population. PMID:26294193

Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculosis

PubMed Central

2013-01-01

Background We earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM). Methods Healthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method. Results MDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05). Conclusion The results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007). PMID:23590701

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

PubMed

Di Grazia, Antonio; Cappiello, Floriana; Imanishi, Akiko; Mastrofrancesco, Arianna; Picardo, Mauro; Paus, Ralf; Mangoni, Maria Luisa

2015-01-01

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025-4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

PubMed

Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

2017-10-01

In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Human Antimicrobial Peptides and Proteins

PubMed Central

Wang, Guangshun

2014-01-01

As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs) play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized medicine to combat drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively, multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP, isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able to induce the expression of antimicrobial peptides, opening new avenues to the development of anti-infectious drugs. PMID:24828484

Generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide LL-37 treated monocytes.

PubMed

Zhang, Zhifang; Shively, John E

2010-11-15

Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin), which recruits circulating monocytes during injury, may play a role in bone repair. Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM). In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC) lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC), osteonectin (ON), bone sialoprotein II (BSP II), osteopontin (OP), RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP), and cathepsin K (CK). Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte derived lineage and their bone forming phenotype. These cells may have wide ranging implications in the clinic including repair of broken bones and treatment of osteoporosis.

Cathelicidin Insufficiency in Patients with Fatal Leptospirosis.

PubMed

Lindow, Janet C; Wunder, Elsio A; Popper, Stephen J; Min, Jin-Na; Mannam, Praveen; Srivastava, Anup; Yao, Yi; Hacker, Kathryn P; Raddassi, Khadir; Lee, Patty J; Montgomery, Ruth R; Shaw, Albert C; Hagan, Jose E; Araújo, Guilherme C; Nery, Nivison; Relman, David A; Kim, Charles C; Reis, Mitermayer G; Ko, Albert I

2016-11-01

Leptospirosis causes significant morbidity and mortality worldwide; however, the role of the host immune response in disease progression and high case fatality (>10-50%) is poorly understood. We conducted a multi-parameter investigation of patients with acute leptospirosis to identify mechanisms associated with case fatality. Whole blood transcriptional profiling of 16 hospitalized Brazilian patients with acute leptospirosis (13 survivors, 3 deceased) revealed fatal cases had lower expression of the antimicrobial peptide, cathelicidin, and chemokines, but more abundant pro-inflammatory cytokine receptors. In contrast, survivors generated strong adaptive immune signatures, including transcripts relevant to antigen presentation and immunoglobulin production. In an independent cohort (23 survivors, 22 deceased), fatal cases had higher bacterial loads (P = 0.0004) and lower anti-Leptospira antibody titers (P = 0.02) at the time of hospitalization, independent of the duration of illness. Low serum cathelicidin and RANTES levels during acute illness were independent risk factors for higher bacterial loads (P = 0.005) and death (P = 0.04), respectively. To investigate the mechanism of cathelicidin in patients surviving acute disease, we administered LL-37, the active peptide of cathelicidin, in a hamster model of lethal leptospirosis and found it significantly decreased bacterial loads and increased survival. Our findings indicate that the host immune response plays a central role in severe leptospirosis disease progression. While drawn from a limited study size, significant conclusions include that poor clinical outcomes are associated with high systemic bacterial loads, and a decreased antibody response. Furthermore, our data identified a key role for the antimicrobial peptide, cathelicidin, in mounting an effective bactericidal response against the pathogen, which represents a valuable new therapeutic approach for leptospirosis.

Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection.

PubMed

Bucki, Robert; Niemirowicz, Katarzyna; Wnorowska, Urszula; Byfield, Fitzroy J; Piktel, Ewelina; Wątek, Marzena; Janmey, Paul A; Savage, Paul B

2015-10-01

Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

pH Dependent Antimicrobial Peptides and Proteins, Their Mechanisms of Action and Potential as Therapeutic Agents

PubMed Central

Malik, Erum; Dennison, Sarah R.; Harris, Frederick; Phoenix, David A.

2016-01-01

Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms, and here we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic, although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure/function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era. PMID:27809281

Membrane selectivity and disordering mechanism of antimicrobial peptide protegrin-1

NASA Astrophysics Data System (ADS)

Ishitsuka, Yuji

Protegrin-1 (PG-1) is a beta-sheet antimicrobial peptide (AMP), a class of peptides innate to various organisms and functions as a defense agent against harmful microorganisms by means of membrane disordering. Characteristic chemical and structural properties of AMPs allow selective interaction against invaders' cell membranes. Despite their enormous biomedical potential, progress towards developing them into therapeutic agents has been hampered by a lack of insight into their mechanism of action. AMP insertion assays using Langmuir monolayers reveal that both electrostatic properties of the lipid head group as well as the packing density of the lipid tail group play important roles in determining the membrane selectivity of AMPs. These results help elucidate how the AMP selectively targets the cell membrane of microorganisms over the cell membrane of the host. In addition, these results also explain the higher hemolytic ability of PG-1 against human red blood cells (RBCs) compared to the hemolytic ability of PG-1 against sheep and pig RBCs. Synchrotron X-ray reflectivity shows that PG-1 penetrates into the lipid layer. Grazing incidence X-ray diffraction and fluorescence microscopy indicate that the insertion of PG-1 disorders tail group packing. Membrane selectivity and insertion location information of AMPs with different primary sequence and secondary structure have been obtained by using a truncated version of PG-1: PC-17, and an alpha-helical AMP, LL-37, respectively. The similarity of the membrane disordering process across these various peptides motivated us to test the membrane disordering effect of molecules designed to mimic these peptides. Peptide-mimics based on meta-phenylene ethynylenes demonstrate similar membrane disordering effects, showing that the potency of AMPs is derived from their overall chemical and structural properties, rather than exact peptide sequence. Atomic force microscopy (AFM) was used to directly image first, the PG-1 concentration-dependent membrane thinning effect, and second, the PG-1-induced structural transformation of a contiguous supported bilayer patch into a porous one. Our results point the membrane disordering mechanism of PG-1 towards the carpet/toroidal model of membrane disordering. Ongoing AMP-related projects are also discussed.

Single-Molecule Resolution of Antimicrobial Peptide Interactions with Supported Lipid A Bilayers.

PubMed

Nelson, Nathaniel; Schwartz, Daniel K

2018-06-05

The molecular interactions between antimicrobial peptides (AMPs) and lipid A-containing supported lipid bilayers were probed using single-molecule total internal reflection fluorescence microscopy. Hybrid supported lipid bilayers with lipid A outer leaflets and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)) inner leaflets were prepared and characterized, and the spatiotemporal trajectories of individual fluorescently labeled LL37 and Melittin AMPs were determined as they interacted with the bilayer surfaces comprising either monophosphoryl or diphosphoryl lipid A (from Escherichia coli) to determine the impact of electrostatic interactions. Large numbers of trajectories were obtained and analyzed to obtain the distributions of surface residence times and the statistics of the spatial trajectories. Interestingly, the AMP species were sensitive to subtle differences in the charge of the lipid, with both peptides diffusing more slowly and residing longer on the diphosphoryl lipid A. Furthermore, the single-molecule dynamics indicated a qualitative difference between the behavior of AMPs on hybrid Lipid A bilayers and on those composed entirely of DOPE. Whereas AMPs interacting with a DOPE bilayer exhibited two-dimensional Brownian diffusion with a diffusion coefficient of ∼1.7 μm 2 /s, AMPs adsorbed to the lipid A surface exhibited much slower apparent diffusion (on the order of ∼0.1 μm 2 /s) and executed intermittent trajectories that alternated between two-dimensional Brownian diffusion and desorption-mediated three-dimensional flights. Overall, these findings suggested that bilayers with lipid A in the outer leaflet, as it is in bacterial outer membranes, are valuable model systems for the study of the initial stage of AMP-bacterium interactions. Furthermore, single-molecule dynamics was sensitive to subtle differences in electrostatic interactions between cationic AMPs and monovalent or divalent anionic lipid A moieties. Copyright © 2018 Biophysical Society. All rights reserved.

Effects of Coal Fly Ash Particulate Matter on the Antimicrobial Activity of Airway Surface Liquid

PubMed Central

Vargas Buonfiglio, Luis G.; Mudunkotuwa, Imali A.; Abou Alaiwa, Mahmoud H.; Vanegas Calderón, Oriana G.; Borcherding, Jennifer A.; Gerke, Alicia K.; Zabner, Joseph; Grassian, Vicki H.

2017-01-01

Background: Sustained exposure to ambient particulate matter (PM) is a global cause of mortality. Coal fly ash (CFA) is a byproduct of coal combustion and is a source of anthropogenic PM with worldwide health relevance. The airway epithelia are lined with fluid called airway surface liquid (ASL), which contains antimicrobial proteins and peptides (AMPs). Cationic AMPs bind negatively charged bacteria to exert their antimicrobial activity. PM arriving in the airways could potentially interact with AMPs in the ASL to affect their antimicrobial activity. Objectives: We hypothesized that PM can interact with ASL AMPs to impair their antimicrobial activity. Methods: We exposed pig and human airway explants, pig and human ASL, and the human cationic AMPs β-defensin-3, LL-37, and lysozyme to CFA or control. Thereafter, we assessed the antimicrobial activity of exposed airway samples using both bioluminescence and standard colony-forming unit assays. We investigated PM-AMP electrostatic interaction by attenuated total reflection Fourier-transform infrared spectroscopy and measuring the zeta potential. We also studied the adsorption of AMPs on PM. Results: We found increased bacterial survival in CFA-exposed airway explants, ASL, and AMPs. In addition, we report that PM with a negative surface charge can adsorb cationic AMPs and form negative particle–protein complexes. Conclusion: We propose that when CFA arrives at the airway, it rapidly adsorbs AMPs and creates negative complexes, thereby decreasing the functional amount of AMPs capable of killing pathogens. These results provide a novel translational insight into an early mechanism for how ambient PM increases the susceptibility of the airways to bacterial infection. https://doi.org/10.1289/EHP876 PMID:28696208

Purification and partial characterization of bacteriocin produced by Lactococcus lactis ssp. lactis LL171.

PubMed

Kumari, Archana; Akkoç, Nefise; Akçelik, Mustafa

2012-04-01

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.

Do antimicrobial peptides PR-39, PMAP-36 and PMAP-37 have any effect on bacterial growth and quality of liquid-stored boar semen?

PubMed

Bussalleu, Eva; Sancho, Sílvia; Briz, Maria D; Yeste, Marc; Bonet, Sergi

2017-02-01

The use of antimicrobial peptides (AMP) has become one of the most promising alternatives to the use of antibiotics (Abs) in semen extender's formulation to overcome the increasing bacterial resistance to antibiotics. However, AMP may impair boar sperm quality, so that their deleterious effects might be higher than their effectiveness against bacteria. Thus, the aim of this study was to determine whether three different AMP, the proline-arginine-rich antimicrobial peptide PR-39 (PR-39), and the porcine myeloid antimicrobial peptides 36 (PMAP-36) and 37 (PMAP-37) had any effect upon boar sperm quality and bacterial growth. For this purpose, three different concentrations of each peptide (1 μM, 10 μM and 20 μM for PR-39 and 0.5 μM, 1 μM and 3 μM for PMAP-36 and PMAP-37) were added to 2 mL of a pool of extended semen with BTS without Abs; two controls, one without AMPs and Abs, and the other with Abs only were used for each peptide (n = 3). Total (TMOT) and progressive (PMOT) sperm motility, sperm viability and bacterial concentration were assessed before the addition of each AMP or Abs and at 1, 3, 6, 8 and 10 days post-addition. For each AMP, results revealed a drop in the TMOT and PMOT in all treatments and controls. In regard to sperm viability, while PR-39 at 10 μM maintained it in values similar to those of the control with Abs and PMAP-36 kept also the sperm viability in a similar fashion to the treatment with Abs, PMAP-37 was more effective in keeping sperm viability than controls (P < 0.05). Whereas PR-39 at 20 μM and PMAP-37 at 3 μM were quite effective in controlling bacterial load, PMAP-36 did not avoid bacterial growth at any concentration tested. In conclusion, taking all results together, PMAP-37 seems to be a suitable candidate to replace Abs in extended semen, as it hardly impairs sperm viability and controls the bacterial load. Nevertheless, further studies are still required to improve its effectiveness. Copyright © 2016. Published by Elsevier Inc.

Identification and quantification of innate immune system mediators in human breast milk.

PubMed

Armogida, Sheila A; Yannaras, Niki M; Melton, Alton L; Srivastava, Maya D

2004-01-01

Breast-feeding decreases the risk of breast cancer in mothers and infection, allergy, and autoimmunity in infants. The presence of mediators of the innate immune system in human milk, including soluble defensins, cathelicidins, and toll-like receptors (TLRs), has not been researched thoroughly. The whey fractions of colostrum and transitional and mature milk (n = 40) from normal mothers (n = 18) and from mothers with autoimmune or allergic diseases (n = 22) were analyzed for defensins by competitive enzyme-linked immunosorbent assay, and expression of messenger RNA (mRNA) for defensins, TLRs, and cathelicidin-derived antimicrobial peptide (LL-37) by cells in breast milk was determined by semiquantitative reverse-transcription-polymerase chain reaction. In whey, human neutrophil-derived a-defensin 1 (HNP-1) and human beta-defensin 2 (HBD-2) were present in the highest concentrations (median, 33.0 and 31.3 microg/mL, respectively), human alpha-defensin 6 (HD-6) was present in moderate amounts (3.1 microg/mL), and HD-5 and HBD-1 were present in the lowest concentrations (2.4 and 1.7 microg/mL, respectively). There was great variability of defensin levels between subjects, but there was no relation to autoimmune or allergic diagnosis. HNP-1, HD-5, and HD-6 were present in significantly higher levels in colostrum than in mature milk. Regarding defensin mRNA expression in the breast milk cells, 95% of the samples (n = 41) were positive for HBD-1, 68% were positive for HD-5, 22% were positive for HBD-3, 15% were positive for HBD-2, 5% were positive for HBD-4, and 2% were positive for HD-6; 88% (14/16) were positive for HNP-1. Breast milk cells also expressed mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9, CD14, and LL-37. Human breast milk contains high concentrations of multiple defensin proteins and cells in breast milk express mRNA for these defensins, multiple TLRs, and LL-37. The innate immune system in breast milk is complex and likely provides protection for maternal breast tissue and the developing digestive tract of newborns.

Investigation on antimicrobial agents of the terrestrial Streptomyces sp. BCC71188.

PubMed

Supong, Khomsan; Sripreechasak, Paranee; Tanasupawat, Somboon; Danwisetkanjana, Kannawat; Rachtawee, Pranee; Pittayakhajonwut, Pattama

2017-01-01

The terrestrial actinomycete strain BCC71188 was identified as Streptomyces by its morphology (having spiral chain spore on the aerial mycelium), chemotaxonomy (containing LL-diaminopimelic acid in the cell wall), and 16S rRNA gene sequence analysis [showing high similarity values compared with Streptomyces samsunensis M1463 T (99.85 %) and Streptomyces malaysiensis NBRC 16446 T (99.40 %)]. The crude extract exhibited antimalarial against Plasmodium falciparum (IC 50 0.19 μg/ml), anti-TB against Mycobacterial tuberculosis (MIC 6.25 μg/ml), and antibacterial against Bacillus cereus (MIC 1.56 μg/ml) activities. Therefore, chemical investigation was conducted by employing bioassay-guided method and led to the isolation of 19 compounds including two cyclic peptides (1-2), five macrolides (3-7), new naphthoquinone (8), nahuoic acid C (9), geldanamycin derivatives (10-13), cyclooctatin (14), germicidins A (15) and C (16), actinoramide A (17), abierixin, and 29-O-methylabierixin. These isolated compounds were evaluated for antimicrobial activity, such as antimalarial, anti-TB, and antibacterial activities, and for cytotoxicity against both cancerous (MCF-7, KB, NCI-H187) and non-cancerous (Vero) cells. Compounds 1-7, 10-14 exhibited antimalarial (IC 50 0.22-7.14 μg/ml), and elaiophylin analogs (4-6) displayed anti-TB (MIC 0.78-12.00 μg/ml) and B. cereus (MIC 0.78-3.13 μg/ml) activities. Compounds 1, 2, 14, and abierixin displayed weak cytotoxicity, indicating a potential for antimicrobial agents.

The cathelicidin protein CRAMP is a potential atherosclerosis self-antigen in ApoE(-/-) mice

PubMed Central

Mihailovic, Peter M.; Lio, Wai Man; Yano, Juliana; Zhao, Xiaoning; Zhou, Jianchang; Chyu, Kuang-Yuh; Shah, Prediman K.; Cercek, Bojan

2017-01-01

Auto-immunity is believed to contribute to inflammation in atherosclerosis. The antimicrobial peptide LL-37, a fragment of the cathelicidin protein precursor hCAP18, was previously identified as an autoantigen in psoriasis. Given the reported link between psoriasis and coronary artery disease, the biological relevance of the autoantigen to atherosclerosis was tested in vitro using a truncated (t) form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE(-/-) mice. Stimulation with tCRAMP resulted in increased CD8+ T cells with Central Memory and Effector Memory phenotypes in ApoE(-/-) mice, differentially activated by feeding with normal chow or high fat diet. Immunization of ApoE(-/-) with different doses of the shortened peptide (Cramp) resulted in differential outcomes with a lower dose reducing atherosclerosis whereas a higher dose exacerbating the disease with increased neutrophil infiltration of the atherosclerotic plaques. Low dose Cramp immunization also resulted in increased splenic CD8+ T cell degranulation and reduced CD11b+CD11c+ conventional dendritic cells (cDCs), whereas high dose increased CD11b+CD11c+ cDCs. Our results identified CRAMP, the mouse homolog of hCAP-18, as a potential self-antigen involved in the immune response to atherosclerosis in the ApoE(-/-) mouse model. PMID:29091929

PepO, a CovRS-controlled endopeptidase, disrupts Streptococcus pyogenes quorum sensing.

PubMed

Wilkening, Reid V; Chang, Jennifer C; Federle, Michael J

2016-01-01

Group A Streptococcus (GAS, Streptococcus pyogenes) is a human-restricted pathogen with a capacity to both colonize asymptomatically and cause illnesses ranging from pharyngitis to necrotizing fasciitis. An understanding of how and when GAS switches between genetic programs governing these different lifestyles has remained an enduring mystery and likely requires carefully tuned environmental sensors to activate and silence genetic schemes when appropriate. Herein, we describe the relationship between the Control of Virulence (CovRS, CsrRS) two-component system and the Rgg2/3 quorum-sensing pathway. We demonstrate that responses of CovRS to the stress signals Mg(2+) and a fragment of the antimicrobial peptide LL-37 result in modulated activity of pheromone signaling of the Rgg2/3 pathway through a means of proteolysis of SHP peptide pheromones. This degradation is mediated by the cytoplasmic endopeptidase PepO, which is the first identified enzymatic silencer of an RRNPP-type quorum-sensing pathway. These results suggest that under conditions in which the virulence potential of GAS is elevated (i.e. enhanced virulence gene expression), cellular responses mediated by the Rgg2/3 pathway are abrogated and allow individuals to escape from group behavior. These results also indicate that Rgg2/3 signaling is instead functional during non-virulent GAS lifestyles. © 2015 John Wiley & Sons Ltd.

Characterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity towards Haemophilus parasuis.

PubMed

Teixeira, Mário Lettieri; Dalla Rosa, Andréia; Brandelli, Adriano

2013-05-01

Haemophilus parasuis is the pathogen that causes Glässer's disease, a major illness affecting young pigs. The aim of this work was to investigate the antagonistic activity of antimicrobial substances produced by Bacillus species against H. parasuis. Among the tested strains, only Bacillus subtilis ATCC 6633 inhibited H. parasuis growth. The antibacterial substance was purified by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-50 and ion-exchange chromatography on DEAE-cellulose. The purification was about 100-fold with a yield of 0.33 %. The purified substance was resistant up to 80 °C and pH ranging 3-7, but the substance lost its activity when it was treated with proteases. The peptide had a molecular mass of 1083 Da and its sequence was determined by MS as NRWCFAGDD, which showed no homology with other known antimicrobial peptides. The complete inhibition of H. parasuis growth was observed at 20 µg peptide ml(-1) after 20 min of exposure. The peptide obtained by chemical synthesis also showed antimicrobial activity on H. parasuis. The identification of antimicrobial substances that can be effective against H. parasuis is very relevant to combat this pathogen that causes important losses in swine production.

Physiological relevance of LL-37 induced bladder inflammation and mast cells.

PubMed

Oottamasathien, Siam; Jia, Wanjian; Roundy, Lindsi McCoard; Zhang, Jianxing; Wang, Li; Ye, Xiangyang; Hill, A Cameron; Savage, Justin; Lee, Wong Yong; Hannon, Ann Marie; Milner, Sylvia; Prestwich, Glenn D

2013-10-01

We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm(2). Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Physiological Relevance of LL-37 Induced Bladder Inflammation and Mast Cells

PubMed Central

Roundy, Lindsi McCoard; Zhang, Jianxing; Wang, Li; Ye, Xiangyang; Hill, A. Cameron; Savage, Justin; Lee, Wong Yong; Hannon, Ann Marie; Milner, Sylvia; Prestwich, Glenn D.

2014-01-01

Purpose We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells. Materials and Methods To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm2. Results Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration. Conclusions Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells. PMID:23313203

Antimicrobial lectin from Schinus terebinthifolius leaf.

PubMed

Gomes, F S; Procópio, T F; Napoleão, T H; Coelho, L C B B; Paiva, P M G

2013-03-01

Schinus terebinthifolius leaves are used for treating human diseases caused by micro-organisms. This work reports the isolation, characterization and antimicrobial activity of S. terebinthifolius leaf lectin (SteLL). The isolation procedure involved protein extraction with 0.15 mol l(-1) NaCl, filtration through activated charcoal and chromatography of the filtrate on a chitin column. SteLL is a 14-kDa glycopeptide with haemagglutinating activity that is inhibited by N-acetyl-glucosamine, not affected by ions (Ca(2+) and Mg(2+)) and stable upon heating (30-100 °C) as well as over the pH 5.0-8.0. The antimicrobial effect of SteLL was evaluated by determining the minimal inhibitory (MIC), bactericide (MBC) and fungicide (MFC) concentrations. Lectin was active against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis and Staphylococcus aureus. Highest bacteriostatic and bactericide effects were detected for Salm. enteritidis (MIC: 0.45 μg ml(-1)) and Staph. aureus (MBC: 7.18 μg ml(-1)), respectively. SteLL impaired the growth (MIC: 6.5 μg ml(-1)) and survival (MFC: 26 μg ml(-1)) of Candida albicans. SteLL, a chitin-binding lectin, purified in milligram quantities, showed antimicrobial activity against medically important bacteria and fungi. SteLL can be considered as a new biomaterial for potential antimicrobial applications. © 2012 The Society for Applied Microbiology.

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis.

PubMed

López-Solanilla, E; García-Olmedo, F; Rodríguez-Palenzuela, P

1998-06-01

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat alpha-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.

Does smoking affect gingival crevicular fluid LL-37 levels following non-surgical periodontal treatment in chronic periodontitis?

PubMed

Türkoğlu, Oya; Eren, Gülnihal; Emingil, Gülnur; Azarsız, Elif; Kutukculer, Necil; Atilla, Gül

2016-01-01

LL-37 contributes to maintaining the balance between health and disease. Smoking is a risk factor for periodontitis that impairs neutrophil functions. The aim of the present study was to comparatively evaluate gingival crevicular fluid (GCF) LL-37 levels in smoker and non-smoker chronic periodontitis (CP) patients and controls, as well as the effect of non-surgical periodontal treatment on GCF LL-37 levels. Thirty-one CP patients (16 smokers, 15 non-smokers) and thirty-one controls (16 smokers, 15 non-smokers) were included in the study. CP patients received non-surgical treatment. GCF LL-37 levels and periodontal parameters were assessed at baseline, 1 and 3 months after completion of non-surgical periodontal treatment. GCF LL-37 levels were analyzed by ELISA. No significant difference was observed in GCF LL-37 levels between smoker and non-smoker controls (p>0.05). Smoker CP group had significantly lower GCF LL-37 level than non-smoker CP group at baseline (p<0.05). GCF LL-37 levels significantly decreased in non-smoker CP group at first week, 1 and 3 months after completion of non-surgical periodontal treatment (p<0.05) although no significant decrease in GCF LL-37 levels was observed in smoker CP group (p>0.05). Periodontal parameters were correlated with GCF LL-37 levels in non-smoker CP group (p<0.05), but not in smoker CP group (p>0.05). GCF LL-37 levels do not seem to be affected from smoking in periodontal health. However, smoking might have a suppressive effect on GCF LL-37 levels in CP. Non-surgical treatment is effective in decreasing GCF LL-37 levels in non-smoker CP patients but not in smokers with CP. Copyright © 2015. Published by Elsevier Ltd.

Modulating the internalization of bacille Calmette-Guérin by cathelicidin in bladder cancer cells.

PubMed

Choi, Se Young; Kim, Soon-Ja; Chi, Byung Hoon; Kwon, Jong Kyou; Chang, In Ho

2015-04-01

To confirm the role of cathelicidin (LL-37) in the internalization of bacille Calmette-Guérin (BCG) into bladder cancer cells. Enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction analysis evaluated the changes in protein and messenger ribonucleic acid (RNA) expression with BCG incubation after LL-37 pretreatment in 5637 and T24 human bladder cancer cells. The internalization rate was evaluated by a double immunofluorescence assay, and confocal microscopy confirmed the function of LL-37 in BCG internalization. We also investigated the difference in internalization rates and cell viability between LL-37, anti-LL-37 antibody, and LL-37 plus anti-LL-37 antibody. The levels of LL-37 increased after BCG exposure in bladder cancer cells in dose- and time-dependent manners. Increasing LL-37 levels using recombinant LL-37 protein further dose dependently decreased BCG internalization in both cell lines. The internalization rates of BCG after LL-37 instillation were lower compared with the controls, and the internalization rate of BCG after anti-LL-37 antibody instillation was significantly higher compared with the controls in both cell lines (P <.05). Viability of LL-37 plus BCG group was higher compared with the BCG-alone group. The anti-LL-37 antibody plus BCG group had decreased cell viability compared with the BCG-alone group in both cell lines. Bladder cancer cells produce cathelicidin when infected with BCG and upregulate cathelicidin to defend against BCG by inhibiting its internalization. Blocking the action of cathelicidin may increase the internalization and effectiveness of BCG in reducing bladder cancer cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.

PubMed

Hwang, John H; Lyes, Matthew; Sladewski, Katherine; Enany, Shymaa; McEachern, Elisa; Mathew, Denzil P; Das, Soumita; Moshensky, Alexander; Bapat, Sagar; Pride, David T; Ongkeko, Weg M; Crotty Alexander, Laura E

2016-06-01

Electronic (e)-cigarette use is rapidly rising, with 20 % of Americans ages 25-44 now using these drug delivery devices. E-cigarette users expose their airways, cells of host defense, and colonizing bacteria to e-cigarette vapor (EV). Here, we report that exposure of human epithelial cells at the air-liquid interface to fresh EV (vaped from an e-cigarette device) resulted in dose-dependent cell death. After exposure to EV, cells of host defense-epithelial cells, alveolar macrophages, and neutrophils-had reduced antimicrobial activity against Staphylococcus aureus (SA). Mouse inhalation of EV for 1 h daily for 4 weeks led to alterations in inflammatory markers within the airways and elevation of an acute phase reactant in serum. Upon exposure to e-cigarette vapor extract (EVE), airway colonizer SA had increased biofilm formation, adherence and invasion of epithelial cells, resistance to human antimicrobial peptide LL-37, and up-regulation of virulence genes. EVE-exposed SA were more virulent in a mouse model of pneumonia. These data suggest that e-cigarettes may be toxic to airway cells, suppress host defenses, and promote inflammation over time, while also promoting virulence of colonizing bacteria. Acute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV.

Latent viral reactivation is associated with changes in plasma antimicrobial protein concentrations during long-duration spaceflight

NASA Astrophysics Data System (ADS)

Spielmann, G.; Laughlin, M. S.; Kunz, H.; Crucian, B. E.; Quiriarte, H. D.; Mehta, S. K.; Pierson, D. L.; Simpson, R. J.

2018-05-01

Long duration spaceflights are associated with profound dysregulation of the immune system and latent viral reactivations. However, little is known on the impact of long duration spaceflight on innate immunity which raises concerns on crewmembers' ability to fight infections during a mission. The aim of this study was to determine the effects of spaceflight on plasma antimicrobial proteins (AMPs) and how these changes impact latent herpesvirus reactivations. Plasma, saliva and urine samples were obtained from 23 crewmembers before, during and after a 6-month mission on the International Space Station (ISS). Plasma AMP concentrations were determined by ELISA, and saliva Epstein-Barr virus (EBV) and varicella zoster virus (VZV) and urine cytomegalovirus (CMV) DNA levels were quantified by Real-Time PCR. There was a non-significant increase in plasma HNP1-3 and LL-37 during the early and middle stages of the missions, which was significantly associated with changes in viral DNA during and after spaceflight. Plasma HNP1-3 and Lysozyme increased at the late mission stages in astronauts who had exhibited EBV and VZV reactivations during the early flight stages. Following return to Earth and during recovery, HNP1-3 and lysozyme concentrations were associated with EBV and VZV viral DNA levels, reducing the magnitude of viral reactivation. Reductions in plasma LL-37 upon return were associated with greater CMV reactivation. This study shows that biomarkers of innate immunity appeared to be partially restored after 6-months in space and suggests that following adaptation to the space environment, plasma HNP1-3 and lysozyme facilitate the control of EBV and VZV reactivation rate and magnitude in space and upon return on earth. However, the landing-associated decline in plasma LL-37 may enhance the rate of CMV reactivation in astronauts following spaceflight, potentially compromising crewmember health after landing.

Characterization of the branched antimicrobial peptide M6 by analyzing its mechanism of action and in vivo toxicity.

PubMed

Pini, Alessandro; Giuliani, Andrea; Falciani, Chiara; Fabbrini, Monica; Pileri, Silvia; Lelli, Barbara; Bracci, Luisa

2007-06-01

We analyzed functional activity of the antimicrobial peptide M6 in vitro and in vivo. The peptide was identified by our group by phage library selection, rational modification and synthesis in a tetrabranched form (Pini et al., Antimicrob. Agents Chemother. 2005; 49: 2665-72). We found that it binds lipopolysaccharide, causes perforation of cell membranes without destroying external cell morphology and strongly binds DNA. The latter feature suggests that it could inhibit metabolic pathways, blocking DNA replication and/or transcription. We also observed that M6 does not stimulate humoral immune response when repeatedly administered to animals. We also analyzed M6 toxicity when administered to animals by intraperitoneal or by intravenous injection, determining a preliminary LD50 (125 and 37.5 mg/kg, respectively), which suggested that M6 could be used in vivo. These features make the antimicrobial branched peptide M6 a promising candidate for the development of a new antibacterial drug. Copyright (c) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Real-World Efficacy of Azelaic Acid 15% Gel for the Reduction of Inflammatory Lesions of Rosacea.

PubMed

Wirth, P J; Henderson Berg, M H; Sadick, N

2017-11-01

Approximately 16 million Americans have rosacea, an inflammatory cutaneous disorder with central facial erythema, papules, pustules, telangiectasia, flushing, and swelling being among the more commonly recognized features. Overexpression of cathelicidin peptide LL-37 has been implicated in the pathophysiology of rosacea. Azelaic acid has been found to inhibit the pathologic expression of cathelicidin, as well as the hyperactive protease activity that cleaves cathelicidin into LL-37. Given these findings, a small prospective, open-label, interventional trial was undertaken to assess the effects of azelaic acid 15% gel on inflammatory lesions of papulopustular rosacea in a real-world setting. Use of azelaic acid was associated with a significant reduction in inflammatory lesions, which persisted beyond the active treatment phase. Overall, azelaic acid 15% gel is an appropriate initial topical therapy for the treatment of moderate facial rosacea.

Minocycline modulates NFκB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.

PubMed

Guerra, Alberto Daniel; Rose, Warren E; Hematti, Peiman; Kao, W John

2017-07-21

Mesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds. Modulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague-Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student's t test was used to determine p values for all assays with multiple or two conditions, respectively. Minocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH 2 -terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization. Minocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.

Role of C-terminal heptapeptide in pore-forming activity of antimicrobial agent, gaegurin 4.

PubMed

Kim, H J; Kim, S S; Lee, M H; Lee, B J; Ryu, P D

2004-10-01

Gaegurin 4 (GGN4) is an antimicrobial peptide of 37 amino acids isolated from the skin of a frog, Rana rugosa. GGN4 has a disulfide bond between the residues 31 and 37, which is highly conserved among the antimicrobial peptides isolated from skin of the genus, Rana. However, the role of this C-terminal heptapeptide motif is not well understood. In this work, we compared the membrane effects of the full-length GGN4 (C37) and GGN4 1-30 (C30), which is devoid of the C-terminal seven amino acids to elucidate the function of the C-terminal motif. C37 induced significantly larger membrane conductance (>10x) in the model lipid bilayers formed with acidic and neutral phospholipids and larger K+ efflux from gram-positive (>30x) and gram-negative bacteria. However, the pores induced by C37 and C30 were not different in their permeability to K+ over Cl- (permeability ratio of K+ to Cl- = 4.8-7.1). In addition, the pore-forming effect of C37 or C30 in acidic membranes was not different from that in neutral membranes. Furthermore, C37-induced K+ efflux was not significantly decreased by the reducing agent, dithiothreitol. The results indicate that C-terminal heptapeptide sequence plays an important role in maintaining the high pore-forming activity of GGN4, but does not participate in forming GGN4-induced pore structure. The disulfide bond in this region does not appear critical for such high ionophoric activity of GGN4.

Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque.

PubMed

Malcolm, J; Sherriff, A; Lappin, D F; Ramage, G; Conway, D I; Macpherson, L M D; Culshaw, S

2014-12-01

Secretion of antimicrobial proteins (AMPs) and salivary antibodies can modify biofilm formation at host body surfaces. In adolescents, associations have been reported between dental caries and salivary AMPs. AMPs demonstrate direct antimicrobial effects at high concentrations, and at lower more physiological concentrations they mediate changes in host cell defenses, which may alter the local environment and indirectly shape local biofilm formation. The expression of salivary AMPs in preschool children, at an age when the oral bacteria are known to change, has not been investigated. We sought to investigate salivary AMP expression in the context of previously well-documented changes in the oral cavities of this age group including salivary immunoglobulin A (IgA), oral bacteria and dental caries. Dental plaque and saliva were collected from 57 children aged 12-24 months at baseline, of whom 23 children were followed-up at 3 years of age. At each time, saliva was assessed for LL37, human neutrophil peptides 1-3, calprotectin, lactoferrin, salivary IgA, total plaque bacteria and Streptococcus mutans. Over time, concentrations of AMPs, S. mutans and bacteria-specific salivary IgA increased. Caries experience was also recorded when children were 3 years old. Concentrations of AMPs were highest in the saliva of 3-year-old children with the greatest burden of S. mutans. These data suggest that salivary AMPs are variable over time and between individuals, and are linked with bacterial colonization. At follow up, the majority of children remained caries free. Larger longitudinal studies are required to confirm whether salivary AMP levels are predictive of caries and whether their modulation offers therapeutic benefit. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of the antimicrobial peptide protegrine 1 on sperm viability and bacterial load of boar seminal doses.

PubMed

Sancho, S; Briz, M; Yeste, M; Bonet, S; Bussalleu, E

2017-10-01

The presence of bacteria adversely affects boar sperm quality of seminal doses intended for artificial insemination. Currently, the most common measure to prevent bacteriospermia is the addition of antibiotics in semen extenders; however, mounting evidence shows that microbial resistance exists. A promising alternative to replace antibiotics are antimicrobial peptides. In this study, the effects of the antimicrobial peptide protegrine 1 (PG1) on the sperm viability and bacterial load of boar seminal doses were evaluated. Three different concentrations of PG1 (2.5, 25 and 100 μg/ml) were tested over a storing period of 10 days at 17°C. Sperm viability was analysed by fluorescence microscopy (SYBR14/propidium iodide), and bacterial load was assessed by plating 100 μl of each sample in Luria-Bertani medium and incubated at 37°C for 72 hr under aerobic conditions. Protegrine 1 was effective in controlling the bacterial load in all the assessed concentrations (p < .05), reaching the lowest values at the highest concentrations of the antimicrobial peptide. Nevertheless, sperm viability was significantly (p < .05) reduced by all tested concentrations of this peptide, the most cytotoxic effects being observed at the highest PG1 concentrations. Despite these results, the use of PG1 as an alternative to antibiotics cannot be totally discarded, as further studies using the truncated form of this peptide are needed. © 2017 Blackwell Verlag GmbH.

Coloured cornea replacements with anti-infective properties: expanding the safe use of silver nanoparticles in regenerative medicine

NASA Astrophysics Data System (ADS)

Alarcon, E. I.; Vulesevic, B.; Argawal, A.; Ross, A.; Bejjani, P.; Podrebarac, J.; Ravichandran, R.; Phopase, J.; Suuronen, E. J.; Griffith, M.

2016-03-01

Despite the broad anti-microbial and anti-inflammatory properties of silver nanoparticles (AgNPs), their use in bioengineered corneal replacements or bandage contact lenses has been hindered due to their intense yellow coloration. In this communication, we report the development of a new strategy to pre-stabilize and incorporate AgNPs with different colours into collagen matrices for fabrication of corneal implants and lenses, and assessed their in vitro and in vivo activity.Despite the broad anti-microbial and anti-inflammatory properties of silver nanoparticles (AgNPs), their use in bioengineered corneal replacements or bandage contact lenses has been hindered due to their intense yellow coloration. In this communication, we report the development of a new strategy to pre-stabilize and incorporate AgNPs with different colours into collagen matrices for fabrication of corneal implants and lenses, and assessed their in vitro and in vivo activity. Electronic supplementary information (ESI) available: Collagen hydrogel, moulded as a cornea, prepared containing collagen protected AgNPs and representative images for collagen hydrogels, moulded as corneas, containing Blue AgNPs either unprotected or protected with LL37-SH; representative TEM images for Green-1 AgNPs prepared in this work; changes on surface plasmon band after synthesis for Green-2 AgNPs without LL37-SH; representative picture of the powder obtained for Green-1 AgNPs capped with LL37-SH after 72 h lyophilization, see main text; representative TEM images for Blue and Green-1 AgNPs prepared in this work; absorption spectra for the supernatants for collagen hydrogels containing Blue AgNPs; absorbance at 600 nm of PAO1 cultures prepared in 25% LB media incubated for 14 h at 37 °C in the presence of different concentrations of AgNPs, Green-1 or Blue, or silver nitrate; HECC cell density (cells per cm2) measured on gels with and without silver nitrate after 1 day of cell incubation; total silver concentration in tissue surrounding the implant area, see experimental, measured at 24 and 72 h post surgery; concentration of mouse IL-6 in homogenized skin tissue extracted from gel implant region after 24 h and 72 h. See DOI: 10.1039/c6nr01339b

Influence of Amphibian Antimicrobial Peptides and Short Lipopeptides on Bacterial Biofilms Formed on Contact Lenses

PubMed Central

Maciejewska, Magdalena; Bauer, Marta; Neubauer, Damian; Kamysz, Wojciech; Dawgul, Malgorzata

2016-01-01

The widespread use of contact lenses is associated with several complications, including ocular biofilm-related infections. They are very difficult to manage with standard antimicrobial therapies, because bacterial growth in a biofilm is associated with an increased antibiotic resistance. The principal aim of this study was to evaluate the efficacy of antimicrobial peptides (AMPs) in eradication of bacterial biofilms formed on commercially available contact lenses. AMPs were synthesized according to Fmoc/tBu chemistry using the solid-phase method. Minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of the compounds were determined. Anti-biofilm activity of the antimicrobial peptides determined at different temperatures (25 °C and 37 °C) were compared with the effectiveness of commercially available contact lens solutions. All of the tested compounds exhibited stronger anti-biofilm properties as compared to those of the tested lens solutions. The strongest activity of AMPs was noticed against Gram-positive strains at a temperature of 25 °C. Conclusions: The results of our experiments encourage us toward further studies on AMPs and their potential application in the prophylaxis of contact lens-related eye infections. PMID:28773992

Studies on tridecaptin B(1), a lipopeptide with activity against multidrug resistant Gram-negative bacteria.

PubMed

Cochrane, Stephen A; Lohans, Christopher T; van Belkum, Marco J; Bels, Manon A; Vederas, John C

2015-06-07

Previously other groups had reported that Paenibacillus polymyxa NRRL B-30507 produces SRCAM 37, a type IIA bacteriocin with antimicrobial activity against Campylobacter jejuni. Genome sequencing and isolation of antimicrobial compounds from this P. polymyxa strain show that the antimicrobial activity is due to polymyxins and tridecaptin B1. The complete structural assignment, synthesis, and antimicrobial profile of tridecaptin B1 is reported, as well as the putative gene cluster responsible for its biosynthesis. This peptide displays strong activity against multidrug resistant Gram-negative bacteria, a finding that is timely to the current problem of antibiotic resistance.

Ability of innate defence regulator peptides IDR-1002, IDR-HH2 and IDR-1018 to protect against Mycobacterium tuberculosis infections in animal models.

PubMed

Rivas-Santiago, Bruno; Castañeda-Delgado, Julio E; Rivas Santiago, Cesar E; Waldbrook, Matt; González-Curiel, Irma; León-Contreras, Juan C; Enciso-Moreno, Jose Antonio; del Villar, Victor; Mendez-Ramos, Jazmin; Hancock, Robert E W; Hernandez-Pando, Rogelio

2013-01-01

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15-30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB.

Antimicrobial peptides as natural bio-preservative to enhance the shelf-life of food.

PubMed

Rai, Mahendra; Pandit, Raksha; Gaikwad, Swapnil; Kövics, György

2016-09-01

Antimicrobial peptides (AMPs) are diverse group of natural proteins present in animals, plants, insects and bacteria. These peptides are responsible for defense of host from pathogenic organisms. Chemical, enzymatic and recombinant techniques are used for the synthesis of antimicrobial peptides. These peptides have been found to be an alternative to the chemical preservatives. Currently, nisin is the only antimicrobial peptide, which is widely utilized in the preservation of food. Antimicrobial peptides can be used alone or in combination with other antimicrobial, essential oils and polymeric nanoparticles to enhance the shelf-life of food. This review presents an overview on different types of antimicrobial peptides, purification techniques, mode of action and application in food preservation.

Emerging Roles for MAS-Related G Protein-Coupled Receptor-X2 in Host Defense Peptide, Opioid, and Neuropeptide-Mediated Inflammatory Reactions.

PubMed

Ali, Hydar

2017-01-01

Mast cells (MCs) are tissue-resident immune cells that contribute to host defense but are best known for their roles in allergic and inflammatory diseases. In humans, MCs are divided into two subtypes based on the protease content of their secretory granules. Thus, human lung MCs contain only tryptase and are known as MC T , whereas skin MCs contain both tryptase and chymase and are known as MC TC . Patients with severe asthma display elevated MCs in the lung, which undergo phenotypic change from MC T to MC TC . Although the human genome contains four Mas related G protein coupled receptor X (MRGPRX) genes, an important feature of MC TC is that they selectively express MRGPRX2. It is activated by antimicrobial host defense peptides such as human β-defensins and the cathelicidin LL-37 and likely contributes to host defense. MRGPRX2 is also a receptor for the neuropeptide substance P, major basic protein, eosinophil peroxidase, opioids, and many FDA-approved cationic drugs. Increased expression of MRGPRX2 or enhanced downstream signaling likely contributes to chronic inflammatory diseases such as rosacea, atopic dermatitis, chronic urticaria, and severe asthma. In this chapter, I will discuss the expression profile and function of MRGPRX1-4 and review the emerging roles of MRGPRX2 on host defense, chronic inflammatory diseases, and drug-induced pseudoallergic reactions. I will also examine the novel aspects of MRGPRX2 signaling in MCs as it related to degranulation and review the mechanisms of its regulation. © 2017 Elsevier Inc. All rights reserved.

Lactose in Human Breast Milk an Inducer of Innate Immunity with Implications for a Role in Intestinal Homeostasis

PubMed Central

Printz, Gordana; Yoshio, Hiroyuki; Alvelius, Gunvor; Lagercrantz, Hugo; Strömberg, Roger; Jörnvall, Hans; Gudmundsson, Gudmundur H.; Agerberth, Birgitta

2013-01-01

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant. PMID:23326523

Host-pathogen interplay of Haemophilus ducreyi.

PubMed

Janowicz, Diane M; Li, Wei; Bauer, Margaret E

2010-02-01

Haemophilus ducreyi, the causative agent of the sexually transmitted infection chancroid, is primarily a pathogen of human skin. During infection, H. ducreyi thrives extracellularly in a milieu of professional phagocytes and other antibacterial components of the innate and adaptive immune responses. This review summarizes our understanding of the interplay between this pathogen and its host that leads to development and persistence of disease. H. ducreyi expresses key virulence mechanisms to resist host defenses. The secreted LspA proteins are tyrosine-phosphorylated by host kinases, which may contribute to their antiphagocytic effector function. The serum resistance and adherence functions of DsrA map to separate domains of this multifunctional virulence factor. An influx transporter protects H. ducreyi from killing by the antimicrobial peptide LL37. Regulatory genes have been identified that may coordinate virulence factor expression during disease. Dendritic cells and natural killer cells respond to H. ducreyi and may be involved in determining the differential outcomes of infection observed in humans. A human model of H. ducreyi infection has provided insights into virulence mechanisms that allow this human-specific pathogen to survive immune pressures. Components of the human innate immune system may also determine the ultimate fate of H. ducreyi infection by driving either clearance of the organism or an ineffective response that allows disease progression.

Fatty acid conjugation enhances the activities of antimicrobial peptides.

PubMed

Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

2013-04-01

Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

Antimicrobial Lactoferrin Peptides: The Hidden Players in the Protective Function of a Multifunctional Protein

PubMed Central

Sinha, Mau; Kaushik, Sanket; Kaur, Punit; Singh, Tej P.

2013-01-01

Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin. PMID:23554820

Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides.

PubMed

Lázár, Viktória; Martins, Ana; Spohn, Réka; Daruka, Lejla; Grézal, Gábor; Fekete, Gergely; Számel, Mónika; Jangir, Pramod K; Kintses, Bálint; Csörgő, Bálint; Nyerges, Ákos; Györkei, Ádám; Kincses, András; Dér, András; Walter, Fruzsina R; Deli, Mária A; Urbán, Edit; Hegedűs, Zsófia; Olajos, Gábor; Méhi, Orsolya; Bálint, Balázs; Nagy, István; Martinek, Tamás A; Papp, Balázs; Pál, Csaba

2018-06-01

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide-antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.

Francisella philomiragia Infection and Lethality in Mammalian Tissue Culture Cell Models, Galleria mellonella, and BALB/c Mice

PubMed Central

Propst, Crystal N.; Pylypko, Stephanie L.; Blower, Ryan J.; Ahmad, Saira; Mansoor, Mohammad; van Hoek, Monique L.

2016-01-01

Francisella (F.) philomiragia is a Gram-negative bacterium with a preference for brackish environments that has been implicated in causing bacterial infections in near-drowning victims. The purpose of this study was to characterize the ability of F. philomiragia to infect cultured mammalian cells, a commonly used invertebrate model, and, finally, to characterize the ability of F. philomiragia to infect BALB/c mice via the pulmonary (intranasal) route of infection. This study shows that F. philomiragia infects J774A.1 murine macrophage cells, HepG2 cells and A549 human Type II alveolar epithelial cells. However, replication rates vary depending on strain at 24 h. F. philomiragia infection after 24 h was found to be cytotoxic in human U937 macrophage-like cells and J774A.1 cells. This is in contrast to the findings that F. philomiragia was non-cytotoxic to human hepatocellular carcinoma cells, HepG2 cells and A549 cells. Differential cytotoxicity is a point for further study. Here, it was demonstrated that F. philomiragia grown in host-adapted conditions (BHI, pH 6.8) is sensitive to levofloxacin but shows increased resistance to the human cathelicidin LL-37 and murine cathelicidin mCRAMP when compared to related the Francisella species, F. tularensis subsp. novicida and F. tularensis subsp. LVS. Previous findings that LL-37 is strongly upregulated in A549 cells following F. tularensis subsp. novicida infection suggest that the level of antimicrobial peptide expression is not sufficient in cells to eradicate the intracellular bacteria. Finally, this study demonstrates that F. philomiragia is lethal in two in vivo models; Galleria mellonella via hemocoel injection, with a LD50 of 1.8 × 103, and BALB/c mice by intranasal infection, with a LD50 of 3.45 × 103. In conclusion, F. philomiragia may be a useful model organism to study the genus Francisella, particularly for those researchers with interest in studying microbial ecology or environmental strains of Francisella. Additionally, the Biosafety level 2 status of F. philomiragia makes it an attractive model for virulence and pathogenesis studies. PMID:27252681

Ability of Innate Defence Regulator Peptides IDR-1002, IDR-HH2 and IDR-1018 to Protect against Mycobacterium tuberculosis Infections in Animal Models

PubMed Central

Rivas-Santiago, Bruno; Castañeda-Delgado, Julio E.; Rivas Santiago, Cesar E.; Waldbrook, Matt; González-Curiel, Irma; León–Contreras, Juan C.; Enciso-Moreno, Jose Antonio; del Villar, Victor; Mendez-Ramos, Jazmin; Hancock, Robert E. W.; Hernandez-Pando, Rogelio

2013-01-01

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15–30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB. PMID:23555622

A simple and low-cost platform technology for producing pexiganan antimicrobial peptide in E. coli.

PubMed

Zhao, Chun-Xia; Dwyer, Mirjana Dimitrijev; Yu, Alice Lei; Wu, Yang; Fang, Sheng; Middelberg, Anton P J

2015-05-01

Antimicrobial peptides, as a new class of antibiotics, have generated tremendous interest as potential alternatives to classical antibiotics. However, the large-scale production of antimicrobial peptides remains a significant challenge. This paper reports a simple and low-cost chromatography-free platform technology for producing antimicrobial peptides in Escherichia coli (E. coli). A fusion protein comprising a variant of the helical biosurfactant protein DAMP4 and the known antimicrobial peptide pexiganan is designed by joining the two polypeptides, at the DNA level, via an acid-sensitive cleavage site. The resulting DAMP4(var)-pexiganan fusion protein expresses at high level and solubility in recombinant E. coli, and a simple heat-purification method was applied to disrupt cells and deliver high-purity DAMP4(var)-pexiganan protein. Simple acid cleavage successfully separated the DAMP4 variant protein and the antimicrobial peptide. Antimicrobial activity tests confirmed that the bio-produced antimicrobial peptide has the same antimicrobial activity as the equivalent product made by conventional chemical peptide synthesis. This simple and low-cost platform technology can be easily adapted to produce other valuable peptide products, and opens a new manufacturing approach for producing antimicrobial peptides at large scale using the tools and approaches of biochemical engineering. © 2014 Wiley Periodicals, Inc.

Antimicrobial lipids: novel innate defense molecules are elevated in sinus secretions of patients with chronic rhinosinusitis.

PubMed

Lee, Jivianne T; Jansen, Mike; Yilma, Abebayehu N; Nguyen, Angels; Desharnais, Robert; Porter, Edith

2010-01-01

Airway secretions possess intrinsic antimicrobial properties that contribute to the innate host defense of the respiratory tract. These microbicidal capabilities have largely been attributed to the presence of antibacterial polypeptides. However, recent investigation has indicated that host-derived lipids including cholesteryl esters also exhibit antimicrobial properties. The purpose of this study was to determine whether sinus secretions contain such antimicrobial lipids and to compare the lipid composition in patients with and without chronic rhinosinusitis (CRS). Maxillary sinus fluid was obtained via antral lavage from subjects with (seven patients) and without (nine patients) a history of CRS. After specimen collection, total lipid was extracted according to Bligh and Dyer (Bligh EG and Dyer WJ, A rapid method of total lipid extraction and purification, Can J Biochem Physiol 37:911-918, 1959) and lipid profiles were obtained by reverse phase high-performance liquid chromatography on an amide-embedded C18 column. In addition, the neutrophil-specific antimicrobial peptides human neutrophil peptides 1-3 (HNP1-3) were quantified by Western immunoblotting. Lipids, including cholesteryl esters, were identified in the maxillary sinus secretions of patients with and without CRS. However, levels of lipid composition differed between the two groups with CRS patients exhibiting greater amounts of all classes of lipids, reaching over 10-fold higher concentration when compared with non-CRS patients. This increase was independent of HNP1-3 content. Sinus secretions of patients with CRS appear to show elevated levels of antimicrobial lipids compared with controls independent from neutrophil influx. This up-regulation suggests that host-derived lipids act as mediators of mucosal immunity in CRS. Further study is necessary to determine if such antimicrobial lipids function alone or synergistically with antibacterial peptides in conferring such inherent microbicidal properties.

Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria.

PubMed

Leptihn, Sebastian; Har, Jia Yi; Chen, Jianzhu; Ho, Bow; Wohland, Thorsten; Ding, Jeak Ling

2009-05-11

Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial. Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level. Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process: 1) Binding, mediated mainly by charged residues in the peptide; 2) Peptide association, as peptide concentration increases evidenced by a change in diffusive behavior; 3) Membrane disruption, during which lipopolysaccharide is not released; and 4) Lysis, by leakage of cytosolic content through large membrane defects.

Perspectives and Peptides of the Next Generation

NASA Astrophysics Data System (ADS)

Brogden, Kim A.

Shortly after their discovery, antimicrobial peptides from prokaryotes and eukaryotes were recognized as the next potential generation of pharmaceuticals to treat antibiotic-resistant bacterial infections and septic shock, to preserve food, or to sanitize surfaces. Initial research focused on identifying the spectrum of antimicrobial agents, determining the range of antimicrobial activities against bacterial, fungal, and viral pathogens, and assessing the antimicrobial activity of synthetic peptides versus their natural counterparts. Subsequent research then focused on the mechanisms of antimicrobial peptide activity in model membrane systems not only to identify the mechanisms of antimicrobial peptide activity in microorganisms but also to discern differences in cytotoxicity for prokaryotic and eukaryotic cells. Recent, contemporary work now focuses on current and future efforts to construct hybrid peptides, peptide congeners, stabilized peptides, peptide conjugates, and immobilized peptides for unique and specific applications to control the growth of microorganisms in vitro and in vivo.

Antimicrobial peptides and proteins of the horse - insights into a well-armed organism

PubMed Central

2011-01-01

Antimicrobial peptides play a pivotal role as key effectors of the innate immune system in plants and animals and act as endogenous antibiotics. The molecules exhibit an antimicrobial activity against bacteria, viruses, and eukaryotic pathogens with different specificities and potencies depending on the structure and amino-acid composition of the peptides. Several antimicrobial peptides were comprehensively investigated in the last three decades and some molecules with remarkable antimicrobial properties have reached the third phase of clinical studies. Next to the peptides themselves, numerous organisms were examined and analyzed regarding their repertoire of antimicrobial peptides revealing a huge number of candidates with potencies and properties for future medical applications. One of these organisms is the horse, which possesses numerous peptides that are interesting candidates for therapeutical applications in veterinary medicine. Here we summarize investigations and knowledge on equine antimicrobial peptides, point to interesting candidates, and discuss prospects for therapeutical applications. PMID:21888650

Why Does the Healthy Cornea Resist Pseudomonas aeruginosa Infection?

PubMed Central

Evans, David J.; Fleiszig, Suzanne M. J.

2013-01-01

Purpose To provide our perspective on why the cornea is resistant to infection based on our research results with Pseudomonas aeruginosa. Perspective We focus on our current understanding of the interplay between bacteria, tear fluid and the corneal epithelium that determine health as the usual outcome, and propose a theoretical model for how contact lens wear might change those interactions to enable susceptibility to P. aeruginosa infection. Methods Use of “null-infection” in vivo models, cultured human corneal epithelial cells, contact lens-wearing animal models, and bacterial genetics help to elucidate mechanisms by which P. aeruginosa survive at the ocular surface, adheres, and traverses multilayered corneal epithelia. These models also help elucidate the molecular mechanisms of corneal epithelial innate defense. Results and Discussion Tear fluid and the corneal epithelium combine to make a formidable defense against P. aeruginosa infection of the cornea. Part of that defense involves the expression of antimicrobials such as β-defensins, the cathelicidin LL-37, cytokeratin-derived antimicrobial peptides, and RNase7. Immunomodulators such as SP-D and ST2 also contribute. Innate defenses of the cornea depend in part on MyD88, a key adaptor protein of TLR and IL-1R signaling, but the basal lamina represents the final barrier to bacterial penetration. Overcoming these defenses involves P. aeruginosa adaptation, expression of the type three secretion system, proteases, and P. aeruginosa biofilm formation on contact lenses. Conclusion After more than two decades of research focused on understanding how contact lens wear predisposes to P. aeruginosa infection, our working hypothesis places blame for microbial keratitis on bacterial adaptation to ocular surface defenses, combined with changes to the biochemistry of the corneal surface caused by trapping bacteria and tear fluid against the cornea under the lens. PMID:23601656

Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.

PubMed

Pestrak, Matthew J; Chaney, Sarah B; Eggleston, Heather C; Dellos-Nolan, Sheri; Dixit, Sriteja; Mathew-Steiner, Shomita S; Roy, Sashwati; Parsek, Matthew R; Sen, Chandan K; Wozniak, Daniel J

2018-02-01

Pseudomonas aeruginosa causes devastating infections in immunocompromised individuals. Once established, P. aeruginosa infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of P. aeruginosa, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1ΔwspF, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in P. aeruginosa-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H2O2 and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.

Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.

PubMed

Mallbris, Lotus; Carlén, Lina; Wei, Tianling; Heilborn, Johan; Nilsson, Margareta Frohm; Granath, Fredrik; Ståhle, Mona

2010-05-01

Reduced production of antimicrobial peptides was proposed to contribute to susceptibility for skin infections in atopic dermatitis (AD). Focusing on the human cathelicidin protein, hCAP18, the aim of the present study was to explore whether reduced hCAP18 expression is a constitutive trait in AD and if established inducers affect the expression of hCAP18 in the skin of AD. First, we compared levels of hCAP18 mRNA between lesional skin in AD and psoriasis and verified significantly lower expression of hCAP18 mRNA in AD. In non-lesional skin, however, there was no difference between AD, psoriasis and healthy, indicating that there is no constitutive defect in the production of hCAP18 in AD patients. In healthy skin, hCAP18 was reported to be rapidly induced following wounding and here we verified this pattern in healthy controls and in psoriasis. In AD lesions, however, the expression of hCAP18 mRNA was markedly suppressed following wounding. Obviously, the inflammation in AD lesions neutralizes the expected induction of hCAP18 and even induces suppression. Notably, the mechanism to upregulate hCAP18 following vitamin D treatment was functional in lesional as well as in non-lesional AD indicating that the CAMP gene is normally regulated in this respect. In addition, cultured primary keratinocytes from non-lesional skin of psoriasis, AD and healthy skin, upregulated hCAP18mRNA following treatment with vitamin D. Itching is a hallmark of AD and scratching inevitably injures the skin. Failure to upregulate hCAP18 in eczema following injury is likely to affect antimicrobial protection and tissue repair in AD.

Antimicrobial Peptides in Reptiles

PubMed Central

van Hoek, Monique L.

2014-01-01

Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

PubMed

Kraszewska, Joanna; Beckett, Michael C; James, Tharappel C; Bond, Ursula

2016-07-15

Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

PubMed

Iwakoshi-Ukena, Eiko; Ukena, Kazuyoshi; Okimoto, Aiko; Soga, Miyuki; Okada, Genya; Sano, Naomi; Fujii, Tamotsu; Sugawara, Yoshiaki; Sumida, Masayuki

2011-04-01

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus. Copyright © 2010 Elsevier Inc. All rights reserved.

Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations.

PubMed

Heljo, P; Ross, A; Zarraga, I E; Pappenberger, A; Mahler, H-C

2015-10-01

Antimicrobial preservatives are known to interact with proteins and potentially affect their stability in aqueous solutions. In this systematic study, the interactions of a model peptide with three commonly used preservatives, benzyl alcohol, phenol and m-cresol, were evaluated. The impact on peptide oligomerization was studied using GC-MALS, SEC-MALS and DLS, antimicrobial efficiency of different formulations were studied using the Ph. Eur. antimicrobial efficacy test, and the molecular adsorption of preservative molecules on reversible peptide oligomers was monitored using NMR. The hydrodynamic radius and molar mass of the peptide oligomers was shown to clearly increase in the presence of m-cresol but less significantly with phenol and benzyl alcohol. The increase in size was most likely caused by peptide self-interactions becoming more attractive, leading to reversible oligomerization. On the other hand, increasing the concentration of peptide in multi-dose formulations led to reduced molecular mobility and decreased antimicrobial efficacy of all preservatives. Peptide-preservative interactions not only affect peptide self-interactions, but also antimicrobial efficiency of the preservatives and are thus of significant relevance. Adsorption of preservatives on oligomeric states of peptides is proposed as a mechanism to explain this reduced antimicrobial efficacy.

Nanostructures as promising tools for delivery of antimicrobial peptides.

PubMed

Brandelli, A

2012-07-01

Antimicrobial peptides have been extensively investigated for their potential applications as therapeutics and food biopreservatives. The antimicrobial activity may be impaired by the susceptibility for proteolytic degradation and undesirable interactions of the antimicrobial peptide in the biological environment. Development of nanostructures for entrapment and delivery of antimicrobial peptides may represent an alternative to the direct application of these substances. Lipid nanovesicles have been developed for encapsulation of antimicrobial peptides. Phosphatidylcholine is often employed in liposome manufacture, which is mostly achieved by the thin-film hydration method. Nanofibers may allow different physical modes of drug loading, including direct adsorption on the nanofiber surface or the assembly of drug-loaded nanoparticles. Self-assembled peptides reveal attractive features as nanostructures for applications in drug delivery and promising as antimicrobial agent for treatment of brain infections. Magnetic nanoparticles and nanotubules are also potential structures for entrapment of antimicrobial peptides. Nanoparticles can be also chemically modified with specific cell surface ligands to enhance cell adhesion and site specific delivery. This article reviews the most important nanostructures as promising tools for peptide delivery systems.

New milk protein-derived peptides with potential antimicrobial activity: an approach based on bioinformatic studies.

PubMed

Dziuba, Bartłomiej; Dziuba, Marta

2014-08-20

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

PubMed Central

Dziuba, Bartłomiej; Dziuba, Marta

2014-01-01

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins. PMID:25141106

Influence of Dimerization of Lipopeptide Laur-Orn-Orn-Cys-NH2 and an N-terminal Peptide of Human Lactoferricin on Biological Activity.

PubMed

Kamysz, Elżbieta; Sikorska, Emilia; Dawgul, Małgorzata; Tyszkowski, Rafał; Kamysz, Wojciech

Lactoferrin (LF) is a naturally occurring antimicrobial peptide that is cleaved by pepsin to lactoferricin (LFcin). LFcin has an enhanced antimicrobial activity as compared to that of LF. Recently several hetero- and homodimeric antimicrobial peptides stabilized by a single disulfide bond linking linear polypeptide chains have been discovered. We have demonstrated that the S-S bond heterodimerization of lipopeptide Laur-Orn-Orn-Cys-NH 2 (peptide III) and the synthetic N -terminal peptide of human lactoferricin (peptide I) yields a dimer (peptide V), which is almost as microbiologically active as the more active monomer and at the same time it is much less toxic. Furthermore, it has been found that the S-S bond homodimerization of both peptide I and peptide III did not affect antimicrobial and haemolytic activity of the compounds. The homo- and heterodimerization of peptides I and III resulted in either reduction or loss of antifungal activity. This work suggests that heterodimerization of antimicrobial lipopeptides via intermolecular disulfide bond might be a powerful modification deserving consideration in the design of antimicrobial peptides.

Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

PubMed Central

Haynie, S L; Crum, G A; Doele, B A

1995-01-01

A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and Secreted Aspartyl Proteinase (SAPS) 2, 4, 5 and 6 Expressions

DTIC Science & Technology

2013-04-01

1 AWARD NUMBER: W81XWH-12-2-0025 TITLE: Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition...drugs using various synthetic and naturally occurring antimicrobial molecules. Natural antimicrobial peptides , such as defensins produced by...These antimicrobial peptides generally exhibit selective toxicity for microorganisms and show fewer propensities to induce microbial resistance

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

PubMed

Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

2013-01-01

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Virtual screening of a milk peptide database for the identification of food-derived antimicrobial peptides.

PubMed

Liu, Yufang; Eichler, Jutta; Pischetsrieder, Monika

2015-11-01

Milk provides a wide range of bioactive substances, such as antimicrobial peptides and proteins. Our study aimed to identify novel antimicrobial peptides naturally present in milk. The components of an endogenous bovine milk peptide database were virtually screened for charge, amphipathy, and predicted secondary structure. Thus, 23 of 248 screened peptides were identified as candidates for antimicrobial effects. After commercial synthesis, their antimicrobial activities were determined against Escherichia coli NEB5α, E. coli ATCC25922, and Bacillus subtilis ATCC6051. In the tested concentration range (<2 mM), bacteriostatic activity of 14 peptides was detected including nine peptides inhibiting both Gram-positive and Gram-negative bacteria. The most effective fragment was TKLTEEEKNRLNFLKKISQRYQKFΑLPQYLK corresponding to αS2 -casein151-181 , with minimum inhibitory concentration (MIC) of 4.0 μM against B. subtilis ATCC6051, and minimum inhibitory concentrations of 16.2 μM against both E. coli strains. Circular dichroism spectroscopy revealed conformational changes of most active peptides in a membrane-mimic environment, transitioning from an unordered to α-helical structure. Screening of food peptide databases by prediction tools is an efficient method to identify novel antimicrobial food-derived peptides. Milk-derived antimicrobial peptides may have potential use as functional food ingredients and help to understand the molecular mechanisms of anti-infective milk effects. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli

PubMed Central

Lewis, Steven B.; Prior, Alison; Ellis, Samuel J.; Cook, Vivienne; Chan, Simon S. M.; Gelson, William; Schüller, Stephanie

2016-01-01

Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8. PMID:27446815

Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and Secreted Aspartyl Proteinase (SAPS) 2, 4, 5 and 6 Expressions

DTIC Science & Technology

2015-04-01

AWARD NUMBER: W81XWH-12-2-0025 TITLE: Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and...REPORT TYPE Annual 3. DATES COVERED 1 Apr 2014 - 31 Mar 2015 4. TITLE AND SUBTITLE Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue...of new antifungal drugs using various synthetic and naturally occurring antimicrobial molecules. Natural antimicrobial peptides , such as defensins

The Potential of Antimicrobial Peptides as Biocides

PubMed Central

Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

2011-01-01

Antimicrobial peptides constitute a diverse class of naturally occurring antimicrobial molecules which have activity against a wide range of pathogenic microorganisms. Antimicrobial peptides are exciting leads in the development of novel biocidal agents at a time when classical antibiotics are under intense pressure from emerging resistance, and the global industry in antibiotic research and development stagnates. This review will examine the potential of antimicrobial peptides, both natural and synthetic, as novel biocidal agents in the battle against multi-drug resistant pathogen infections. PMID:22072905

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry.

PubMed

Bishop, Barney M; Juba, Melanie L; Russo, Paul S; Devine, Megan; Barksdale, Stephanie M; Scott, Shaylyn; Settlage, Robert; Michalak, Pawel; Gupta, Kajal; Vliet, Kent; Schnur, Joel M; van Hoek, Monique L

2017-04-07

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins.

PubMed

D'Este, Francesca; Benincasa, Monica; Cannone, Giuseppe; Furlan, Michela; Scarsini, Michele; Volpatti, Donatella; Gennaro, Renato; Tossi, Alessandro; Skerlavaj, Barbara; Scocchi, Marco

2016-12-01

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant β-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serum Stabilities of Short Tryptophan- and Arginine-Rich Antimicrobial Peptide Analogs

PubMed Central

Nguyen, Leonard T.; Chau, Johnny K.; Perry, Nicole A.; de Boer, Leonie; Zaat, Sebastian A. J.; Vogel, Hans J.

2010-01-01

Background Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the “antimicrobial centre” of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library. Methodology/Principal Findings HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum. Conclusions/Significance Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications. PMID:20844765

Hydrocarbon-stapled lipopeptides exhibit selective antimicrobial activity.

PubMed

Jenner, Zachary B; Crittenden, Christopher M; Gonzalez, Martín; Brodbelt, Jennifer S; Bruns, Kerry A

2017-05-01

Antimicrobial peptides (AMPs) occur widely in nature and have been studied for their therapeutic potential. AMPs are of interest due to the large number of possible chemical structural combinations using natural and unnatural amino acids, with varying effects on their biological activities. Using physicochemical properties from known naturally occurring amphipathic cationic AMPs, several hydrocarbon-stapled lipopeptides (HSLPs) were designed, synthesized, and tested for antimicrobial properties. Peptides were chemically modified by N-terminal acylation, C-terminal amidation, and some were hydrocarbon stapled by intramolecular olefin metathesis. The effects of peptide length, amphipathic character, and stapling on antimicrobial activity were tested against Escherichia coli, three species of Gram-positive bacteria (Staphylococcus aureus, Bacillus megaterium, and Enterococcus faecalis), and two strains of Candida albicans. Peptides were shown to disrupt liposomes of different phospholipid composition, as measured by leakage of a fluorescent compound from vesicles. Peptides with (S)-2-(4'-pentenyl)-alanine substituted for l-alanine in a reference peptide showed a marked increase in antimicrobial activity, hemolysis, and membrane disruption. Stapled peptides exhibited slightly higher antimicrobial potency; those with greatest hydrophobic character showed the greatest hemolysis and liposome leakage, but lower antimicrobial activity. The results support a model of HSLPs as membrane-disruptive AMPs with potent antimicrobial activity and relatively low hemolytic potential at biologically active peptide concentrations. © 2017 Wiley Periodicals, Inc.

Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

PubMed

Shin, Areum; Lee, Eunjung; Jeon, Dasom; Park, Young-Guen; Bang, Jeong Kyu; Park, Yong-Sun; Shin, Song Yub; Kim, Yangmee

2015-06-30

Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

LL-37 Recruits Immunosuppressive Regulatory T Cells to Ovarian Tumors

DTIC Science & Technology

2009-11-01

receptor. Western blot analysis of MSC lysates showed that ERK-1 and -2 are robustly phosphorylated beginning 10 minutes after LL-37 treatment and...Carretero, Escamez et al. 2008; von Haussen, Koczulla et al. 2008). Western blot analysis of LL-37-treated SK-OV-3 cell lysates showed the robust...mesenchymal stem cells in the treatment of gliomas ." Cancer Res 65(8): 3307-18. Studeny, M., F. C. Marini, et al. (2004). "Mesenchymal stem cells: potential

[BIOLOGICAL ACTIVITY OF ANTIMICROBIAL PEPTIDES FROM CHICKENS THROMBOCYTES].

PubMed

Sycheva, M V; Vasilchenko, A S; Rogozhin, E A; Pashkova, T M; Popova, L P; Kartashova, O L

2016-01-01

Isolation and study of biological activity of antimicrobial peptides from chickens thrombocytes. Peptides from chickens thrombocytes, obtained by reverse-phase high-performance liquid chromatography method with stepped and linear gradients of concentration increase of the organic solvent were used in the study. Their antimicrobial activity was determined by microtitration method in broth; mechanism of biological effect--by using fluorescent spectroscopy method with DNA-tropic dyes. Individual fractions of peptides were isolated from chickens thrombocytes, that possess antimicrobial activity against Staphylococcus aureus P209 and Escherichia coli K12. A disruption of integrity of barrier structures of microorganisms under the effect of thrombocyte antimicrobial peptides and predominance of cells with damaged membrane in the population of E. coli was established. The data obtained on antimicrobial activity and mechanism of bactericidal effect of the peptide fractions from chickens thrombocytes isolated for the first time expand the understanding of functional properties of chickens thrombocytes and open a perspective for their further study with the aim of use as antimicrobial means.

Dynamical and phase behavior of a phospholipid membrane altered by an antimicrobial peptide at low concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mamontov, Eugene; Tyagi, M.; Qian, Shuo

Here we discuss that the mechanism of action of antimicrobial peptides is traditionally attributed to the formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert a similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature nomore » longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides.« less

Antimicrobial activity of antihypertensive food-derived peptides and selected alanine analogues.

PubMed

McClean, Stephen; Beggs, Louise B; Welch, Robert W

2014-03-01

This study evaluated four food-derived peptides with known antihypertensive activities for antimicrobial activity against pathogenic microorganisms, and assessed structure-function relationships using alanine analogues. The peptides (EVSLNSGYY, barley; PGTAVFK, soybean; TTMPLW, α-casein; VHLPP, α-zein) and the six alanine substitution peptides of PGTAVFK were synthesised, characterised and evaluated for antimicrobial activity using the bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus and the yeast, Candida albicans. The peptides TTMPLW and PGTAVFK inhibited growth of all four microorganisms tested, with activities of a similar order of magnitude to ampicillin and ethanol controls. EVSLNSGYY inhibited the growth of the bacteria, but VHLPP showed no antimicrobial activity. The alanine analogue, PGAAVFK showed the highest overall antimicrobial activity and PGTAVFA showed no activity; overall, the activities of the analogues were consistent with their structures. Some peptides with antihypertensive activity also show antimicrobial activity, suggesting that food-derived peptides may exert beneficial effects via a number of mechanisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dynamical and phase behavior of a phospholipid membrane altered by an antimicrobial peptide at low concentration

DOE PAGES

Mamontov, Eugene; Tyagi, M.; Qian, Shuo; ...

2016-05-27

Here we discuss that the mechanism of action of antimicrobial peptides is traditionally attributed to the formation of pores in the lipid cell membranes of pathogens, which requires a substantial peptide to lipid ratio. However, using incoherent neutron scattering, we show that even at a concentration too low for pore formation, an archetypal antimicrobial peptide, melittin, disrupts the regular phase behavior of the microscopic dynamics in a phospholipid membrane, dimyristoylphosphatidylcholine (DMPC). At the same time, another antimicrobial peptide, alamethicin, does not exert a similar effect on the DMPC microscopic dynamics. The melittin-altered lateral motion of DMPC at physiological temperature nomore » longer resembles the fluid-phase behavior characteristic of functional membranes of the living cells. The disruptive effect demonstrated by melittin even at low concentrations reveals a new mechanism of antimicrobial action relevant in more realistic scenarios, when peptide concentration is not as high as would be required for pore formation, which may facilitate treatment with antimicrobial peptides.« less

Structure-activity studies and therapeutic potential of host defense peptides of human thrombin.

PubMed

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur

2011-06-01

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

Recent updates of marine antimicrobial peptides.

PubMed

Semreen, Mohammad H; El-Gamal, Mohammed I; Abdin, Shifaa; Alkhazraji, Hajar; Kamal, Leena; Hammad, Saba; El-Awady, Faten; Waleed, Dima; Kourbaj, Layal

2018-03-01

Antimicrobial peptides are group of proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens. This class of compounds contributed to solving the microbial resistance dilemma that limited the use of many potent antimicrobial agents. The marine environment is known to be one of the richest sources for antimicrobial peptides, yet this environment is not fully explored. Hence, the scientific research attention should be directed toward the marine ecosystem as enormous amount of useful discoveries could be brought to the forefront. In the current article, the marine antimicrobial peptides reported from mid 2012 to 2017 have been reviewed.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

PubMed

Li, Wenyi; Tailhades, Julien; O'Brien-Simpson, Neil M; Separovic, Frances; Otvos, Laszlo; Hossain, M Akhter; Wade, John D

2014-10-01

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

Production of the antimicrobial peptides Caseicin A and B by Bacillus isolates growing on sodium caseinate.

PubMed

Kent, R M; Guinane, C M; O'Connor, P M; Fitzgerald, G F; Hill, C; Stanton, C; Ross, R P

2012-08-01

The aim of this study was to identify Bacillus isolates capable of degrading sodium caseinate and subsequently to generate bioactive peptides with antimicrobial activity. Sodium caseinate (2.5% w/v) was inoculated separately with 16 Bacillus isolates and allowed to ferment overnight. Protein breakdown in the fermentates was analysed using gel permeation-HPLC (GP-HPLC) and screened for peptides (<3-kDa) with MALDI-TOF mass spectrometry. Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR), two previously characterized antimicrobial peptides, were identified in the fermentates of both Bacillus cereus and Bacillus thuringiensis isolates. The caseicin peptides were subsequently purified by RP-HPLC and antimicrobial assays indicated that the peptides maintained the previously identified inhibitory activity against the infant formula pathogen Cronobacter sakazakii. We report a new method using Bacillus sp. to generate two previously characterized antimicrobial peptides from casein. This study highlights the potential to exploit Bacillus sp. or the enzymes they produce for the generation of bioactive antimicrobial peptides from bovine casein. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

NASA Astrophysics Data System (ADS)

Avitabile, Concetta; D'Andrea, Luca Domenico; Romanelli, Alessandra

2014-03-01

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

Bovine and human lactoferricin peptides: chimeras and new cyclic analogs.

PubMed

Arias, Mauricio; McDonald, Lindsey J; Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

2014-10-01

Lactoferrin (LF) is an important antimicrobial and immune regulatory protein present in neutrophils and most exocrine secretions of mammals. The antimicrobial activity of LF has been related to the presence of an antimicrobial peptide sequence, called lactoferricin (LFcin), located in the N-terminal region of the protein. The antimicrobial activity of bovine LFcin is considerably stronger than the human version. In this work, chimera peptides combining segments of bovine and human LFcin were generated in order to study their antimicrobial activity and mechanism of action. In addition, the relevance of the conserved disulfide bridge and the resulting cyclic structure of both LFcins were analyzed by using "click chemistry" and sortase A-catalyzed cyclization of the peptides. The N-terminal region of bovine LFcin (residues 17-25 of bovine LF) proved to be very important for the antimicrobial activity of the chimera peptides against E. coli, when combined with the C-terminal region of human LFcin. Similarly the cyclic bovine LFcin analogs generated by "click chemistry" and sortase A preserved the antimicrobial activity of the original peptide, showing the significance of these two techniques in the design of cyclic antimicrobial peptides. The mechanism of action of bovine LFcin and its active derived peptides was strongly correlated with membrane leakage in E. coli and up to some extent with the ability to induce vesicle aggregation. This mechanism was also preserved under conditions of high ionic strength (150 mM NaCl) illustrating the importance of these peptides in a more physiologically relevant system.

C. albicans Growth, Transition, Biofilm Formation, and Gene Expression Modulation by Antimicrobial Decapeptide KSL-W

DTIC Science & Technology

2013-11-07

Simon Theberge1, Abdelhabib Semlali1,2, Abdullah Alamri1, Kai P Leung3 and Mahmoud Rouabhia1* Abstract Background: Antimicrobial peptides have been the... peptides , including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought...the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy. Keywords: Antimicrobial peptide , KSL-W, C. albicans, Growth

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.

PubMed

Dolashka, Pavlina; Moshtanska, Vesela; Borisova, Valika; Dolashki, Aleksander; Stevanovic, Stefan; Dimanov, Tzvetan; Voelter, Wolfgang

2011-07-01

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. Copyright © 2011 Elsevier Inc. All rights reserved.

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

PubMed

Astafieva, A A; Rogozhin, E A; Odintsova, T I; Khadeeva, N V; Grishin, E V; Egorov, Ts A

2012-08-01

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini

DTIC Science & Technology

2014-02-20

spectroscopy was applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial ...modified antimicrobial peptide cecropin P1 (CP1) was chemically immobilized onto SAM with a maleimide terminal group. Two important characteristics...applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial peptide cecropin

Probing Protein Sequences as Sources for Encrypted Antimicrobial Peptides

PubMed Central

Brand, Guilherme D.; Magalhães, Mariana T. Q.; Tinoco, Maria L. P.; Aragão, Francisco J. L.; Nicoli, Jacques; Kelly, Sharon M.; Cooper, Alan; Bloch, Carlos

2012-01-01

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources. PMID:23029273

Direct effects of fermented cow's milk product with Lactobacillus paracasei CBA L74 on human enterocytes.

PubMed

Paparo, L; Aitoro, R; Nocerino, R; Fierro, C; Bruno, C; Canani, R Berni

2018-01-29

Cow's milk fermented with Lactobacillus paracasei CBA L74 (FM-CBAL74) exerts a preventive effect against infectious diseases in children. We evaluated if this effect is at least in part related to a direct modulation of non-immune and immune defence mechanisms in human enterocytes. Human enterocytes (Caco-2) were stimulated for 48 h with FM-CBAL74 at different concentrations. Cell growth was assessed by colorimetric assay; cell differentiation (assessed by lactase expression), tight junction proteins (zonula occludens1 and occludin), mucin 2, and toll-like receptor (TRL) pathways were analysed by real-time PCR; innate immunity peptide synthesis, beta-defensin-2 (HBD-2) and cathelicidin (LL-37) were evaluated by ELISA. Mucus layer thickness was analysed by histochemistry. FMCBA L74 stimulated cell growth and differentiation, tight junction proteins and mucin 2 expression, and mucus layer thickness in a dose-dependent fashion. A significant stimulation of HBD-2 and LL-37 synthesis, associated with a modulation of TLR pathway, was also observed. FM-CBAL74 regulates non-immune and immune defence mechanisms through a direct interaction with the enterocytes. These effects could be involved in the preventive action against infectious diseases demonstrated by this fermented product in children.

Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity

NASA Astrophysics Data System (ADS)

Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.; Dong, He

2015-11-01

Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05233e

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna, Katsuwonus pelamis.

PubMed

Seo, Jung-Kil; Lee, Min Jeong; Go, Hye-Jin; Kim, Yeon Jun; Park, Nam Gyu

2014-02-01

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2-17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1-12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish. Copyright © 2014 Elsevier Ltd. All rights reserved.

In silico optimization of a guava antimicrobial peptide enables combinatorial exploration for peptide design.

PubMed

Porto, William F; Irazazabal, Luz; Alves, Eliane S F; Ribeiro, Suzana M; Matos, Carolina O; Pires, Állan S; Fensterseifer, Isabel C M; Miranda, Vivian J; Haney, Evan F; Humblot, Vincent; Torres, Marcelo D T; Hancock, Robert E W; Liao, Luciano M; Ladram, Ali; Lu, Timothy K; de la Fuente-Nunez, Cesar; Franco, Octavio L

2018-04-16

Plants are extensively used in traditional medicine, and several plant antimicrobial peptides have been described as potential alternatives to conventional antibiotics. However, after more than four decades of research no plant antimicrobial peptide is currently used for treating bacterial infections, due to their length, post-translational modifications or  high dose requirement for a therapeutic effect . Here we report the design of antimicrobial peptides derived from a guava glycine-rich peptide using a genetic algorithm. This approach yields guavanin peptides, arginine-rich α-helical peptides that possess an unusual hydrophobic counterpart mainly composed of tyrosine residues. Guavanin 2 is characterized as a prototype peptide in terms of structure and activity. Nuclear magnetic resonance analysis indicates that the peptide adopts an α-helical structure in hydrophobic environments. Guavanin 2 is bactericidal at low concentrations, causing membrane disruption and triggering hyperpolarization. This computational approach for the exploration of natural products could be used to design effective peptide antibiotics.

Guanidino Groups Greatly Enhance the Action of Antimicrobial Peptidomimetics Against Bacterial Cytoplasmic Membranes

DTIC Science & Technology

2014-05-28

SECURITY CLASSIFICATION OF: Antimicrobial peptides or their synthetic mimics are a promising class of potential new antibiotics. Herein we assess the...effect of the type of cationic side chain (i.e., guanidino vs. amino groups) on the membrane perturbing mechanism of antimicrobial ?- peptide ...P.O. Box 12211 Research Triangle Park, NC 27709-2211 Antimicrobial peptidomimetics; Peptide –peptoid chimeras; Guanidinium cation; Bacterial

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection

USDA-ARS?s Scientific Manuscript database

Using quantitative PCR technique, the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes [NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein (BPI), cathepsin D, hepcidin, and liver-expressed antimicrobial peptide 2 ...

Searching for Synthetic Antimicrobial Peptides: An Experiment for Organic Chemistry Students

ERIC Educational Resources Information Center

Vasquez, Thomas E., Jr.; Saldan~a, Cristina; Muzikar, Katy A.; Mashek, Debra; Liu, Jane M.

2016-01-01

This laboratory experiment provides undergraduate students enrolled in organic chemistry the opportunity to design and synthesize their own peptide, which is then tested for antimicrobial activity. After reading a primary scientific paper on antimicrobial peptides, students design and synthesize their own hexapeptide that they hypothesize will…

The role of Vitamin D in immuno-inflammatory responses in Ankylosing Spondylitis patients with and without Acute Anterior Uveitis.

PubMed

Mitulescu, T C; Stavaru, C; Voinea, L M; Banica, L M; Matache, C; Predeteanu, D

2016-01-01

Hypothesis: Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS). Aim: The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU). Methods and Results: 25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation. Discussion: Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS. Abbreviations: Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils.

The role of Vitamin D in immuno-inflammatory responses in Ankylosing Spondylitis patients with and without Acute Anterior Uveitis

PubMed Central

Mitulescu, TC; Stavaru, C; Voinea, LM; Banica, LM; Matache, C; Predeteanu, D

2016-01-01

Hypothesis:Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS). Aim:The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU). Methods and Results:25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation. Discussion:Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS. Abbreviations:Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils. PMID:27713770

Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications

PubMed Central

Solstad, Runar Gjerp; Li, Chun; Isaksson, Johan; Johansen, Jostein; Svenson, Johan; Stensvåg, Klara; Haug, Tor

2016-01-01

The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635) was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring. PMID:27007817

Proanthocyanidin-based Endotoxin Removal

DTIC Science & Technology

2014-01-16

5–7]. These various compounds present a range of limitations. Antimicrobial peptides tend to be somewhat unstable, though there are several...Overall, the PACs provided poor capture of both bacterial species when compared with antibodies or even antimicrobial peptides with detection limits for...Frecer, B. Ho, and J.L. Ding, “De Novo Design of Potent Antimicrobial Peptides ,” Antimicrobial Agents and Chemotherapy 48, 3349–3357 (2004). 5. Y.H

Improving short antimicrobial peptides despite elusive rules for activity.

PubMed

Mikut, Ralf; Ruden, Serge; Reischl, Markus; Breitling, Frank; Volkmer, Rudolf; Hilpert, Kai

2016-05-01

Antimicrobial peptides (AMPs) can effectively kill a broad range of life threatening multidrug-resistant bacteria, a serious threat to public health worldwide. However, despite great hopes novel drugs based on AMPs are still rare. To accelerate drug development we studied different approaches to improve the antibacterial activity of short antimicrobial peptides. Short antimicrobial peptides seem to be ideal drug candidates since they can be synthesized quickly and easily, modified and optimized. In addition, manufacturing a short peptide drug will be more cost efficient than long and structured ones. In contrast to longer and structured peptides short AMPs seem hard to design and predict. Here, we designed, synthesized and screened five different peptide libraries, each consisting of 600 9-mer peptides, against Pseudomonas aeruginosa. Each library is presenting a different approach to investigate effectiveness of an optimization strategy. The data for the 3000 peptides were analyzed using models based on fuzzy logic bioinformatics and plausible descriptors. The rate of active or superior active peptides was improved from 31.0% in a semi-random library from a previous study to 97.8% in the best new designed library. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and screening of potent antimicrobial peptides in arthropod genomes.

PubMed

Duwadi, Deepesh; Shrestha, Anishma; Yilma, Binyam; Kozlovski, Itamar; Sa-Eed, Munaya; Dahal, Nikesh; Jukosky, James

2018-05-01

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2-16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13-25 μg/ml against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5 μg/ml against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Antimicrobial peptides: a review of how peptide structure impacts antimicrobial activity

NASA Astrophysics Data System (ADS)

Soares, Jason W.; Mello, Charlene M.

2004-03-01

Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins.

Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coli.

PubMed

Marani, Mariela M; Perez, Luis O; de Araujo, Alyne Rodrigues; Plácido, Alexandra; Sousa, Carla F; Quelemes, Patrick Veras; Oliveira, Mayara; Gomes-Alves, Ana G; Pueta, Mariana; Gameiro, Paula; Tomás, Ana M; Delerue-Matos, Cristina; Eaton, Peter; Camperi, Silvia A; Basso, Néstor G; de Souza de Almeida Leite, Jose Roberto

2017-03-20

Patagonia's biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7μM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian region's unexplored biodiversity as a source for new molecule discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

Deciphering the Magainin Resistance Process of Escherichia coli Strains in Light of the Cytosolic Proteome

PubMed Central

Maria-Neto, Simone; Cândido, Elizabete de Souza; Rodrigues, Diana Ribas; de Sousa, Daniel Amaro; da Silva, Ezequiel Marcelino; de Moraes, Lidia Maria Pepe; Otero-Gonzalez, Anselmo de Jesus; Magalhães, Beatriz Simas; Dias, Simoni Campos

2012-01-01

Antimicrobial peptides (AMPs) are effective antibiotic agents commonly found in plants, animals, and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. It is vital to understand the molecular details that define the mechanism of action of resistance to AMPs for a rational planning of the next antibiotic generation and also to shed some light on the complex AMP mechanism of action. Here, the antibiotic resistance of Escherichia coli ATCC 8739 to magainin I was evaluated in the cytosolic subproteome. Magainin-resistant strains were selected after 10 subsequent spreads at subinhibitory concentrations of magainin I (37.5 mg · liter−1), and their cytosolic proteomes were further compared to those of magainin-susceptible strains through two-dimensional electrophoresis analysis. As a result, 41 differentially expressed proteins were detected by in silico analysis and further identified by tandem mass spectrometry de novo sequencing. Functional categorization indicated an intense metabolic response mainly in energy and nitrogen uptake, stress response, amino acid conversion, and cell wall thickness. Indeed, data reported here show that resistance to cationic antimicrobial peptides possesses a greater molecular complexity than previously supposed, resulting in cell commitment to several metabolic pathways. PMID:22290970

Insights into the Enhanced in vivo Fitness of Neisseria gonorrhoeae Driven by a Fluoroquinolone Resistance-Conferring Mutant DNA Gyrase

DTIC Science & Technology

2015-02-05

enhancement in fitness relative to its parent strain was also more resistant to human and bacterial antimicrobial peptides , including the cathelicidins...32 Deep sequencing data analysis.............................................................................. 33 Antimicrobial peptide ...Sensitivity to cationic antimicrobial peptides ....................................................... 57 In vivo competitive infection in wild-type and

Antimicrobial peptide evolution in the Asiatic honey bee Apis cerana.

PubMed

Xu, Peng; Shi, Min; Chen, Xue-Xin

2009-01-01

The Asiatic honeybee, Apis cerana Fabricius, is an important honeybee species in Asian countries. It is still found in the wild, but is also one of the few bee species that can be domesticated. It has acquired some genetic advantages and significantly different biological characteristics compared with other Apis species. However, it has been less studied, and over the past two decades, has become a threatened species in China. We designed primers for the sequences of the four antimicrobial peptide cDNA gene families (abaecin, defensin, apidaecin, and hymenoptaecin) of the Western honeybee, Apis mellifera L. and identified all the antimicrobial peptide cDNA genes in the Asiatic honeybee for the first time. All the sequences were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). In all, 29 different defensin cDNA genes coding 7 different defensin peptides, 11 different abaecin cDNA genes coding 2 different abaecin peptides, 13 different apidaecin cDNA genes coding 4 apidaecin peptides and 34 different hymenoptaecin cDNA genes coding 13 different hymenoptaecin peptides were cloned and identified from the Asiatic honeybee adult workers. Detailed comparison of these four antimicrobial peptide gene families with those of the Western honeybee revealed that there are many similarities in the quantity and amino acid components of peptides in the abaecin, defensin and apidaecin families, while many more hymenoptaecin peptides are found in the Asiatic honeybee than those in the Western honeybee (13 versus 1). The results indicated that the Asiatic honeybee adult generated more variable antimicrobial peptides, especially hymenoptaecin peptides than the Western honeybee when stimulated by pathogens or injury. This suggests that, compared to the Western honeybee that has a longer history of domestication, selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens.

Screening And Optimizing Antimicrobial Peptides By Using SPOT-Synthesis

NASA Astrophysics Data System (ADS)

López-Pérez, Paula M.; Grimsey, Elizabeth; Bourne, Luc; Mikut, Ralf; Hilpert, Kai

2017-04-01

Peptide arrays on cellulose are a powerful tool to investigate peptide interactions with a number of different molecules, for examples antibodies, receptors or enzymes. Such peptide arrays can also be used to study interactions with whole cells. In this review, we focus on the interaction of small antimicrobial peptides with bacteria. Antimicrobial peptides (AMPs) can kill multidrug-resistant (MDR) human pathogenic bacteria and therefore could be next generation antibiotics targeting MDR bacteria. We describe the screen and the result of different optimization strategies of peptides cleaved from the membrane. In addition, screening of antibacterial activity of peptides that are tethered to the surface is discussed. Surface-active peptides can be used to protect surfaces from bacterial infections, for example implants.

Antimicrobial activity and mechanism of PDC213, an endogenous peptide from human milk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yazhou; Nanjing Maternal and Child Health Medical Institute, Nanjing Maternal and Child Health Hospital, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing; Zhou, Yahui

Human milk has always been considered an ideal source of elemental nutrients to both preterm and full term infants in order to optimally develop the infant's tissues and organs. Recently, hundreds of endogenous milk peptides were identified in human milk. These peptides exhibited angiotensin-converting enzyme inhibition, immunomodulation, or antimicrobial activity. Here, we report the antimicrobial activity and mechanism of a novel type of human antimicrobial peptide (AMP), termed PDC213 (peptide derived from β-Casein 213-226 aa). PDC213 is an endogenous peptide and is present at higher levels in preterm milk than in full term milk. The inhibitory concentration curve and diskmore » diffusion tests showed that PDC213 had obvious antimicrobial against S. aureus and Y. enterocolitica, the common nosocomial pathogens in neonatal intensive care units (NICUs). Fluorescent dye methods, electron microscopy experiments and DNA-binding activity assays further indicated that PDC213 can permeabilize bacterial membranes and cell walls rather than bind intracellular DNA to kill bacteria. Together, our results suggest that PDC213 is a novel type of AMP that warrants further investigation. - Highlights: • PDC213 is an endogenous peptide presenting higher levels in preterm milk. • PDC213 showed obvious antimicrobial against S. aereus and Y. enterocolitica. • PDC213 can permeabilize bacterial membranes and cell walls to kill bacterias. • PDC213 is a novel type of antimicrobial peptides worthy further investigation.« less

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

PubMed

Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

2017-12-22

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions.

PubMed

Gusmão, Karla A G; Dos Santos, Daniel M; Santos, Virgílio M; Cortés, María Esperanza; Reis, Pablo V M; Santos, Vera L; Piló-Veloso, Dorila; Verly, Rodrigo M; de Lima, Maria Elena; Resende, Jarbas M

2017-01-01

The availability of antimicrobial peptides from several different natural sources has opened an avenue for the discovery of new biologically active molecules. To the best of our knowledge, only two peptides isolated from the frog Leptodactylus labyrinthicus , namely pentadactylin and ocellatin-F1, have shown antimicrobial activities. Therefore, in order to explore the antimicrobial potential of this species, we have investigated the biological activities and membrane interactions of three peptides isolated from the anuran skin secretion. Three peptide primary structures were determined by automated Edman degradation. These sequences were prepared by solid-phase synthesis and submitted to activity assays against gram-positive and gram-negative bacteria and against two fungal strains. The hemolytic properties of the peptides were also investigated in assays with rabbit blood erythrocytes. The conformational preferences of the peptides and their membrane interactions have been investigated by circular dichroism spectroscopy and liposome dye release assays. The amino acid compositions of three ocellatins were determined and the sequences exhibit 100% homology for the first 22 residues (ocellatin-LB1 sequence). Ocellatin-LB2 carries an extra Asn residue and ocellatin-F1 extra Asn-Lys-Leu residues at C-terminus. Ocellatin-F1 presents a stronger antibiotic potential and a broader spectrum of activities compared to the other peptides. The membrane interactions and pore formation capacities of the peptides correlate directly with their antimicrobial activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. All peptides acquire high helical contents in membrane environments. However, ocellatin-F1 shows in average stronger helical propensities. The obtained results indicate that the three extra amino acid residues at the ocellatin-F1 C-terminus play an important role in promoting stronger peptide-membrane interactions and antimicrobial properties. The extra Asn-23 residue present in ocellatin-LB2 sequence seems to decrease its antimicrobial potential and the strength of the peptide-membrane interactions.

Encrypted Antimicrobial Peptides from Plant Proteins.

PubMed

Ramada, M H S; Brand, G D; Abrão, F Y; Oliveira, M; Filho, J L Cardozo; Galbieri, R; Gramacho, K P; Prates, M V; Bloch, C

2017-10-16

Examples of bioactive peptides derived from internal sequences of proteins are known for decades. The great majority of these findings appear to be fortuitous rather than the result of a deliberate and methodological-based enterprise. In the present work, we describe the identification and the biological activities of novel antimicrobial peptides unveiled as internal fragments of various plant proteins founded on our hypothesis-driven search strategy. All putative encrypted antimicrobial peptides were selected based upon their physicochemical properties that were iteratively selected by an in-house computer program named Kamal. The selected peptides were chemically synthesized and evaluated for their interaction with model membranes. Sixteen of these peptides showed antimicrobial activity against human and/or plant pathogens, some with a wide spectrum of activity presenting similar or superior inhibition efficacy when compared to classical antimicrobial peptides (AMPs). These original and previously unforeseen molecules constitute a broader and undisputable set of evidences produced by our group that illustrate how the intragenic concept is a workable reality and should be carefully explored not only for microbicidal agents but also for many other biological functions.

Antimicrobial Peptides of Meat Origin - An In silico and In vitro Analysis.

PubMed

Keska, Paulina; Stadnik, Joanna

2017-01-01

The aim of this study was to evaluate the antimicrobial activity of meat protein-derived peptides against selected Gram-positive and Gram-negative bacteria. The in silico and in vitro approach was combined to determine the potency of antimicrobial peptides derived from pig (Sus scrofa) and cow (Bos taurus) proteins. The in silico studies consisted of an analysis of the amino acid composition of peptides obtained from the CAMPR database, their molecular weight and other physicochemical properties (isoelectric point, molar extinction coefficient, instability index, aliphatic index, hydropathy index and net charge). The degree of similarity was estimated between the antimicrobial peptide sequences derived from the slaughtered animals and the main meat proteins. Antimicrobial activity of peptides isolated from dry-cured meat products was analysed (in vitro) against two strains of pathogenic bacteria using the disc diffusion method. There was no evidence of growthinhibitory properties of peptides isolated from dry-cured meat products against Escherichia coli K12 ATCC 10798 and Staphylococcus aureus ATCC 25923. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

(Lipo)polysaccharide interactions of antimicrobial peptides.

PubMed

Schmidtchen, Artur; Malmsten, Martin

2015-07-01

Due to rapidly increasing resistance development against conventional antibiotics, as well as problems associated with diseases either triggered or deteriorated by infection, antimicrobial and anti-inflammatory peptides have attracted considerable interest during the last few years. While there is an emerging understanding of the direct antimicrobial function of such peptides through bacterial membrane destabilization, the mechanisms of their anti-inflammatory function are less clear. We here summarize some recent results obtained from our own research on anti-inflammatory peptides, with focus on peptide-(lipo)polysaccharide interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

Antimicrobial and Immunomodulatory Activities of PR-39 Derived Peptides

PubMed Central

Veldhuizen, Edwin J. A.; Schneider, Viktoria A. F.; Agustiandari, Herfita; van Dijk, Albert; Tjeerdsma-van Bokhoven, Johanna L. M.; Bikker, Floris J.; Haagsman, Henk P.

2014-01-01

The porcine cathelicidin PR-39 is a host defence peptide that plays a pivotal role in the innate immune defence of the pig against infections. Besides direct antimicrobial activity, it is involved in immunomodulation, wound healing and several other biological processes. In this study, the antimicrobial- and immunomodulatory activity of PR-39, and N- and C-terminal derivatives of PR-39 were tested. PR-39 exhibited an unexpected broad antimicrobial spectrum including several Gram positive strains such as Bacillus globigii and Enterococcus faecalis. Of organisms tested, only Staphylococcus aureus was insensitive to PR-39. Truncation of PR-39 down to 15 (N-terminal) amino acids did not lead to major loss of activity, while peptides corresponding to the C-terminal part of PR-39 were hampered in their antimicrobial activity. However, shorter peptides were all much more sensitive to inhibition by salt. Active peptides induced ATP leakage and loss of membrane potential in Bacillus globigii and Escherichia coli, indicating a lytic mechanism of action for these peptides. Finally, only the mature peptide was able to induce IL-8 production in porcine macrophages, but some shorter peptides also had an effect on TNF-α production showing differential regulation of cytokine induction by PR-39 derived peptides. None of the active peptides showed high cytotoxicity highlighting the potential of these peptides for use as an alternative to antibiotics. PMID:24755622

Alpha-Melanocyte Stimulating Hormone: An Emerging Anti-Inflammatory Antimicrobial Peptide

PubMed Central

Singh, Madhuri; Mukhopadhyay, Kasturi

2014-01-01

The alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide belonging to the melanocortin family. It is well known for its anti-inflammatory and antipyretic effects and shares several characteristics with antimicrobial peptides (AMPs). There have been some recent reports about the direct antimicrobial activity of α-MSH against various microbes belonging to both fungal and bacterial pathogens. Similar to α-MSH's anti-inflammatory properties, its C-terminal residues also exhibit antimicrobial activity parallel to that of the entire peptide. This review is focused on the current findings regarding the direct antimicrobial potential and immunomodulatory mechanism of α-MSH and its C-terminal fragments, with particular emphasis on the prospects of α-MSH based peptides as a strong anti-infective agent. PMID:25140322

Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿

PubMed Central

He, Jian; Eckert, Randal; Pharm, Thanh; Simanian, Maurice D.; Hu, Chuhong; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2007-01-01

Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents. PMID:17296741

iTRAQ-Based Quantitative Proteomic Analysis of the Antimicrobial Mechanism of Peptide F1 against Escherichia coli.

PubMed

Miao, Jianyin; Chen, Feilong; Duan, Shan; Gao, Xiangyang; Liu, Guo; Chen, Yunjiao; Dixon, William; Xiao, Hang; Cao, Yong

2015-08-19

Antimicrobial peptides have received increasing attention in the agricultural and food industries due to their potential to control pathogens. However, to facilitate the development of novel peptide-based antimicrobial agents, details regarding the molecular mechanisms of these peptides need to be elucidated. The aim of this study was to investigate the antimicrobial mechanism of peptide F1, a bacteriocin found in Tibetan kefir, against Escherichia coli at protein levels using iTRAQ-based quantitative proteomic analysis. In response to treatment with peptide F1, 31 of the 280 identified proteins in E. coli showed alterations in their expression, including 10 down-regulated proteins and 21 up-regulated proteins. These 31 proteins all possess different molecular functions and are involved in different molecular pathways, as is evident in referencing the Kyoto Encyclopedia of Genes and Genomes pathways. Specifically, pathways that were significantly altered in E. coli in response to peptide F1 treatment include the tricarboxylic acid cycle, oxidative phosphorylation, glycerophospholipid metabolism, and the cell cycle-caulobacter pathways, which was also associated with inhibition of the cell growth, induction of morphological changes, and cell death. The results provide novel insights into the molecular mechanisms of antimicrobial peptides.

A lesson from Bombinins H, mildly cationic diastereomeric antimicrobial peptides from Bombina skin.

PubMed

Mangoni, Maria Luisa

2013-12-01

Gene-encoded peptide antibiotics represent fascinating molecules for the development of new antimicrobials with a new mode of action: and one of the richest sources is amphibian skin. In particular, the skin of the fire-bellied toad Bombina genus contains mildly cationic antimicrobial peptides (AMPs), named bombinins H, with attractive properties. Indeed, some members of this peptide family coexist in skin secretions as isomers in which a single D-amino acid (alloisoleucine or leucine) is incorporated as a result of a post-translational modification of the respective gene-encoded Lamino acid. Here, a brief overview of the genes coding for these peptides, their spectrum of antimicrobial activities, mechanism of action and interactions with biological or model membranes is reported. Remarkably, a single D-amino acid substitution represents a unique approach developed by Nature not only to modulate the peptide stability in vivo, but also to confer the all-L peptide and its diastereomer distinctive biological features. Overall, such findings should assist in the generation of new peptide-based anti-infective agents, which are urgently needed because of the growing emergence of microbial strains resistant to conventional antimicrobials.

Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

PubMed

Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

2017-02-23

Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

Insights into Antimicrobial Peptides from Spiders and Scorpions

PubMed Central

Wang, Xiuqing; Wang, Guangshun

2015-01-01

The venoms of spiders and scorpions contain a variety of chemical compounds. Antimicrobial peptides (AMPs) from these organisms were first discovered in the 1990s. As of May 2015, there were 42 spider’s and 63 scorpion’s AMPs in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP). These peptides have demonstrated broad or narrow-spectrum activities against bacteria, fungi, viruses, and parasites. In addition, they can be toxic to cancer cells, insects and erythrocytes. To provide insight into such an activity spectrum, this article discusses the discovery, classification, structure and activity relationships, bioinformatics analysis, and potential applications of spider and scorpion AMPs. Our analysis reveals that, in the case of linear peptides, spiders use both glycine-rich and helical peptide models for defense, whereas scorpions use two distinct helical peptide models with different amino acid compositions to exert the observed antimicrobial activities and hemolytic toxicity. Our structural bioinformatics study improves the knowledge in the field and can be used to design more selective peptides to combat tumors, parasites, and viruses. PMID:27165405

Hevein-Like Antimicrobial Peptides of Plants.

PubMed

Slavokhotova, A A; Shelenkov, A A; Andreev, Ya A; Odintsova, T I

2017-12-01

Plant antimicrobial peptides represent one of the evolutionarily oldest innate immunity components providing the first line of host defense to pathogen attacks. This review is dedicated to a small, currently actively studied family of hevein-like peptides that can be found in various monocot and dicot plants. The review thoroughly describes all known peptides belonging to this family including data on their structures, functions, and antimicrobial activity. The main features allowing to assign these peptides to a separate family are given, and the specific characteristics of each peptide are described. Further, the mode of action for hevein-like peptides, their role in plant immune system, and the applications of these molecules in biotechnology and medicine are considered.

Antimicrobial Peptides from Fish

PubMed Central

Masso-Silva, Jorge A.; Diamond, Gill

2014-01-01

Antimicrobial peptides (AMPs) are found widely distributed through Nature, and participate in the innate host defense of each species. Fish are a great source of these peptides, as they express all of the major classes of AMPs, including defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins. As with other species, the fish peptides exhibit broad-spectrum antimicrobial activity, killing both fish and human pathogens. They are also immunomodulatory, and their genes are highly responsive to microbes and innate immuno-stimulatory molecules. Recent research has demonstrated that some of the unique properties of fish peptides, including their ability to act even in very high salt concentrations, make them good potential targets for development as therapeutic antimicrobials. Further, the stimulation of their gene expression by exogenous factors could be useful in preventing pathogenic microbes in aquaculture. PMID:24594555

Molecular Design, Structures, and Activity of Antimicrobial Peptide-Mimetic Polymers

PubMed Central

Takahashi, Haruko; Palermo, Edmund F.; Yasuhara, Kazuma; Caputo, Gregory A.

2014-01-01

There is an urgent need for new antibiotics which are effective against drug-resistant bacteria without contributing to resistance development. We have designed and developed antimicrobial copolymers with cationic amphiphilic structures based on the mimicry of naturally occurring antimicrobial peptides. These copolymers exhibit potent antimicrobial activity against a broad spectrum of bacteria including methicillin-resistant Staphylococcus aureus with no adverse hemolytic activity. Notably, these polymers also did not result in any measurable resistance development in E. coli. The peptide-mimetic design principle offers significant flexibility and diversity in the creation of new antimicrobial materials and their potential biomedical applications. PMID:23832766

Inhibitors of signal peptide peptidase (SPP) affect HSV-1 infectivity in vitro and in vivo

PubMed Central

Allen, Sariah J.; Mott, Kevin R.; Ghiasi, Homayon

2014-01-01

Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology. PMID:24768597

Design of Embedded-Hybrid Antimicrobial Peptides with Enhanced Cell Selectivity and Anti-Biofilm Activity

PubMed Central

Xu, Wei; Zhu, Xin; Tan, Tingting; Li, Weizhong; Shan, Anshan

2014-01-01

Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents. PMID:24945359

Novel antimicrobial peptides isolated from the skin secretions of Hainan odorous frog, Odorrana hainanensis.

PubMed

Wang, Hui; Yu, Zhijun; Hu, Yuhong; Li, Fengjiao; Liu, Limeng; Zheng, Hongyuan; Meng, Hao; Yang, Shujie; Yang, Xiaolong; Liu, Jingze

2012-06-01

Long time geographical isolation of Hainan Island from the China continent has resulted in appearance of many novel frog species. As one of them, Hainan odorous frog, Odorrana hainanensis possesses some special antimicrobial peptides distinct from those found in other Odorrana. In this study, three antimicrobial peptides have been purified and characterized from the skin secretion of O. hainanensis. With the similarity to the temporin family, two peptides are characterized by amidated C-terminals, so they are named as temporin-HN1 (AILTTLANWARKFL-NH(2)) and temporin-HN2 (NILNTIINLAKKIL-NH(2)). The third antimicrobial peptide belongs to the brevinin-1 family which is widely distributed in Eurasian ranids, and thus, it is named as brevinin-1HN1 (FLPLIASLAANFVPKIFCKITKKC). Furthermore, after sequencing 68 clones, eight cDNAs encoding antimicrobial peptide precursors were cloned from the skin-derived cDNA library of O. hainanensis. These eight cDNAs can encode seven mature antimicrobial peptides including the above three, as well as brevinin-1V, brevinin-2HS2, odorranain-A6, and odorranain-B1. Twelve different species of microorganisms were chosen, including Gram-positive, Gram-negative and fungi, to test the antimicrobial activities of temporin-HN1, temporin-HN2, brevinin-1HN1, brevinin-1V, and brevinin-2HS2. The result shows that, in addition to their activities against Gram-positive bacteria, temporin-HN1 and temporin-HN2 also possess activities against some Gram-negative bacteria and fungi. However, the two antimicrobial peptides, brevinin-1HN1 and brevinin-1V of the brevinin-1 family have stronger antimicrobial activities than temporin-HN1 and temporin-HN2 of the temporin family. Brevinin-1HN1 possesses activity against Staphylococcus aureus (ATCC25923), Rhodococcus rhodochrous X15, and Slime mould 090223 at the concentration of 1.2 μM. Copyright © 2012 Elsevier Inc. All rights reserved.

Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity.

PubMed

Wang, Yang; Chen, Jianbo; Zheng, Xin; Yang, Xiaoli; Ma, Panpan; Cai, Ying; Zhang, Bangzhi; Chen, Yuan

2014-12-01

Currently, novel antibiotics are urgently required to combat the emergence of drug-resistant bacteria. Antimicrobial peptides with membrane-lytic mechanism of action have attracted considerable interest. Anoplin, a natural α-helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin-4 composed of D-amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin-4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin-4 treatment relative to anoplin. In conclusion, anoplin-4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2.

PubMed

Wang, Jiarong; Li, Yan; Wang, Xiaoming; Chen, Wei; Sun, Hongbin; Wang, Junfeng

2014-11-01

Lipopolysaccharide (LPS), the important component of the outer membrane of Gram-negative bacteria, contributes to the integrity of the outer membrane and protects the cell against bactericidal agents, including antimicrobial peptides. However, the mechanisms of interaction between antimicrobial peptides and LPS are not clearly understood. Halictines-2 (HAL-2), one of the novel antimicrobial peptides, was isolated from the venom of the eusocial bee Halictus sexcinctus. HAL-2 has exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria and even against cancer cells. Here, we studied the interactions between HAL-2 and LPS to elucidate the antibacterial mechanism of HAL-2 in vitro. Our results show that HAL-2 adopts a significant degree of β-strand structure in the presence of LPS. LPS is capable of inducing HAL-2 amyloid formation, which may play a vital role in its antimicrobial activity. Copyright © 2014 Elsevier B.V. All rights reserved.

An antimicrobial bicyclic peptide from chemical space against multidrug resistant Gram-negative bacteria.

PubMed

Di Bonaventura, Ivan; Baeriswyl, Stéphane; Capecchi, Alice; Gan, Bee-Ha; Jin, Xian; Siriwardena, Thissa N; He, Runze; Köhler, Thilo; Pompilio, Arianna; Di Bonaventura, Giovanni; van Delden, Christian; Javor, Sacha; Reymond, Jean-Louis

2018-05-15

We used the concept of chemical space to explore a virtual library of bicyclic peptides formed by double thioether cyclization of a precursor linear peptide, and identified an antimicrobial bicyclic peptide (AMBP) with remarkable activity against several MDR strains of Acinetobacter baumannii and Pseudomonas aeruginosa.

Bionano Interaction Study on Antimicrobial Star-Shaped Peptide Polymer Nanoparticles.

PubMed

Lam, Shu J; Wong, Edgar H H; O'Brien-Simpson, Neil M; Pantarat, Namfon; Blencowe, Anton; Reynolds, Eric C; Qiao, Greg G

2016-12-14

'Structurally nanoengineered antimicrobial peptide polymers' (SNAPPs), in the form of star-shaped peptide polymer nanoparticles, have been recently demonstrated as a new class of antimicrobial agents with superior in vitro and in vivo efficacy against Gram-negative pathogens, including multidrug-resistant species. Herein, we present a detailed bionano interaction study on SNAPPs by assessing their antimicrobial activities against several Gram-negative bacteria in complex biological matrices. Simulated body fluid and animal serum were used as test media to reveal factors that influence the antimicrobial efficacy of SNAPPs. With the exception of Acinetobacter baumannii, the presence of divalent cations at physiological concentrations reduced the antimicrobial efficacy of SNAPPs from minimum inhibitory concentrations (MICs) within the nanomolar range (40-300 nM) against Escherichia coli, Pseudomanas aeruginosa, and Klebsiella pneumoniae to 0.6-4.7 μM. By using E. coli as a representative bacterial species, we demonstrated that the reduction in activity was due to a decrease in the ability of SNAPPs to cause outer and inner membrane disruption. This effect could be reversed through coadministration with a chelating agent. Interestingly, the potency of SNAPPs against A. baumannii was retained even under high salt concentrations. The presence of serum proteins was also found to affect the interaction of SNAPPs with bacterial membranes, possibly through intermolecular binding. Collectively, this study highlights the need to consider the possible interactions of (bio)molecules present in vivo with any new antimicrobial agent under development. We also demonstrate that outer membrane disruption/destabilization is an important but hitherto under-recognized target for the antimicrobial action of peptide-based agents, such as antimicrobial peptides (AMPs). Overall, the findings presented herein could aid in the design of more efficient peptide-based antimicrobial agents with uncompromised potency even under physiological conditions.

Identification of Peptides in Flowers of Sambucus nigra with Antimicrobial Activity against Aquaculture Pathogens.

PubMed

Álvarez, Claudio Andrés; Barriga, Andrés; Albericio, Fernando; Romero, María Soledad; Guzmán, Fanny

2018-04-27

The elder ( Sambucus spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of Sambucus nigra L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent

NASA Astrophysics Data System (ADS)

Liu, Lihong; Xu, Kaijin; Wang, Huaying; Jeremy Tan, P. K.; Fan, Weimin; Venkatraman, Subbu S.; Li, Lanjuan; Yang, Yi-Yan

2009-07-01

Antimicrobial cationic peptides are of interest because they can combat multi-drug-resistant microbes. Most peptides form α-helices or β-sheet-like structures that can insert into and subsequently disintegrate negatively charged bacterial cell surfaces. Here, we show that a novel class of core-shell nanoparticles formed by self-assembly of an amphiphilic peptide have strong antimicrobial properties against a range of bacteria, yeasts and fungi. The nanoparticles show a high therapeutic index against Staphylococcus aureus infection in mice and are more potent than their unassembled peptide counterparts. Using Staphylococcus aureus-infected meningitis rabbits, we show that the nanoparticles can cross the blood-brain barrier and suppress bacterial growth in infected brains. Taken together, these nanoparticles are promising antimicrobial agents that can be used to treat brain infections and other infectious diseases.

Protocols to test the activity of antimicrobial peptides against the honey bee pathogen Paenibacillus larvae.

PubMed

Khilnani, Jasmin C; Wing, Helen J

2015-10-01

Paenibacillus larvae is the causal agent of the honey bee disease American Foulbrood. Two enhanced protocols that allow the activity of antimicrobial peptides to be tested against P. larvae are presented. Proof of principle experiments demonstrate that the honey bee antimicrobial peptide defensin 1 is active in both assays. Copyright © 2015 Elsevier B.V. All rights reserved.

Structure-activity relationships of an antimicrobial peptide plantaricin s from two-peptide class IIb bacteriocins.

PubMed

Soliman, Wael; Wang, Liru; Bhattacharjee, Subir; Kaur, Kamaljit

2011-04-14

Class IIb bacteriocins are ribosomally synthesized antimicrobial peptides comprising two different peptides synergistically acting in equal amounts for optimal potency. In this study, we demonstrate for the first time potent (nanomolar) antimicrobial activity of a representative class IIb bacteriocin, plantaricin S (Pls), against four pathogenic gram-positive bacteria, including Listeria monocytogenes. The structure-activity relationships for Pls were studied using activity assays, circular dichroism (CD), and molecular dynamics (MD) simulations. The two Pls peptides and five Pls derived fragments were synthesized. The CD spectra of the Pls and selected fragments revealed helical conformations in aqueous 2,2,2-trifluoroethanol. The MD simulations showed that when the two Pls peptides are in antiparallel orientation, the helical regions interact and align, mediated by strong attraction between conserved GxxxG/AxxxA motifs. The results strongly correlate with the antimicrobial activity suggesting that helix-helix alignment of the two Pls peptides and interaction between the conserved motifs are crucial for interaction with the target cell membrane.

Cloning, expression, and purification of a new antimicrobial peptide gene from Musca domestica larva.

PubMed

Pei, Zhihua; Sun, Xiaoning; Tang, Yan; Wang, Kai; Gao, Yunhang; Ma, Hongxia

2014-10-01

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram- bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study. Copyright © 2014 Elsevier B.V. All rights reserved.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

PubMed Central

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-01-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities. PMID:26633506

Phylloseptin-PBa--A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity.

PubMed

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-12-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

An enhancer peptide for membrane-disrupting antimicrobial peptides

PubMed Central

2010-01-01

Background NP4P is a synthetic peptide derived from a natural, non-antimicrobial peptide fragment (pro-region of nematode cecropin P4) by substitution of all acidic amino acid residues with amides (i.e., Glu → Gln, and Asp → Asn). Results In the presence of NP4P, some membrane-disrupting antimicrobial peptides (ASABF-α, polymyxin B, and nisin) killed microbes at lower concentration (e.g., 10 times lower minimum bactericidal concentration for ASABF-α against Staphylococcus aureus), whereas NP4P itself was not bactericidal and did not interfere with bacterial growth at ≤ 300 μg/mL. In contrast, the activities of antimicrobial agents with a distinct mode of action (indolicidin, ampicillin, kanamycin, and enrofloxacin) were unaffected. Although the membrane-disrupting activity of NP4P was slight or undetectable, ASABF-α permeabilized S. aureus membranes with enhanced efficacy in the presence of NP4P. Conclusions NP4P selectively enhanced the bactericidal activities of membrane-disrupting antimicrobial peptides by increasing the efficacy of membrane disruption against the cytoplasmic membrane. PMID:20152058

Buwchitin: a ruminal peptide with antimicrobial potential against Enterococcus faecalis

NASA Astrophysics Data System (ADS)

Oyama, Linda B.; Crochet, Jean-Adrien; Edwards, Joan E.; Girdwood, Susan E.; Cookson, Alan R.; Fernandez-Fuentes, Narcis; Hilpert, Kai; Golyshin, Peter N.; Golyshina, Olga V.; Privé, Florence; Hess, Matthias; Mantovani, Hilario C.; Creevey, Christopher J.; Huws, Sharon A.

2017-07-01

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Employing the promiscuity of lantibiotic biosynthetic machineries to produce novel antimicrobials.

PubMed

Montalbán-López, Manuel; van Heel, Auke J; Kuipers, Oscar P

2017-01-01

As the number of new antibiotics that reach the market is decreasing and the demand for them is rising, alternative sources of novel antimicrobials are needed. Lantibiotics are potent peptide antimicrobials that are ribosomally synthesized and stabilized by post-translationally introduced lanthionine rings. Their ribosomal synthesis and enzymatic modifications provide excellent opportunities to design and engineer a large variety of novel antimicrobial compounds. The research conducted in this area demonstrates that the modularity present in both the peptidic rings as well as in the combination of promiscuous modification enzymes can be exploited to further increase the diversity of lantibiotics. Various approaches, where the modifying enzymes and corresponding leader peptides are decoupled from their natural core peptide and integrated in designed plug-and-play production systems, enable the production of modified peptides that are either derived from vast genomic data or designed using functional parts from a wide diversity of core peptides. These approaches constitute a powerful discovery platform to develop novel antimicrobials with high therapeutic potential. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cyclic Peptides as Novel Therapeutic Microbicides: Engineering of Human Defensin Mimetics.

PubMed

Falanga, Annarita; Nigro, Ersilia; De Biasi, Margherita Gabriella; Daniele, Aurora; Morelli, Giancarlo; Galdiero, Stefania; Scudiero, Olga

2017-07-20

Cyclic peptides are receiving significant attention thanks to their antimicrobial activity and high serum stability, which is useful to develop and design novel antimicrobial agents. Antimicrobial peptides appear to be key components of innate defences against bacteria, viruses, and fungi. Among the others, defensins possess a strong microbicidial activity. Defensins are cationic and amphipathic peptides with six cysteine residues connected by three disulfide bonds found in plants, insects, and mammals; they are divided in three families: α-, β-, and θ-defensins. α-Defensins are contained in the primary granules of human neutrophils; β-defensins are expressed in human epithelia; and θ-defensins are pseudo-cyclic defensins not found in humans, but in rhesus macaques. The structural diversities among the three families are reflected in a different antimicrobial action as well as in serum stability. The engineering of these peptides is an exciting opportunity to obtain more functional antimicrobial molecules highlighting their potential as therapeutic agents. The present review reports the most recent advances in the field of cyclic peptides with a specific regard to defensin analogs.

Conformational study of melectin and antapin antimicrobial peptides in model membrane environments

NASA Astrophysics Data System (ADS)

Kocourková, Lucie; Novotná, Pavlína; Čujová, Sabína; Čeřovský, Václav; Urbanová, Marie; Setnička, Vladimír

2017-01-01

Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.

Chemical Cleavage of an Asp-Cys Sequence Allows Efficient Production of Recombinant Peptides with an N-Terminal Cysteine Residue.

PubMed

Pane, Katia; Verrillo, Mariavittoria; Avitabile, Angela; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Di Maro, Antimo; Rega, Camilla; Amoresano, Angela; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

2018-04-18

Peptides with an N-terminal cysteine residue allow site-specific modification of proteins and peptides and chemical synthesis of proteins. They have been widely used to develop new strategies for imaging, drug discovery, diagnostics, and chip technologies. Here we present a method to produce recombinant peptides with an N-terminal cysteine residue as a convenient alternative to chemical synthesis. The method is based on the release of the desired peptide from a recombinant fusion protein by mild acid hydrolysis of an Asp-Cys sequence. To test the general validity of the method we prepared four fusion proteins bearing three different peptides (20-37 amino acid long) at the C-terminus of a ketosteroid isomerase-derived and two Onconase-derived carriers for the production of toxic peptides in E. coli. The chosen peptides were (C)GKY20, an antimicrobial peptide from the C-terminus of human thrombin, (C)ApoB L , an antimicrobial peptide from an inner region of human Apolipoprotein B, and (C)p53pAnt, an anticancer peptide containing the C-terminal region of the p53 protein fused to the cell penetrating peptide Penetratin. Cleavage efficiency of Asp-Cys bonds in the four fusion proteins was studied as a function of pH, temperature, and incubation time. In spite of the differences in the amino acid sequence (GTGDCGKY, GTGDCHVA, GSGTDCGSR, SQGSDCGSR) we obtained for all the proteins a cleavage efficiency of about 70-80% after 24 h incubation at 60 °C and pH 2. All the peptides were produced with very good yield (5-16 mg/L of LB cultures), high purity (>96%), and the expected content of free thiol groups (1 mol per mole of peptide). Furthermore, (C)GKY20 was modified with PyMPO-maleimide, a commercially available fluorophore bearing a thiol reactive group, and with 6-hydroxy-2-cyanobenzothiazole, a reagent specific for N-terminal cysteines, with yields of 100% thus demonstrating that our method is very well suited for the production of fully reactive peptides with an N-terminal cysteine residue.

Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer.

PubMed

Proaño-Bolaños, Carolina; Zhou, Mei; Wang, Lei; Coloma, Luis A; Chen, Tianbao; Shaw, Chris

2016-09-02

Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20-32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD- and the sequence motifs -VALGAVSK- or -GKAAL(N/G/S) (V/A)V- in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3-fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11μM). CZS-1 reached 100% haemolysis at 120.87μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin.

PubMed

Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

2012-03-01

Lactoferricin and lactoferrampin are two antimicrobial peptides found in the N-terminal lobe of bovine lactoferrin with broad spectrum antimicrobial activity against a range of Gram-positive and Gram-negative bacteria as well as Candida albicans. A heterodimer comprised of lactoferrampin joined to a fragment of lactoferricin was recently reported in which these two peptides were joined at their C-termini through the two amino groups of a single Lys residue (Bolscher et al., 2009, Biochimie 91(1):123-132). This hybrid peptide, termed LFchimera, has significantly higher antimicrobial activity compared to the individual peptides or an equimolar mixture of the two. In this work, the underlying mechanism behind the increased antibacterial activity of LFchimera was investigated. Differential scanning calorimetry studies demonstrated that all the peptides influenced the thermotropic phase behaviour of anionic phospholipid suspensions. Calcein leakage and vesicle fusion experiments with anionic liposomes revealed that LFchimera had enhanced membrane perturbing properties compared to the individual peptides. Peptide structures were evaluated using circular dichroism and NMR spectroscopy to gain insight into the structural features of LFchimera that contribute to the increased antimicrobial activity. The NMR solution structure, determined in a miscible co-solvent mixture of chloroform, methanol and water, revealed that the Lys linkage increased the helical content in LFchimera compared to the individual peptides, but it did not fix the relative orientations of lactoferricin and lactoferrampin with respect to each other. The structure of LFchimera provides insight into the conformation of this peptide in a membranous environment and improves our understanding of its antimicrobial mechanism of action. Copyright © 2011 Elsevier B.V. All rights reserved.

Enhancement of the anti-inflammatory activity of temporin-1Tl-derived antimicrobial peptides by tryptophan, arginine and lysine substitutions.

PubMed

Rajasekaran, Ganesan; Kamalakannan, Radhakrishnan; Shin, Song Yub

2015-10-01

Temporin-1Tl (TL) is a 13-residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti-inflammatory activity. To develop novel AMP with improved anti-inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin-resistant Staphylococcus aureus strains compared with TL. TL-1 and TL-4 showed a little increase in antimicrobial selectivity, while TL-2 and TL-3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti-inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor-α (TNF-α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti-inflammatory activity is as follows: TL-2 ≈ TL-3 ≈ TL-4 > TL-1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti-inflammatory activity. These results apparently suggest that the anti-inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti-inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram-negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Antimicrobial potency of cationic antimicrobial peptides can be predicted from their amino acid composition: Application to the detection of "cryptic" antimicrobial peptides.

PubMed

Pane, Katia; Durante, Lorenzo; Crescenzi, Orlando; Cafaro, Valeria; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Izzo, Viviana; Di Donato, Alberto; Notomista, Eugenio

2017-04-21

Cationic antimicrobial peptides (CAMPs) are essential components of innate immunity. Here we show that antimicrobial potency of CAMPs is linearly correlated to the product C m H n L where C is the net charge of the peptide, H is a measure of its hydrophobicity and L its length. Exponents m and n define the relative contribution of charge and hydrophobicity to the antimicrobial potency. Very interestingly the values of m and n are strain specific. The ratio n/(m+n) can vary between ca. 0.5 and 1, thus indicating that some strains are sensitive to highly charged peptides, whereas others are particularly susceptible to more hydrophobic peptides. The slope of the regression line describing the correlation "antimicrobial potency"/"C m H n L product" changes from strain to strain indicating that some strains acquired a higher resistance to CAMPs than others. Our analysis provides also an effective computational strategy to identify CAMPs included inside the structure of larger proteins or precursors, which can be defined as "cryptic" CAMPs. We demonstrate that it is not only possible to identify and locate with very good precision the position of cryptic peptides, but also to analyze the internal structure of long CAMPs, thus allowing to draw an accurate map of the molecular determinants of their antimicrobial activity. A spreadsheet, provided in the Supplementary material, allows performing the analysis of protein sequences. Our strategy is also well suited to analyze large pools of sequences, thus significantly improving the identification of new CAMPs and the study of innate immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

PubMed

Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu

2016-01-01

The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide-based antimicrobial biomaterials. Further exploration of these AMPs will allow for diverse biotechnological and clinical applications such as biomedical coating, food storaging, and animal feeding. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Novel formulations for antimicrobial peptides.

PubMed

Carmona-Ribeiro, Ana Maria; de Melo Carrasco, Letícia Dias

2014-10-09

Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

Novel Formulations for Antimicrobial Peptides

PubMed Central

Carmona-Ribeiro, Ana Maria; Carrasco, Letícia Dias de Melo

2014-01-01

Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy. PMID:25302615

Investigation of the antimicrobial activities of Snakin-Z, a new cationic peptide derived from Zizyphus jujuba fruits.

PubMed

Daneshmand, Fatemeh; Zare-Zardini, Hadi; Ebrahimi, Leila

2013-01-01

Snakin-Z is a novel antimicrobial peptide (AMP) that is identified from the fruit of Zizyphus jujuba. This peptide is composed of 31 amino acids which is determined with the sequence of CARLNCVPKGTSGNTETCPCYASLHSCRKYG and molecular weight of 3318.82 Da. Snakin-Z is not identical to any AMP in the peptide database. According to this study, Snakin-Z potentially has antimicrobial property against bacteria and fungi. Minimal inhibitory concentration (MIC) value of this peptide is suitable for antimicrobial activity. We assessed that Snakin-Z could affect Phomopsis azadirachtae with the MIC value of 7.65 μg/mL and vice versa Staphylococcus aureus with the MIC value of 28.8 μg/mL. Interestingly, human red blood cells also showed good tolerance to the Snakin-Z. On the basis of this study, Snakin-Z can be an appropriate candidate for therapeutic applications in the future due to its antimicrobial property.

The novel antimicrobial peptides from skin of Chinese broad-folded frog, Hylarana latouchii (Anura:Ranidae).

PubMed

Wang, Hui; Lu, Yi; Zhang, Xiuqing; Hu, Yuhong; Yu, Haining; Liu, Jingze; Sun, Junshe

2009-02-01

Broad-folded frogs (Hylarana latouchii), one member of 12 species of the genus Hylarana in the Chinese frog fauna, are widely distributed in the South of China. In this study, we purified and characterized three antimicrobial peptides from the skin secretion of H. latouchii. Five different cDNA fragments encoding the precursors of these antimicrobial peptides were cloned, and five mature antimicrobial peptides belonging to two different families were deduced from the five cDNAs. Structural characterization of the mature peptides had identified them as members of the brevinin-1 and temporin families. They were named brevinin-1LTa (FFGTALKIAANVLPTAICKILKKC), brevinin-1LTb (FFGTALKIAANILPTAICKILKKC), temporin-LTa (FFPLVLGALGSILPKIF-NH(2)), temporin-LTb (FIITGLVRGLTKLF-NH(2)) and temorin-LTc (SLSRFLSFLKIVYPPAF-NH(2)). Brevinin-1LTa, temporin-LTa, temporin-LTb and temporin-LTc with different antimicrobial activities induced significant morphological alterations of the tested microbial surfaces as shown by scanning electron microscopy, which indicated strong membrane disruption.

The Vibrio cholerae VprA-VprB Two-Component System Controls Virulence Through Endotoxin Modification

DTIC Science & Technology

2014-12-23

antimicrobial peptides of the innate immune system bind to the membrane of Gram-negative pathogens via conserved, surface-exposed lipopolysaccharide (LPS... antimicrobial peptide polymyxin. However, the regulatory mechanisms of lipid A modification in V. 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE...12211 Research Triangle Park, NC 27709-2211 bacterial cell surface, host immune system, cationic antimicrobial peptides , lipid A, LPS REPORT

Labeled Antimicrobial Peptides for Detection of Microorganisms

DTIC Science & Technology

2008-12-01

1. INTRODUCTION Antimicrobial peptides (AMPs) are part of the innate defense system found in all organisms to protect them from microbial infection...2005) with antimicrobial activity against predominantly gram-negative bacteria. SMAP29 is from the cathelicidin family of peptides found in sheep ...in buffer, milk and apple juice. Cells were grown and prepared in PBST as described above. 20 III anti-£. coli 0157 paramagnetic Dyna-beads (Dynal

Brazilian Kefir-Fermented Sheep's Milk, a Source of Antimicrobial and Antioxidant Peptides.

PubMed

de Lima, Meire Dos Santos Falcão; da Silva, Roberto Afonso; da Silva, Milena Fernandes; da Silva, Paulo Alberto Bezerra; Costa, Romero Marcos Pedrosa Brandão; Teixeira, José António Couto; Porto, Ana Lúcia Figueiredo; Cavalcanti, Maria Taciana Holanda

2017-12-28

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep's milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep's milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep's milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep's milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens.

PubMed

Lavine, M D; Chen, G; Strand, M R

2005-12-01

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A (Pi-cecA), lebocin (Pi-leb) and lysozyme (Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Antimicrobial preservative use in parenteral products: past and present.

PubMed

Meyer, Brian K; Ni, Alex; Hu, Binghua; Shi, Li

2007-12-01

The following review provides a comprehensive summary of antimicrobial preservatives that are commonly used in licensed parenteral products to date. The information reviewed includes the general properties of the preservatives, the doses and frequency of their use, the classes of the preserved products (peptide, protein, vaccine, and small molecule products), the interactions with other formulation components, and the criteria commonly used for their selection in parental product formulations. It was revealed that phenol and benzyl alcohol are the two most common antimicrobial preservatives used in peptide and protein products, while phenoxyethanol is the most frequently used preservative in vaccines. Benzyl alcohol or a combination of methylparaben and propylparaben are generally found in small molecule parenteral formulations. The key criteria for antimicrobial preservative selection are the preservative's dose, antimicrobial functionality, and effect on the active ingredient. Additionally, the use of spectroscopic techniques (circular dicroism (CD) and fluorescence) and differential scanning calorimetry (DSC) were identified as common techniques used in evaluating an antimicrobial preservative for its impact on the conformational stability of peptide, protein, and vaccine antigens. The future use of preservatives is also discussed, including antimicrobial agents such as peptides, and regulatory requirements for antimicrobial effectiveness testing. (c) 2007 Wiley-Liss, Inc.

Low-Level Tragus Stimulation for the Treatment of Ischemia and Reperfusion Injury in Patients With ST-Segment Elevation Myocardial Infarction: A Proof-of-Concept Study.

PubMed

Yu, Lilei; Huang, Bing; Po, Sunny S; Tan, Tuantuan; Wang, Menglong; Zhou, Liping; Meng, Guannan; Yuan, Shenxu; Zhou, Xiaoya; Li, Xuefei; Wang, Zhuo; Wang, Songyun; Jiang, Hong

2017-08-14

The aim of this study was to investigate whether low-level tragus stimulation (LL-TS) treatment could reduce myocardial ischemia-reperfusion injury in patients with ST-segment elevation myocardial infarction (STEMI). The authors' previous studies suggested that LL-TS could reduce the size of myocardial injury induced by ischemia. Patients who presented with STEMI within 12 h of symptom onset, treated with primary percutaneous coronary intervention, were randomized to the LL-TS group (n = 47) or the control group (with sham stimulation [n = 48]). LL-TS, 50% lower than the electric current that slowed the sinus rate, was delivered to the right tragus once the patients arrived in the catheterization room and lasted for 2 h after balloon dilatation (reperfusion). All patients were followed for 7 days. The occurrence of reperfusion-related arrhythmia, blood levels of creatine kinase-MB, myoglobin, N-terminal pro-B-type natriuretic peptide and inflammatory markers, and echocardiographic characteristics were evaluated. The incidence of reperfusion-related ventricular arrhythmia during the first 24 h was significantly attenuated by LL-TS. In addition, the area under the curve for creatine kinase-MB and myoglobin over 72 h was smaller in the LL-TS group than the control group. Furthermore, blood levels of inflammatory markers were decreased by LL-TS. Cardiac function, as demonstrated by the level of N-terminal pro-B-type natriuretic peptide, the left ventricular ejection fraction, and the wall motion index, was markedly improved by LL-TS. LL-TS reduces myocardial ischemia-reperfusion injury in patients with STEMI. This proof-of-concept study raises the possibility that this noninvasive strategy may be used to treat patients with STEMI undergoing primary percutaneous coronary intervention. Copyright © 2017. Published by Elsevier Inc.

Engineering antimicrobial peptides with improved antimicrobial and hemolytic activities.

PubMed

Zhao, Jun; Zhao, Chao; Liang, Guizhao; Zhang, Mingzhen; Zheng, Jie

2013-12-23

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water-membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide-lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs.

Antimicrobial peptides and plant disease control.

PubMed

Montesinos, Emilio

2007-05-01

Several diseases caused by viruses, bacteria and fungi affect plant crops, resulting in losses and decreasing the quality and safety of agricultural products. Plant disease control relies mainly on chemical pesticides that are currently subject to strong restrictions and regulatory requirements. Antimicrobial peptides are interesting compounds in plant health because there is a need for new products in plant protection that fit into the new regulations. Living organisms secrete a wide range of antimicrobial peptides produced through ribosomal (defensins and small bacteriocins) or non-ribosomal synthesis (peptaibols, cyclopeptides and pseudopeptides). Several antimicrobial peptides are the basis for the design of new synthetic analogues, have been expressed in transgenic plants to confer disease protection or are secreted by microorganisms that are active ingredients of commercial biopesticides.

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

PubMed

Taylor, Karen; Clarke, David J; McCullough, Bryan; Chin, Wutharath; Seo, Emily; Yang, De; Oppenheim, Joost; Uhrin, Dusan; Govan, John R W; Campopiano, Dominic J; MacMillan, Derek; Barran, Perdita; Dorin, Julia R

2008-03-14

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.

A folding-dependent mechanism of antimicrobial peptide resistance to degradation unveiled by solution structure of distinctin

PubMed Central

Raimondo, Domenico; Andreotti, Giuseppina; Saint, Nathalie; Amodeo, Pietro; Renzone, Giovanni; Sanseverino, Marina; Zocchi, Ivana; Molle, Gerard; Motta, Andrea; Scaloni, Andrea

2005-01-01

Many bioactive peptides, presenting an unstructured conformation in aqueous solution, are made resistant to degradation by posttranslational modifications. Here, we describe how molecular oligomerization in aqueous solution can generate a still unknown transport form for amphipathic peptides, which is more compact and resistant to proteases than forms related to any possible monomer. This phenomenon emerged from 3D structure, function, and degradation properties of distinctin, a heterodimeric antimicrobial compound consisting of two peptide chains linked by a disulfide bond. After homodimerization in water, this peptide exhibited a fold consisting of a symmetrical full-parallel four-helix bundle, with a well secluded hydrophobic core and exposed basic residues. This fold significantly stabilizes distinctin against proteases compared with other linear amphipathic peptides, without affecting its antimicrobial, hemolytic, and ion-channel formation properties after membrane interaction. This full-parallel helical orientation represents a perfect compromise between formation of a stable structure in water and requirement of a drastic structural rearrangement in membranes to elicit antimicrobial potential. Thus, distinctin can be claimed as a prototype of a previously unrecognized class of antimicrobial derivatives. These results suggest a critical revision of the role of peptide oligomerization whenever solubility or resistance to proteases is known to affect biological properties. PMID:15840728

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

PubMed

Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

2016-08-10

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1-35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Antimicrobial peptides from skin secretions of Hypsiboas pulchellus (Anura: Hylidae).

PubMed

Siano, Alvaro; Húmpola, María Verónica; de Oliveira, Eliandre; Albericio, Fernando; Simonetta, Arturo C; Lajmanovich, Rafael; Tonarelli, Georgina G

2014-04-25

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 μM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 μM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides.

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

Controlling the Biomimetic Implant Interface: Modulating Antimicrobial Activity by Spacer Design

NASA Astrophysics Data System (ADS)

Wisdom, Cate; Vanoosten, Sarah Kay; Boone, Kyle W.; Khvostenko, Dmytro; Arnold, Paul M.; Snead, Malcolm L.; Tamerler, Candan

2016-08-01

Surgical site infection is a common cause of post-operative morbidity, often leading to implant loosening, ultimately requiring revision surgery, increased costs and worse surgical outcomes. Since implant failure starts at the implant surface, creating and controlling the bio-material interface will play a critical role in reducing infection while improving host cell-to-implant interaction. Here, we engineered a biomimetic interface based upon a chimeric peptide that incorporates a titanium binding peptide (TiBP) with an antimicrobial peptide (AMP) into a single molecule to direct binding to the implant surface and deliver an antimicrobial activity against S. mutans and S. epidermidis, two bacteria which are linked with clinical implant infections. To optimize antimicrobial activity, we investigated the design of the spacer domain separating the two functional domains of the chimeric peptide. Lengthening and changing the amino acid composition of the spacer resulted in an improvement of minimum inhibitory concentration by a three-fold against S. mutans. Surfaces coated with the chimeric peptide reduced dramatically the number of bacteria, with up to a nine-fold reduction for S. mutans and a 48-fold reduction for S. epidermidis. Ab initio predictions of antimicrobial activity based on structural features were confirmed. Host cell attachment and viability at the biomimetic interface were also improved compared to the untreated implant surface. Biomimetic interfaces formed with this chimeric peptide offer interminable potential by coupling antimicrobial and improved host cell responses to implantable titanium materials, and this peptide based approach can be extended to various biomaterials surfaces.

Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants

PubMed Central

Yazici, Hilal; O'Neill, Mary B.; Kacar, Turgay; Wilson, Brandon R.; Oren, E. Emre; Sarikaya, Mehmet; Tamerler, Candan

2016-01-01

Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property. PMID:26795060

Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

PubMed

Yazici, Hilal; O'Neill, Mary B; Kacar, Turgay; Wilson, Brandon R; Oren, E Emre; Sarikaya, Mehmet; Tamerler, Candan

2016-03-02

Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

Characterization of the Antimicrobial Peptide Penisin, a Class Ia Novel Lantibiotic from Paenibacillus sp. Strain A3

PubMed Central

Baindara, Piyush; Chaudhry, Vasvi; Mittal, Garima; Liao, Luciano M.; Matos, Carolina O.; Khatri, Neeraj; Franco, Octavio L.; Patil, Prabhu B.

2015-01-01

Attempts to isolate novel antimicrobial peptides from microbial sources have been on the rise recently, despite their low efficacy in therapeutic applications. Here, we report identification and characterization of a new efficient antimicrobial peptide from a bacterial strain designated A3 that exhibited highest identity with Paenibacillus ehimensis. Upon purification and subsequent molecular characterization of the antimicrobial peptide, referred to as penisin, we found the peptide to be a bacteriocin-like peptide. Consistent with these results, RAST analysis of the entire genome sequence revealed the presence of a lantibiotic gene cluster containing genes necessary for synthesis and maturation of a lantibiotic. While circular dichroism and one-dimension nuclear magnetic resonance experiments confirmed a random coil structure of the peptide, similar to other known lantibiotics, additional biochemical evidence suggests posttranslational modifications of the core peptide yield six thioether cross-links. The deduced amino acid sequence of the putative biosynthetic gene penA showed approximately 74% similarity with elgicin A and 50% similarity with the lantibiotic paenicidin A. Penisin effectively killed methicillin-resistant Staphylococcus aureus (MRSA) and did not exhibit hemolysis activity. Unlike other lantibiotics, it effectively inhibited the growth of Gram-negative bacteria. Furthermore, 80 mg/kg of body weight of penisin significantly reduced bacterial burden in a mouse thigh infection model and protected BALB/c mice in a bacteremia model entailing infection with Staphylococcus aureus MTCC 96, suggesting that it could be a promising new antimicrobial peptide. PMID:26574006

Antimicrobial Peptides Derived from Fusion Peptides of Influenza A Viruses, a Promising Approach to Designing Potent Antimicrobial Agents.

PubMed

Wang, Jingyu; Zhong, Wenjing; Lin, Dongguo; Xia, Fan; Wu, Wenjiao; Zhang, Heyuan; Lv, Lin; Liu, Shuwen; He, Jian

2015-10-01

The emergence and dissemination of antibiotic-resistant bacterial pathogens have spurred the urgent need to develop novel antimicrobial agents with different mode of action. In this respect, we turned several fusogenic peptides (FPs) derived from the hemagglutinin glycoproteins (HAs) of IAV into potent antibacterials by replacing the negatively or neutrally charged residues of FPs with positively charged lysines. Their antibacterial activities were evaluated by testing the MICs against a panel of bacterial strains including S. aureus, S. mutans, P. aeruginosa, and E. coli. The results showed that peptides HA-FP-1, HA-FP-2-1, and HA-FP-3-1 were effective against both Gram-positive and Gram-negative bacteria with MICs ranging from 1.9 to 16.0 μm, while the toxicities toward mammalian cells were low. In addition, the mode of action and the secondary structure of these peptides were also discussed. These data not only provide several potent peptides displaying promising potential in development as broad antimicrobial agents, but also present a useful strategy in designing new antimicrobial agents. © 2015 John Wiley & Sons A/S.

Antimicrobial activity and mechanism of PDC213, an endogenous peptide from human milk.

PubMed

Sun, Yazhou; Zhou, Yahui; Liu, Xiao; Zhang, Fan; Yan, Linping; Chen, Ling; Wang, Xing; Ruan, Hongjie; Ji, Chenbo; Cui, Xianwei; Wang, Jiaqin

2017-02-26

Human milk has always been considered an ideal source of elemental nutrients to both preterm and full term infants in order to optimally develop the infant's tissues and organs. Recently, hundreds of endogenous milk peptides were identified in human milk. These peptides exhibited angiotensin-converting enzyme inhibition, immunomodulation, or antimicrobial activity. Here, we report the antimicrobial activity and mechanism of a novel type of human antimicrobial peptide (AMP), termed PDC213 (peptide derived from β-Casein 213-226 aa). PDC213 is an endogenous peptide and is present at higher levels in preterm milk than in full term milk. The inhibitory concentration curve and disk diffusion tests showed that PDC213 had obvious antimicrobial against S. aureus and Y. enterocolitica, the common nosocomial pathogens in neonatal intensive care units (NICUs). Fluorescent dye methods, electron microscopy experiments and DNA-binding activity assays further indicated that PDC213 can permeabilize bacterial membranes and cell walls rather than bind intracellular DNA to kill bacteria. Together, our results suggest that PDC213 is a novel type of AMP that warrants further investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use.

PubMed

Games, Patrícia Dias; daSilva, Elói Quintas Gonçalves; Barbosa, Meire de Oliveira; Almeida-Souza, Hebréia Oliveira; Fontes, Patrícia Pereira; deMagalhães, Marcos Jorge; Pereira, Paulo Roberto Gomes; Prates, Maura Vianna; Franco, Gloria Regina; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Baracat-Pereira, Maria Cristina

2016-12-15

Antimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves ('Magali R') are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses. Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature. Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessary.

De-Novo Design of Antimicrobial Peptides for Plant Protection

PubMed Central

Zeitler, Benjamin; Herrera Diaz, Areli; Dangel, Alexandra; Thellmann, Martha; Meyer, Helge; Sattler, Michael; Lindermayr, Christian

2013-01-01

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents. PMID:23951222

De-novo design of antimicrobial peptides for plant protection.

PubMed

Zeitler, Benjamin; Herrera Diaz, Areli; Dangel, Alexandra; Thellmann, Martha; Meyer, Helge; Sattler, Michael; Lindermayr, Christian

2013-01-01

This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of "healthy" food, these peptides might serve as templates for novel antibacterial and antifungal agents.

Small angle X-ray scattering as a high-throughput method to classify antimicrobial modes of action.

PubMed

von Gundlach, A R; Garamus, V M; Gorniak, T; Davies, H A; Reischl, M; Mikut, R; Hilpert, K; Rosenhahn, A

2016-05-01

Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

Conlon, J Michael; Al-Ghaferi, Nadia; Abraham, Bency; Leprince, Jérôme

2007-08-01

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.

Biosynthesis of the Polycyclic Antimicrobial Peptides Lacticin 481, Haloduracin, and Cinnamycin

ERIC Educational Resources Information Center

Cooper, Lisa E.

2009-01-01

Lantibiotics are bacterial-derived polycyclic antimicrobial peptides. They are genetically encoded and ribosomally synthesized as precursor peptides containing a structural region that undergoes post-translational modification and a leader sequence that is not modified. Specific serine and threonine residues in the pre-lantibiotic structural…

Antibiofilm and Antimicrobial Efficacy of DispersinB (registered trademark)-KSL-W Peptide-Based Wound Gel Against Chronic Wound Infection Associated Bacteria

DTIC Science & Technology

2014-01-21

Antibiofilm and Antimicrobial Efficacy of DispersinB-KSL-W Peptide-Based Wound Gel Against Chronic Wound Infection Associated Bacteria Purushottam V...major contributors to the slow or non-healing chronic wounds such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers. Being a protected...combination of DispersinB and KSL-W peptide showed synergistic antibiofilm and antimicrobial activity against chronic wound infection associated

Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax).

PubMed

Álvarez, Claudio Andrés; Acosta, Félix; Montero, Daniel; Guzmán, Fanny; Torres, Elisa; Vega, Belinda; Mercado, Luis

2016-08-01

The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species. Copyright © 2016 Elsevier Ltd. All rights reserved.

Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

PubMed

Sousa, Juliana C; Berto, Raquel F; Gois, Elicélia A; Fontenele-Cardi, Nauíla C; Honório, José E R; Konno, Katsuhiro; Richardson, Michael; Rocha, Marcos F G; Camargo, Antônio A C M; Pimenta, Daniel C; Cardi, Bruno A; Carvalho, Krishnamurti M

2009-07-01

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

A Rigidity-Enhanced Antimicrobial Activity: A Case for Linear Cationic α-Helical Peptide HP(2–20) and Its Four Analogues

PubMed Central

Liu, Li; Fang, Ying; Huang, Qingsheng; Wu, Jianhua

2011-01-01

Linear cationic α-helical antimicrobial peptides are referred to as one of the most likely substitutes for common antibiotics, due to their relatively simple structures (≤40 residues) and various antimicrobial activities against a wide range of pathogens. Of those, HP(2–20) was isolated from Helicobacter pylori ribosomal protein. To reveal a mechanical determinant that may mediate the antimicrobial activities, we examined the mechanical properties and structural stabilities of HP(2–20) and its four analogues of same chain length by steered molecular dynamics simulation. The results indicated the following: the resistance of H-bonds to the tensile extension mediated the early extensive stage; with the loss of H-bonds, the tensile force was dispensed to prompt the conformational phase transition; and Young's moduli (N/m2) of the peptides were about 4∼8×109. These mechanical features were sensitive to the variation of the residue compositions. Furthermore, we found that the antimicrobial activity is rigidity-enhanced, that is, a harder peptide has stronger antimicrobial activity. It suggests that the molecular spring constant may be used to seek a new structure-activity relationship for different α-helical peptide groups. This exciting result was reasonably explained by a possible mechanical mechanism that regulates both the membrane pore formation and the peptide insertion. PMID:21283643

Transcriptional Profiling of Mycobacterium tuberculosis Exposed to In Vitro Lysosomal Stress

PubMed Central

Lin, Wenwei; de Sessions, Paola Florez; Teoh, Garrett Hor Keong; Mohamed, Ahmad Naim Nazri; Zhu, Yuan O.; Koh, Vanessa Hui Qi; Ang, Michelle Lay Teng; Dedon, Peter C.; Hibberd, Martin Lloyd

2016-01-01

Increasing experimental evidence supports the idea that Mycobacterium tuberculosis has evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge of M. tuberculosis response to the hostile lysosomal environment, we profiled the global transcriptional activity of M. tuberculosis when exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication, M. tuberculosis expressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified the glgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection and in vitro stress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding gene Rv1258c was selected for validation. An M. tuberculosis ΔRv1258c mutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1. PMID:27324481

Molecular target of synthetic antimicrobial oligomer in bacterial membranes

NASA Astrophysics Data System (ADS)

Yang, Lihua; Gordon, Vernita; Som, Abhigyan; Cronan, John; Tew, Gregory; Wong, Gerard

2008-03-01

Antimicrobial peptides comprises a key component of innate immunity for a wide range of multicellular organisms. It has been shown that natural antimicrobial peptides and their synthetic analogs have demonstrated broad-spectrum antimicrobial activity via permeating bacterial membranes selectively. Synthetic antimicrobials with tunable structure and toxicological profiles are ideal for investigations of selectivity mechanisms. We investigate interactions and self-assembly using a prototypical family of antimicrobials based on phenylene ethynylene. Results from synchrotron small angle x-ray scattering (SAXS) results and in vitro microbicidal assays on genetically modified `knock-out' bacteria will be presented.

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin

PubMed Central

Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed

2015-01-01

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat. PMID:25956765

Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

PubMed

Singh, Shalini; Papareddy, Praveen; Kalle, Martina; Schmidtchen, Artur; Malmsten, Martin

2013-11-01

Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. Copyright © 2013. Published by Elsevier B.V.

Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide against Pseudomonas spp.

PubMed Central

Eckert, Randal; Qi, Fengxia; Yarbrough, Daniel K.; He, Jian; Anderson, Maxwell H.; Shi, Wenyuan

2006-01-01

Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designed Pseudomonas-specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics against Pseudomonas spp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminate Pseudomonas spp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics. PMID:16569868

In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

PubMed Central

Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian

2014-01-01

To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections. PMID:24982064

Long-pulsed 1064-nm Nd: YAG laser ameliorates LL-37-induced rosacea-like skin lesions through promoting collagen remodeling in BALB/c mice.

PubMed

Kim, Miri; Kim, Jongsic; Jeong, Seo-Won; Jo, Hyunmu; Park, Hyun Jeong

2018-02-01

Long-pulsed 1064-nm neodymium: yttrium-aluminum-garnet laser (LPND) effectively treats rosacea, although the underlying mechanism is unclear, to evaluate the histological effects and molecular mechanism of LPND on LL-37-induced rosacea-like skin lesions in mice. Intradermal injection of LL-37 was performed into the dorsal skin of BALB/c mice (n = 30) twice a day for 2 days. Fifteen mice were treated with LPND. After 48 h, the excised skin sample was stained for histology and type I collagen; transforming growth factor (TGF)-β, matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase (TIMP)-1, tumor necrosis factor (TNF)-α, and interleukin (IL)-1α mRNA levels were determined by real-time RT-PCR. Intradermal injection of LL-37 induced rosacea-like clinical features. LPND treatment significantly reduced erythema and increased dermal collagen production. Levels of Type I collagen, TGF-β, and MMP-1 mRNA were significantly higher in LPND-treated mice than in untreated mice. LPND may improve rosacea by ameliorating dermal connective tissue disorganization and elastosis through MMP-mediated dermal collagen remodeling.

Antimicrobial activity and interactions of cationic peptides derived from Galleria mellonella cecropin D-like peptide with model membranes.

PubMed

Oñate-Garzón, José; Manrique-Moreno, Marcela; Trier, Steven; Leidy, Chad; Torres, Rodrigo; Patiño, Edwin

2017-03-01

Antimicrobial peptides are effector molecules of the innate immune system against invading pathogens. The cationic charge in their structures has a strong correlation with antimicrobial activity, being responsible for the initial electrostatic interaction between peptides and the anionic microbial surface. This paper contains evidence that charge modification in the neutral peptide Gm cecropin D-like (WT) improved the antimicrobial activity of the modified peptides. Two cationic peptides derived from WT sequence named as ΔM1 and ΔM2, with net charge of +5 and +9, respectively, showed at least an eightfold increase in their antimicrobial activity in comparison to WT. The mechanism of action of these peptides was investigated using small unilamellar vesicles (SUVs) as model membranes. To study permeabilization effects of the peptides on cell membranes, entrapped calcein liposomes were used and the results showed that all peptides induced calcein release from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) SUVs, whereas in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), POPC/POPG and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG SUVs, only ΔM1 and ΔM2 induced a notable permeabilization. In addition, interactions of these peptides with phospholipids at the level of the glycerol backbone and hydrophobic domain were studied through observed changes in generalized polarization and fluorescence anisotropy using probes such as Laurdan and DPH, respectively. The results suggest that peptides slightly ordered the bilayer structure at the level of glycerol backbone and on the hydrophobic core in 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) SUVs, whereas in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/DMPG SUVs, only ΔM1 and ΔM2 peptides increased the order of bilayers. Thus, peptides would be inducing clustering of phospholipids creating phospholipid domains with a higher phase transition temperature.

A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs.

PubMed

Gordon, Y Jerold; Romanowski, Eric G; McDermott, Alison M

2005-07-01

Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Literature review. Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

USDA-ARS?s Scientific Manuscript database

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobia...

Purification, characterization and application of a novel antimicrobial peptide from Andrias davidianus blood.

PubMed

Pei, J; Feng, Z; Ren, T; Sun, H; Han, H; Jin, W; Dang, J; Tao, Y

2018-01-01

The Andrias davidianus has been known as a traditional Chinese medicine for a long time. Its blood is considered as a waste or by-product of the meat production industry. Although there are reports on isolation of the antimicrobial peptides from different resources, there are no reports of their isolation from A. davidianus blood. In this work, an antimicrobial peptide, andricin B, was isolated from the blood of A. davidianus by an innovative method in which the magnetic liposome adsorption was combined with reversed-phase high-performance liquid chromatography. The structure, antimicrobial activity and safety of andricin B were further investigated. Amino acid sequence was determined by N-terminal sequencing and found to be Gly-Leu-Thr-Arg-Leu-Phe-Ser-Val-Ile-Lys. Circular dichroism (CD) spectra and prediction of three-dimensional structure by bioinformatics software suggested the presence of a well-defined random coil conformation. Andricin B was found to be active against all bacteria tested in this study as well as some fungi. The minimum inhibitory concentrations (MICs) were in the range 8-64 μg ml -1 . Moreover, the haemolytic testing also suggested that andricin B could be considered safe at the MICs. Finally, andricin B was shown to inhibit the growth of Staphylococcus aureus in the cooked meat of A. davidianus. This study shows that andricin B is a promising novel antimicrobial peptide that may provide further insights towards the development of new drugs. This is the pioneer study on screening and isolation of antimicrobial peptide from the blood of Andrias davidianus. Here, we have developed a novel method by combining magnetic liposomes adsorption with reversed-phase high-performance liquid chromatography to purify and screen the antimicrobial peptides. From this screen, we identified a novel antimicrobial peptide which we name as andricin B. Andricin B is unique as it checks the growth of both Gram-positive and Gram-negative bacteria as well as few fungal species. © 2017 The Society for Applied Microbiology.

Different Interfacial Behaviors of N- and C-Terminus Cysteine-Modified Cecropin P1 Chemically Immobilized onto Polymer Surface

DTIC Science & Technology

2013-08-06

naturally occurring antimicrobial peptides (AMPs) have been studied as an alternative with a broad range of activity and binding affinity toward...microorganisms.10−16 For example, chemically immo- bilized cecropin P1, cecropin A, cecropin B, and other antimicrobial peptides have demonstrated promise for...Autom. 2006, 11 (6), 341−351. (10) Gregory, K.; Mello, C. M. Immobilization of Escherichia coli cells by use of the antimicrobial peptide cecropin P1

Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide

PubMed Central

Eckert, Randal ; He, Jian; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2006-01-01

Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora. PMID:17060534

Cationic peptides from peptic hydrolysates of rice endosperm protein exhibit antimicrobial, LPS-neutralizing, and angiogenic activities.

PubMed

Taniguchi, Masayuki; Kawabe, Junya; Toyoda, Ryu; Namae, Toshiki; Ochiai, Akihito; Saitoh, Eiichi; Tanaka, Takaaki

2017-11-01

In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte-free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC 50 value of 87μM. However, all five cationic peptides exhibited LPS-neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus.

PubMed

Grafskaia, Ekaterina N; Polina, Nadezhda F; Babenko, Vladislav V; Kharlampieva, Daria D; Bobrovsky, Pavel A; Manuvera, Valentin A; Farafonova, Tatyana E; Anikanov, Nikolay A; Lazarev, Vassili N

2018-04-01

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A.

PubMed

Sathyan, Naveen; Philip, Rosamma; Chaithanya, E R; Anil Kumar, P R; Sanjeevan, V N; Singh, I S Bright

2013-01-01

Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates.

Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A

PubMed Central

Sathyan, Naveen; Philip, Rosamma; Chaithanya, E. R.; Anil Kumar, P. R.; Sanjeevan, V. N.; Singh, I. S. Bright

2013-01-01

Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates. PMID:27398241

The Neuroendocrine Peptide Catestatin Is a Cutaneous Antimicrobial and Induced in the Skin after Injury

PubMed Central

Radek, Katherine A.; Lopez-Garcia, Belen; Hupe, Melanie; Niesman, Ingrid R.; Elias, Peter M.; Taupenot, Laurent; Mahata, Sushil K.; O’Connor, Daniel T.; Gallo, Richard L.

2009-01-01

Epithelia establish a microbial barrier against infection through the production of antimicrobial peptides (AMPs). In this study, we investigated whether catestatin (Cst), a peptide derived from the neuroendocrine protein chromogranin A (CHGA), is a functional AMP and is present in the epidermis. We show that Cst is antimicrobial against relevant skin microbes, including Gram-positive and Gram-negative bacteria, yeast, and fungi. The antimicrobial mechanism of Cst was found to be similar to other AMPs, as it was dependent on bacterial charge and growth conditions, and induced membrane disruption. The potential relevance of Cst against skin pathogens was supported by the observation that CHGA was expressed in keratinocytes. In human skin, CHGA was found to be proteolytically processed into the antimicrobial fragment Cst, thus enabling its AMP function. Furthermore, Cst expression in murine skin increased in response to injury and infection, providing potential for increased protection against infection. These data demonstrate that a neuroendocrine peptide has antimicrobial function against a wide assortment of skin pathogens and is upregulated upon injury, thus demonstrating a direct link between the neuroendocrine and cutaneous immune systems. PMID:18185531

Expression analysis and identification of antimicrobial peptide transcripts from six North American frog species

USGS Publications Warehouse

Robertson, Laura S.; Fellers, Gary M.; Marranca, Jamie Marie; Kleeman, Patrick M.

2013-01-01

Frogs secrete antimicrobial peptides onto their skin. We describe an assay to preserve and analyze antimicrobial peptide transcripts from field-collected skin secretions that will complement existing methods for peptide analysis. We collected skin secretions from 4 North American species in the field in California and 2 species in the laboratory. Most frogs appeared healthy after release; however, Rana boylii in the Sierra Nevada foothills, but not the Coast Range, showed signs of morbidity and 2 died after handling. The amount of total RNA extracted from skin secretions was higher in R. boylii and R. sierrae compared to R. draytonii, and much higher compared to Pseudacris regilla. Interspecies variation in amount of RNA extracted was not explained by size, but for P. regilla it depended upon collection site and date. RNA extracted from skin secretions from frogs handled with bare hands had poor quality compared to frogs handled with gloves or plastic bags. Thirty-four putative antimicrobial peptide precursor transcripts were identified. This study demonstrates that RNA extracted from skin secretions collected in the field is of high quality suitable for use in sequencing or quantitative PCR (qPCR). However, some species do not secrete profusely, resulting in very little extracted RNA. The ability to measure transcript abundance of antimicrobial peptides in field-collected skin secretions complements proteomic analyses and may provide insight into transcriptional mechanisms that could affect peptide abundance.

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

In Silico Template Selection of Short Antimicrobial Peptide Viscotoxin for Improving Its Antimicrobial Efficiency in Development of Potential Therapeutic Drugs.

PubMed

Senthilkumar, B; Rajasekaran, R

2017-03-01

Rapid increase in antibiotic resistance has posed a worldwide threat, due to increased mortality, morbidity, and expenditure caused by antibiotic-resistant microbes. Recent development of the antimicrobial peptides like viscotoxin (Vt) has been successfully comprehended as a substitute for classical antibiotics. A structurally stable peptide, Vt can enhance antimicrobial property and can be used for various developmental purposes. Thus, structural stability among the antimicrobial peptides, Vt A1 (3C8P), A2 (1JMN), A3 (1ED0), B (1JMP), and C (1ORL) of Viscus album was computationally analyzed. In specific, the static confirmation of VtA3 showed high number of intramolecular interactions, along with an increase in hydrophobicity than others comparatively. Further, conformational sampling was used to analyze various geometrical parameters such as root mean square deviation, root mean square fluctuation, radius of gyration, and ovality which also revealed the structural stability of VtA3. Moreover, the statistically validated contours of surface area, lipophilicity, and distance constraints of disulfide bonds also supported the priority of VtA3 with respect to stability. Finally, the functional activity of peptides was accessed by computing their free energy of membrane association and membrane interactions, which defined VtA3 as functionally stable. Currently, peptide-based antibiotics and nanoparticles have attracted the pharmaceutical industries for their potential therapeutic applications. Thereby, it is proposed that viscotoxin A3 (1ED0) could be used as a preeminent template for scaffolding potentially efficient antimicrobial peptide-based drugs and nanomaterials in future.

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

PubMed

Park, Yoonkyung; Park, Seong-Cheol; Park, Hae-Kyun; Shin, Song Yub; Kim, Yangmee; Hahm, Kyung-Soo

2007-01-01

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.

Biological Applications of Designed Hairpin Peptides: As Antimicrobials and as Inhibitors of Amyloidogenesis

NASA Astrophysics Data System (ADS)

Sivanesam, Kalkena

More than 40 diseases have been associated with the misfolding of peptides (or proteins) that form fibrils with a very specific morphology. These peptides classified as amyloidogenic peptides have been implicated in the development of Alzheimer's Disease, Parkinson's Disease, Type II Diabetes, Hungtinton's Disease etc. To date, these diseases have no cure, only therapies that can ameliorate the symptoms to a degree. Inhibition of the amyloidogenesis of these peptides has been proposed as a possible treatment option. While small molecules have been heavily tested as inhibitors of amyloidogenesis, peptides have emerged as potential inhibitors. In this work, the ability of a set of designed hairpin peptides to inhibit the amyloidogenesis of two different systems, alpha-synuclein (implicated in Parkinson's Disease) and human amylin (implicated in Type II Diabetes) is tested. Using circular dichroism and thioflavin T fluorescence, the ability of these peptides to inhibit amyloidogenesis is tested. The binding loci of these inhibitors to alpha-synuclein are also explored. The use of peptides as antimicrobials on the other hand is not a novel concept. However, most antimicrobial peptides, both natural and designed, rely heavily on covalent stabilizations in order to maintain secondary structure. In this study, non-covalent stabilizations are applied to a couple of natural as well as designed antimicrobials in order to study the effects of secondary structure stabilization on biological activity.

Cationic antimicrobial peptides inactivate Shiga toxin-encoding bacteriophages

NASA Astrophysics Data System (ADS)

Del Cogliano, Manuel E.; Hollmann, Axel; Martinez, Melina; Semorile, Liliana; Ghiringhelli, Pablo D.; Maffía, Paulo C.; Bentancor, Leticia V.

2017-12-01

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: 1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, 2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and 3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

PubMed Central

Benincasa, Monica; Lagatolla, Cristina; Dolzani, Lucilla; Milan, Annalisa; Pacor, Sabrina; Liut, Gianfranco; Tossi, Alessandro; Cescutti, Paola; Rizzo, Roberto

2016-01-01

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation. PMID:27681920

Bifidobacterium bifidum in a Rat Model of Necrotizing Enterocolitis: Antimicrobial Peptide and Protein Responses

PubMed Central

Underwood, Mark A.; Kananurak, Anchasa; Coursodon, Christine F.; Adkins-Reick, Camille K.; Chu, Hiutung; Bennett, Stephen H.; Wehkamp, Jan; Castillo, Patricia A.; Leonard, Brian C.; Tancredi, Daniel J.; Sherman, Michael P.; Dvorak, Bohuslav; Bevins, Charles L.

2013-01-01

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear. We report that like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF+BIF). All groups were exposed to asphyxia and cold stress. The expression of lysozyme, secretory phospholipase A2, pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF (NEC) group, compared to the DF and FF+BIF groups where disease was attenuated. We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut. PMID:22322385

Ocellatins: new antimicrobial peptides from the skin secretion of the South American frog Leptodactylus ocellatus (Anura: Leptodactylidae).

PubMed

Nascimento, Anna Christina C; Zanotta, Lanuse C; Kyaw, Cynthia M; Schwartz, Elisabeth N F; Schwartz, Carlos A; Sebben, Antonio; Sousa, Marcelo V; Fontes, Wagner; Castro, Mariana S

2004-11-01

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (1GVVDILKGAGKDLLAHLVGKISEKV25-CONH2), ocellatin 2 (1GVLDIFKDAAKQILAHAAEKQI25-CONH2) and ocellatin 3 (1GVLDILKNAAKNILAHAAEQI21-CONH2) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin.

PubMed

Xiang, Ning; Lyu, Yuan; Zhu, Xiao; Bhunia, Arun K; Narsimhan, Ganesan

2016-11-01

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test. Copyright © 2016 Elsevier Inc. All rights reserved.

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni

PubMed Central

Falkenberg, Shollie M.; Briggs, Robert E.; Tatum, Fred M.; Sacco, Randy E.

2017-01-01

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2–5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10–30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates. PMID:28827826

Antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni.

PubMed

Dassanayake, Rohana P; Falkenberg, Shollie M; Briggs, Robert E; Tatum, Fred M; Sacco, Randy E

2017-01-01

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2-5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10-30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates.

Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

PubMed Central

Lee, Judy T. Y.; Wang, Guangshun; Tam, Yu Tong; Tam, Connie

2016-01-01

Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics. PMID:27891122

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

PubMed

Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

2016-03-02

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships

PubMed Central

Levinson, Pauline; Choi, Robert Y.; Cole, Amy L.; Hirbod, Taha; Rhedin, Samuel; Payne, Barbara; Guthrie, Brandon L.; Bosire, Rose; Cole, Alexander M.; Farquhar, Carey; Broliden, Kristina

2012-01-01

Background HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection. Methodology/Principal Findings Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity. Conclusions/Significance These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules. PMID:22389677

[Application on food preservative of antimicrobial peptides].

PubMed

Zhao, Hongyan; Mu, Yu; Zhao, Baohua

2009-07-01

Antimicrobial peptides are an integral component of the innate immune system, it can counteract outer membrane pathogen such as bacteria, fungi, viruses, protozoan and so on. Owing to the sterilization and innocuity, it has the potential to be crude food preservative. In this paper the uses of antibacterial peptides in the food preservative were analyzed.

Identification of novel Amurin-2 variants from the skin secretion of Rana amurensis, and the design of cationicity-enhanced analogues.

PubMed

Zhang, Luyao; Chen, Xiaoling; Zhang, Ying; Ma, Chengbang; Xi, Xinping; Wang, Lei; Zhou, Mei; Burrows, James F; Chen, Tianbao

2018-03-18

Rana amurensis is important in Chinese medicine as its skin secretions contain abundant bioactive peptides. Here, we have identified the antimicrobial peptide Amurin-2 and three highly-conserved variants, Amurin-2a, Amurin-2b and Amurin-2c through a combination of molecular cloning and MS/MS fragmentation sequencing. Synthetic replicates of these peptides demonstrate potent antimicrobial activity against S. aureus, whilst some have activity against C.albicans and even resistant bacterial MRSA. Furthermore, two Lys-analogues (K 4 -Amurin-2 and K 11 -Amurin-2) were designed to improve the bioactive function and the antimicrobial activity of K 4 -Amurin-2 against E.coli was enhanced distinctly. In addition, the two modified peptides also showed more potent activity against S. aureus, C. albicans and MRSA strains. Meanwhile, these peptides showed inhibitory effect on the cell viability of several cancer cells. As a result, these structural and functional studies of Amurin-2 variants and analogues could provide insights for future antimicrobial peptide design. Copyright © 2018. Published by Elsevier Inc.

Membrane interaction of chrysophsin-1, a histidine-rich antimicrobial peptide from red sea bream.

PubMed

Mason, A James; Bertani, Philippe; Moulay, Gilles; Marquette, Arnaud; Perrone, Barbara; Drake, Alex F; Kichler, Antoine; Bechinger, Burkhard

2007-12-25

Chrysophsin-1 is an amphipathic alpha-helical antimicrobial peptide produced in the gill cells of red sea bream. The peptide has broad range activity against both Gram-positive and Gram-negative bacteria but is more hemolytic than other antimicrobial peptides such as magainin. Here we explore the membrane interaction of chrysophsin-1 and determine its toxicity, in vitro, for human lung fibroblasts to obtain a mechanism for its antimicrobial activity and to understand the role of the unusual C-terminal RRRH sequence. At intermediate peptide concentrations, solid-state NMR methods reveal that chrysophsin-1 is aligned parallel to the membrane surface and the lipid acyl chains in mixed model membranes are destabilized, thereby being in agreement with models where permeabilization is an effect of transient membrane disruption. The C-terminal RRRH sequence was shown to have a large effect on the insertion of the peptide into membranes with differing lipid compositions and was found to be crucial for pore formation and toxicity of the peptide to fibroblasts. The combination of biophysical data and cell-based assays suggests likely mechanisms involved in both the antibiotic and toxic activity of chrysophsins.

Antibacterial and anti-inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis.

PubMed

Bezzerri, Valentino; Avitabile, Concetta; Dechecchi, Maria Cristina; Lampronti, Ilaria; Borgatti, Monica; Montagner, Giulia; Cabrini, Giulio; Gambari, Roberto; Romanelli, Alessandra

2014-10-01

Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1β, IL-6 and tumor necrosis factor-α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

A novel chimeric peptide with antimicrobial activity.

PubMed

Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

2015-04-01

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Archetypal tryptophan-rich antimicrobial peptides: properties and applications.

PubMed

Shagaghi, Nadin; Palombo, Enzo A; Clayton, Andrew H A; Bhave, Mrinal

2016-02-01

Drug-resistant microorganisms ('superbugs') present a serious challenge to the success of antimicrobial treatments. Subsequently, there is a crucial need for novel bio-control agents. Many antimicrobial peptides (AMPs) show a broad-spectrum activity against bacteria, fungi or viruses and are strong candidates to complement or substitute current antimicrobial agents. Some AMPs are also effective against protozoa or cancer cells. The tryptophan (Trp)-rich peptides (TRPs) are a subset of AMPs that display potent antimicrobial activity, credited to the unique biochemical properties of tryptophan that allow it to insert into biological membranes. Further, many Trp-rich AMPs cross bacterial membranes without compromising their integrity and act intracellularly, suggesting interactions with nucleic acids and enzymes. In this work, we overview some archetypal TRPs derived from natural sources, i.e., indolicidin, tritrpticin and lactoferricin, summarising their biochemical properties, structures, antimicrobial activities, mechanistic studies and potential applications.

Predictive Model of Linear Antimicrobial Peptides Active against Gram-Negative Bacteria.

PubMed

Vishnepolsky, Boris; Gabrielian, Andrei; Rosenthal, Alex; Hurt, Darrell E; Tartakovsky, Michael; Managadze, Grigol; Grigolava, Maya; Makhatadze, George I; Pirtskhalava, Malak

2018-05-29

Antimicrobial peptides (AMPs) have been identified as a potential new class of anti-infectives for drug development. There are a lot of computational methods that try to predict AMPs. Most of them can only predict if a peptide will show any antimicrobial potency, but to the best of our knowledge, there are no tools which can predict antimicrobial potency against particular strains. Here we present a predictive model of linear AMPs being active against particular Gram-negative strains relying on a semi-supervised machine-learning approach with a density-based clustering algorithm. The algorithm can well distinguish peptides active against particular strains from others which may also be active but not against the considered strain. The available AMP prediction tools cannot carry out this task. The prediction tool based on the algorithm suggested herein is available on https://dbaasp.org.

On the Functional Overlap between Complement and Anti-Microbial Peptides.

PubMed

Zimmer, Jana; Hobkirk, James; Mohamed, Fatima; Browning, Michael J; Stover, Cordula M

2014-01-01

Intriguingly, activated complement and anti-microbial peptides share certain functionalities; lytic, phagocytic, and chemo-attractant activities and each may, in addition, exert cell instructive roles. Each has been shown to have distinct LPS detoxifying activity and may play a role in the development of endotoxin tolerance. In search of the origin of complement, a functional homolog of complement C3 involved in opsonization has been identified in horseshoe crabs. Horseshoe crabs possess anti-microbial peptides able to bind to acyl chains or phosphate groups/saccharides of endotoxin, LPS. Complement activity as a whole is detectable in marine invertebrates. These are also a source of anti-microbial peptides with potential pharmaceutical applicability. Investigating the locality for the production of complement pathway proteins and their role in modulating cellular immune responses are emerging fields. The significance of local synthesis of complement components is becoming clearer from in vivo studies of parenchymatous disease involving specifically generated, complement-deficient mouse lines. Complement C3 is a central component of complement activation. Its provision by cells of the myeloid lineage varies. Their effector functions in turn are increased in the presence of anti-microbial peptides. This may point to a potentiating range of activities, which should serve the maintenance of health but may also cause disease. Because of the therapeutic implications, this review will consider closely studies dealing with complement activation and anti-microbial peptide activity in acute inflammation (e.g., dialysis-related peritonitis, appendicitis, and ischemia).

A Review of Antimicrobial Peptides and Their Therapeutic Potential as Anti-Infective Drugs

PubMed Central

Gordon, Y. Jerold; Romanowski, Eric G.; McDermott, Alison M.

2006-01-01

Purpose. Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Methods. Literature review. Results. Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. Conclusions. While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven. PMID:16020284

Natural antimicrobial peptides as promising anti-HIV candidates

PubMed Central

Wang, Guangshun

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) infection remains to be one of the major global health problems. It is thus necessary to identify novel therapeutic molecules to combat HIV-1. Natural antimicrobial peptides (AMPs) have been recognized as promising templates for developing topical microbicides. This review systematically discusses over 80 anti-HIV peptides annotated in the antimicrobial peptide database (http://aps.unmc.edu/AP). Such peptides have been discovered from bacteria, plants, and animals. Examples include gramicidin and bacteriocins from bacteria, cyclotides from plants, melittins and cecropins from insects, piscidins from fish, ascaphins, caerins, dermaseptins, esculentins, and maximins from amphibians, and cathelicidins and defensins from vertebrates. These peptides appear to work by different mechanisms and could block viral entry in multiple ways. As additional advantages, such anti-HIV peptides may possess other desired features such as antibacterial, antiparasital, spermicidal, and anticancer activity. With continued optimization of peptide stability, production, formulation and delivery methods, it is anticipated that some of these compounds may eventually become new anti-HIV drugs. PMID:26834391

Impedimetric detection of pathogenic Gram-positive bacteria using an antimicrobial peptide from class IIa bacteriocins.

PubMed

Etayash, Hashem; Jiang, Keren; Thundat, Thomas; Kaur, Kamaljit

2014-02-04

Real-time, label-free detection of Gram-positive bacteria with high selectivity and sensitivity is demonstrated using an interdigitated impedimetric array functionalized with naturally produced antimicrobial peptide from class IIa bacteriocins. The antimicrobial peptide, leucocin A, was chemically synthesized and covalently immobilized on interdigitated gold microelectrodes via the interaction between the C-terminal carboxylic acid of the peptide and free amines of a preattached thiolated linker. Exposing the peptide sensor to various concentrations of Gram-positive bacteria generated reproducible impedance spectra that detected peptide-bacteria interactions at a concentration of 1 cell/μL. The peptide sensor also selectively detected Listeria monocytogenes from other Gram-positive strains at a concentration of 10(3) cfu mL(-1). The study highlights that short peptide ligands from bacteriocin class offer high selectivity in bacterial detection and can be used in developing a robust, portable biosensor device to efficiently detect pathogenic Gram-positive bacteria in food samples.

The unconventional antimicrobial peptides of the classical propionibacteria.

PubMed

Faye, Therese; Holo, Helge; Langsrud, Thor; Nes, Ingolf F; Brede, Dag A

2011-02-01

The classical propionibacteria produce genetically unique antimicrobial peptides, whose biological activities are without equivalents, and to which there are no homologous sequences in public databases. In this review, we summarize the genetics, biochemistry, biosynthesis, and biological activities of three extensively studied antimicrobial peptides from propionibacteria. The propionicin T1 peptide constitutes a bona fide example of an unmodified general secretory pathway (sec)-dependent bacteriocin, which is bactericidal towards all tested species of propionibacteria except Propionibacterium freudenreichii. The PAMP antimicrobial peptide represents a novel concept within bacterial antagonism, where an inactive precursor protein is secreted in large amounts, and which activation appears to rely on subsequent processing by proteases in its resident milieu. Propionicin F is a negatively charged bacteriocin that displays an intraspecies bactericidal inhibition spectrum. The biosynthesis of propionicin F appears to proceed through a series of unusual events requiring both N- and C-terminal processing of a precursor protein, which probably requires the radical SAM superfamily enzyme PcfB.

Rational design of anti-microbial peptides with enhanced activity and low cytotoxicity based on the structure of the arginine/histidine-rich peptide, chensinin-1.

PubMed

Shang, D; Sun, Y; Wang, C; Ma, L; Li, J; Wang, X

2012-09-01

To understand the structure-activity relationship of chensinin-1, a anti-microbial peptide (AMP) with an unusual structure, and to develop novel AMPs as therapeutic agents. A series of chensinin-1 analogues were designed and synthesized by one to three replacement of glycines with leucines at the hydrophilic face of chensinin-1 or rearrangement of some of the residues in its sequence. Circular dichroism spectroscopy showed that the analogues adopted α-helical-type conformations in 50% trifluoroethanol/water but adopted β-strand-type conformations in 30 mmol l(-1) sodium dodecyl sulphate. The anti-microbial activities of the peptides against Gram-positive bacteria increased 5- to 30-fold, and these increases paralleled the increases in the peptides' hydrophobicities. Their haemolytic activities also increased. Amphipathicities had little influence on the bactericidal activity of chensinin-1. All peptides caused leakage of calcein entrapped in negatively charged liposomes although with different efficiencies. The peptides did not induce leakage of calcein from uncharged liposomes. Peptide adopted an aperiodic structure can improve the anti-microbial potency by increasing peptide hydrophobicity. Its target is bacteria plasma membrane. Chensinin-1 can act as a new lead molecule for the study of AMPs with atypical structures. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Influence of Bacterial Biofilm Polysaccharide Structure on Interactions with Antimicrobial Peptides: A Study on Klebsiella pneumoniae.

PubMed

Bellich, Barbara; Lagatolla, Cristina; Tossi, Alessandro; Benincasa, Monica; Cescutti, Paola; Rizzo, Roberto

2018-06-06

Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedding cells. They provide advantages to bacteria including protection against antimicrobials. The protection given by biofilms produced by Klebsiella pneumoniae strains towards antimicrobial peptides of the innate immune system was investigated. In particular, the role of matrix bacterial exopolysaccharides was explored. Three clinical strains producing exopolysaccharides with different chemistry were selected and the interaction of purified biofilm polysaccharides with two bovine cathelicidins was studied by circular dichroism spectroscopy and microbiological assays to establish their influence on the peptide’s antimicrobial activity. The spectroscopic data indicated a different extent of interaction with the two peptides, in a manner dependent on their sugar composition, and in particular the presence of rhamnose residues correlated with a lower interaction. The extent of interaction was then related to the protection towards antimicrobial peptides, conferred by the addition of the different exopolysaccharides, in minimum inhibitory concentration (MIC) assays against a reference Escherichia coli strain. Microbiological results were in very good agreement with spectroscopic data, confirming the active role of matrix polysaccharides in determining a biofilm’s protective capacity and indicating lower protection levels afforded by rhamnose containing exopolysaccharides.

Effects of lactoferrin derived peptides on simulants of biological warfare agents.

PubMed

Sijbrandij, Tjitske; Ligtenberg, Antoon J; Nazmi, Kamran; Veerman, Enno C I; Bolscher, Jan G M; Bikker, Floris J

2017-01-01

Lactoferrin (LF) is an important immune protein in neutrophils and secretory fluids of mammals. Bovine LF (bLF) harbours two antimicrobial stretches, lactoferricin and lactoferampin, situated in close proximity in the N1 domain. To mimic these antimicrobial domain parts a chimeric peptide (LFchimera) has been constructed comprising parts of both stretches (LFcin17-30 and LFampin265-284). To investigate the potency of this construct to combat a set of Gram positive and Gram negative bacteria which are regarded as simulants for biological warfare agents, the effect on bacterial killing, membrane permeability and membrane polarity were determined in comparison to the constituent peptides and the native bLF. Furthermore we aimed to increase the antimicrobial potency of the bLF derived peptides by cationic amino acid substitutions. Overall, the bactericidal activity of the peptides could be related to membrane disturbing effects, i.e. membrane permeabilization and depolarization. Those effects were most prominent for the LFchimera. Arginine residues were found to be crucial for displaying antimicrobial activity, as lysine to arginine substitutions resulted in an increased antimicrobial activity, affecting mostly LFampin265-284 whereas arginine to lysine substitutions resulted in a decreased bactericidal activity, predominantly in case of LFcin17-30.

Antimicrobial efficacy of granulysin-derived synthetic peptides in acne vulgaris.

PubMed

Lim, Hee-Sun; Chun, Seung-Min; Soung, Min-Gyu; Kim, Jenny; Kim, Seong-Jin

2015-07-01

Antimicrobial peptides are considered as a potential alternative to antibiotic treatment in acne vulgaris because the development of a resistant strain of Propionibacterium acnes is problematic. Granulysin can be regarded as an ideal substance with which to treat acne because it has antimicrobial and anti-inflammatory effects. This study was performed to explore the effectiveness of granulysin-derived peptides (GDPs) in killing P. acnes in vitro under a standard microbiologic assay and to evaluate their potential use in a topical agent for the treatment of acne vulgaris. Twenty different peptides based on the known sequence of a GDP were synthesized and tested in vitro for antimicrobial activity. Thirty patients with facial acne vulgaris were instructed to apply a topical formulation containing synthetic GDP to acne lesions twice per day for 12 weeks. A newly synthesized peptide in which aspartic acid was substituted with arginine, and methionine was substituted with cysteine, showed the highest antimicrobial activity against P. acnes. Moreover, it was effective against both Gram-positive and Gram-negative bacteria in vitro. After treatment with the topical formulation containing 50 ppm of synthetic peptide for 12 weeks, a significant reduction in the number of pustules was observed, regardless of the increase in the number of comedones. In addition, a significant reduction in the clinical grade of acne based on the Korean Acne Grading System (KAGS) was evident. Synthesized GDP shows strong antimicrobial activity against P. acnes in vitro. The clinical improvement observed suggests a topical formulation containing the GDP has therapeutic potential for the improvement of inflammatory-type acne vulgaris by its antimicrobial activity. © 2015 The International Society of Dermatology.

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

PubMed

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

PubMed Central

Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Small molecule mimics of DFTamP1, a database designed anti-Staphylococcal peptide

PubMed Central

Dong, Yuxiang; Lushnikova, Tamara; Golla, Radha M.; Wang, Xiaofang; Wang, Guangshun

2017-01-01

Antimicrobial peptides (AMPs) are important templates for developing new antimicrobial agents. Previously, we developed a database filtering technology that enabled us to design a potent anti-Staphylococcal peptide DFTamP1. Using this same design approach, we now report the discovery of a new class of bis-indole diimidazolines as AMP small molecule mimics. The best compound killed multiple S. aureus clinical strains in both planktonic and biofilm forms. The compound appeared to target bacterial membranes with antimicrobial activity and membrane permeation ability similar to daptomycin. PMID:28011203

Identification, Characterization, Immunolocalization, and Biological Activity of Lucilin Peptide.

PubMed

Alberto, Tellez German; Alejandra, Zapata Jesica; Johanna, Toro Lily; Carolina, Henao Diana; Pablo, Bedoya Juan; David, Rivera Juan; Valentin, Trujillo Juan; Bruno, Rivas; Lopez, Richard Onalbi Hoyos; Carlos, Castano Jhon

2018-06-08

Maggots from the Lucilia sp. genus are used for debridement of infected and necrotic wounds. Broad-spectrum antimicrobial activity has been described in the excretion/secretions (ES 1 ) of these larvae. This study identifies the genetic sequence of a cecropin-like antimicrobial peptide from Lucilia eximia. Total RNA was extracted and used for PCR-RACE amplification of a cecropin, the native peptide was immunolocalized in the tissues and secretions of the larvae, and a synthetic analog was used to explore its antimicrobial, cytotoxic, LPS neutralizing and wound-healing activities in vitro. The genetic cDNA sequence of a cecropin-like antimicrobial peptide in L. eximia called "Lucilin" was amplified, corresponding to 63 aa completed protein and 40 aa mature peptide; the structure of the mature peptide was predicted as an α-helix. The peptide was immunolocalized in the salivary glands, fat body, the ES, and hemolymph of the maggots. Lucilin synthetic peptide analog was active against E. coli DH10B with a MIC 2 of 7.8 µg/mL, E. coli extended spectrum b-lactamase (ESBL) (MIC: 15.6 µg/mL), and Enterobacter cloacae (MIC: 125 µg/mL), but it was not active against Pseudomonas aeruginosa and Staphylococcus epidermidis; and had no cytotoxic or hemolytic activity. It showed immunomodulatory activity against human peripheral blood mononuclear cells (PBMCs) stimulated with LPS, reducing the TNF-α production when treated at 17 µg/mL and induces cell migration of Hacat at 5 and 50 µg/mL. Lucilin is a cecropin-like peptide from L. eximia with antimicrobial activity against Gram negative bacteria and immunomodulatory activities, decreasing the TNF-α production in PBMCs and inducing cellular migration in human keratinocytes. Copyright © 2018. Published by Elsevier B.V.

Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish.

PubMed

Kütt, Mary-Liis; Stagsted, Jan

2014-09-01

A model system of bovine colostrum and piscidin, a fish-derived antimicrobial peptide, was developed to study potential interactions of antimicrobial peptides in colostrum. We did not detect any antimicrobial activity of colostrum using the radial plate diffusion assay; in fact colostrum completely abrogated activity of added piscidin. This could not be explained by degradation of piscidin by colostrum, which was less than ten percent. We found that colostrum even protected piscidin against degradation by added proteases. We further observed that colostrum and milk rapidly quenched the fluorescence of fluorescein-piscidin but not that of fluorescein. This effect was not seen with BSA and the specific quenching of fluorescein-piscidin by colostrum was saturably inhibited with unlabeled piscidin. Size exclusion chromatography indicated that fluorescein-piscidin bound to casein micelles with no apparent binding to IgG or whey proteins. Further, addition of pure caseins was able to quench fluorescence of fluorescein-piscidin and to inhibit the antimicrobial activity of piscidin. The interaction between caseins and piscidin could be dissociated by guanidine hydrochloride and recovered piscidin had antimicrobial activity against bacteria. Based on our results we propose that caseins could be carriers for antimicrobial peptides in colostrum and milk. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of natural antimicrobial agents to treat dengue infection: In vitro analysis of latarcin peptide activity against dengue virus.

PubMed

Rothan, Hussin A; Bahrani, Hirbod; Rahman, Noorsaadah Abd; Yusof, Rohana

2014-05-31

Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells. The Ltc 1 peptide showed a significantly inhibitory effect against the dengue protease NS2B-NS3pro at 37°C, a physiological human temperature, (IC50, 12.68 ± 3.2 μM), and greater inhibitory effect was observed at 40°C, a temperature similar to a high fever (IC50, 6.58 ± 4.1 μM). A greater reduction in viral load (p.f.u./ml) was observed at simultaneous (0.7 ± 0.3 vs. 7.2 ± 0.5 control) and post-treatment (1.8 ± 0.7 vs. 6.8 ± 0.6 control) compared to the pre-treatment (4.5 ± 0.6 vs. 6.9 ± 0.5 control). Treatment with the Ltc 1 peptide reduced the viral RNA in a dose-dependent manner with EC50 values of 8.3 ± 1.2, 7.6 ± 2.7 and 6.8 ± 2.5 μM at 24, 48 and 72 h, respectively. The Ltc 1 peptide exhibited significant inhibitory effects against dengue NS2B-NS3pro and virus replication in the infected cells. Therefore, further investigation is necessary to develop the Ltc 1 peptide as a new anti-dengue therapeutic.

Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1

PubMed Central

Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

2015-01-01

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484] PMID:26129676

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ribeiro, Marta M.B.; Franquelim, Henri G.; Torcato, Ines M.

Highlights: Black-Right-Pointing-Pointer New kyotorphin derivatives have antimicrobial properties against S. aureus. Black-Right-Pointing-Pointer Atomic force microscopy show membrane disturbing effects of KTP-NH{sub 2} and IbKTP-NH{sub 2}. Black-Right-Pointing-Pointer None of the KTP derivatives are hemolytic. Black-Right-Pointing-Pointer The minimal peptidic sequence with antimicrobial activity is Tyr-Arg, if amidated. -- Abstract: Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was definedmore » as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH{sub 2}, IbKTP, IbKTP-NH{sub 2} - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.« less

Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae).

PubMed

King, Jay D; Leprince, Jérôme; Vaudry, Hubert; Coquet, Laurent; Jouenne, Thierry; Conlon, J Michael

2008-08-01

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Caribbean frog Leptodactylus validus Garman, 1888 led to the identification of three peptides with previously undescribed sequences that were structurally similar to those of antimicrobial peptides isolated from other species of leptodactylid frogs. These paralogs have been termed ocellatin-V1 (GVVDILKGAGKDLLAHALSKLSEKV.NH(2)), ocellatin-V2 (GVLDILKGAGKDLLAHALSKISEKV.NH(2)), and ocellatin-V3 (GVLDILTGAGKDLLAHALSKLSEKV.NH(2)). The very low antimicrobial potency (MIC>200microM) against Escherichia coli and Staphylococcus aureus associated with the peptides is probably a consequence of their lack of amphipathicity and reduced cationicity compared with active members of the ocellatin family from related species.

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

PubMed

Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

2015-07-01

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins

PubMed Central

Brahma, Biswajit; Patra, Mahesh Chandra; Karri, Satyanagalakshmi; Chopra, Meenu; Mishra, Purusottam; De, Bidhan Chandra; Kumar, Sushil; Mahanty, Sourav; Thakur, Kiran; Poluri, Krishna Mohan; Datta, Tirtha Kumar; De, Sachinandan

2015-01-01

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics. PMID:26675301

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

PubMed Central

2012-01-01

Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics. PMID:22947243

Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness.

PubMed

Nadal, Anna; Montero, Maria; Company, Nuri; Badosa, Esther; Messeguer, Joaquima; Montesinos, Laura; Montesinos, Emilio; Pla, Maria

2012-09-04

The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER), analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der) was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII) transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM) plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP), had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM plants expressing, for example, BP100 based on inverted repeats, have adequate agronomic performance and resistant phenotypes as a result of a complex equilibrium between bp100der toxicity to plant cells, antimicrobial activity and transgene-derived plant stress response. It is likely that these results can be extended to other peptides with similar characteristics.

Can antimicrobial peptides scavenge around a cell in less than a second?

PubMed

Chekmenev, Eduard Y; Vollmar, Breanna S; Cotten, Myriam

2010-02-01

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide-lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use (15)N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water-lipid interface. Copyright 2009 Elsevier B.V. All rights reserved.

A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae.

PubMed

Amiche, Mohamed; Ladram, Ali; Nicolas, Pierre

2008-11-01

A growing number of cationic antimicrobial peptides have been isolated from the skin of hylid frogs belonging to the Phyllomedusinae subfamily. The amino acid sequences of these peptides are currently located in several databases under identifiers with no consistent system of nomenclature to describe them. In order to provide a workable terminology for antimicrobial peptides from Phyllomedusid frogs, we have made a systematic effort to collect, analyze, and classify all the Phyllomedusid peptide sequences available in databases. We propose that frogs belonging to the Phyllomedusinae subfamily should be described by the species names set out in Amphibian Species of the World: http://research.amnh.org/herpetology/amphibia/index.php, American Museum of Natural History, New York, USA. Multiple alignments analysis of at least 80 antimicrobial peptides isolated from 12 Phyllomedusinae species were distributed in seven distinct peptide families including dermaseptin, phylloseptin, plasticin, dermatoxin, phylloxin, hyposin and orphan peptides, and will be considered as the name of the headgroup of each family. The parent peptide's name should be followed by the first upper letter of the species for orthologous peptides and publication date determines priority. For example, the abbreviation B for bicolor and H for hypochondrialis. When two species begin with the same letter, two letters in upper case should be used (the first letter followed by the second or the third letter and so on). For example, the abbreviation DI for distincta, DU for duellmani, VA for vaillanti and VN for vanzolinii. Paralogous peptides should bear letter(s) in upper case followed by numbers.

Cell-to-cell contact and antimicrobial peptides play a combined role in the death of Lachanchea thermotolerans during mixed-culture alcoholic fermentation with Saccharomyces cerevisiae.

PubMed

Kemsawasd, Varongsiri; Branco, Patrícia; Almeida, Maria Gabriela; Caldeira, Jorge; Albergaria, Helena; Arneborg, Nils

2015-07-01

The roles of cell-to-cell contact and antimicrobial peptides in the early death of Lachanchea thermotolerans CBS2803 during anaerobic, mixed-culture fermentations with Saccharomyces cerevisiae S101 were investigated using a commercially available, double-compartment fermentation system separated by cellulose membranes with different pore sizes, i.e. 1000 kDa for mixed- and single-culture fermentations, and 1000 and 3.5-5 kDa for compartmentalized-culture fermentations. SDS-PAGE and gel filtration chromatography were used to determine an antimicrobial peptidic fraction in the fermentations. Our results showed comparable amounts of the antimicrobial peptidic fraction in the inner compartments of the mixed-culture and 1000 kDa compartmentalized-culture fermentations containing L. thermotolerans after 4 days of fermentation, but a lower death rate of L. thermotolerans in the 1000 kDa compartmentalized-culture fermentation than in the mixed-culture fermentation. Furthermore, L. thermotolerans died off even more slowly in the 3.5-5 kDa than in the 1000 kDa compartmentalized-culture fermentation, which coincided with the presence of less of the antimicrobial peptidic fraction in the inner compartment of that fermentation than of the 1000 kDa compartmentalized-culture fermentation. Taken together, these results indicate that the death of L. thermotolerans in mixed cultures with S. cerevisiae is caused by a combination of cell-to-cell contact and antimicrobial peptides. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages.

PubMed

Joerger, R D

2003-04-01

Bacteriocins, antimicrobial peptides, and bacteriophage have attracted attention as potential substitutes for, or as additions to, currently used antimicrobial compounds. This publication will review research on the potential application of these alternative antimicrobial agents to poultry production and processing. Bacteriocins are proteinaceous compounds of bacterial origin that are lethal to bacteria other than the producing strain. It is assumed that some of the bacteria in the intestinal tract produce bacteriocins as a means to achieve a competitive advantage, and bacteriocin-producing bacteria might be a desirable part of competitive exclusion preparations. Purified or partially purified bacteriocins could be used as preservatives or for the reduction or elimination of certain pathogens. Currently only nisin, produced by certain strains of Lactococcus lactis subsp. lactis, has regulatory approval for use in certain foods, and its use for poultry products has been studied extensively. Exploration of the application of antimicrobial peptides from sources other than bacteria to poultry has not yet commenced to a significant extent. Evidence for the ability of chickens to produce such antimicrobial peptides has been provided, and it is likely that these peptides play an important role in the defense against various pathogens. Bacteriophages have received renewed attention as possible agents against infecting bacteria. Evidence from several trials indicates that phage therapy can be effective under certain circumstances. Numerous obstacles for the use of phage as antimicrobials for poultry or poultry products remain. Chiefly among them are the narrow host range of many phages, the issue of phage resistance, and the possibility of phage-mediated transfer of genetic material to bacterial hosts. Regulatory issues and the high cost of producing such alternative antimicrobial agents are also factors that might prevent application of these agents in the near future.

N-Acylated and d Enantiomer Derivatives of a Nonamer Core Peptide of Lactoferricin B Showing Improved Antimicrobial Activity

PubMed Central

Wakabayashi, Hiroyuki; Matsumoto, Hiroshi; Hashimoto, Koichi; Teraguchi, Susumu; Takase, Mitsunori; Hayasawa, Hirotoshi

1999-01-01

N-acylated or d enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B. PMID:10223949

N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

PubMed

Wakabayashi, H; Matsumoto, H; Hashimoto, K; Teraguchi, S; Takase, M; Hayasawa, H

1999-05-01

N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

Antiangiogenic activity of the lipophilic antimicrobial peptides from an endophytic bacterial strain isolated from red pepper leaf.

PubMed

Jung, Hye Jin; Kim, Yonghyo; Lee, Hyang Burm; Kwon, Ho Jeong

2015-03-01

The induction of angiogenesis is a crucial step in tumor progression, and therefore, efficient inhibition of angiogenesis is considered a powerful strategy for the treatment of cancer. In the present study, we report that the lipophilic antimicrobial peptides from EML-CAP3, a new endophytic bacterial strain isolated from red pepper leaf (Capsicum annuum L.), exhibit potent antiangiogenic activity both in vitro and in vivo. The newly obtained antimicrobial peptides effectively inhibited the proliferation of human umbilical vein endothelial cells at subtoxic doses. Furthermore, the peptides suppressed the in vitro characteristics of angiogenesis such as endothelial cell invasion and tube formation stimulated by vascular endothelial growth factor, as well as neovascularization of the chorioallantoic membrane of growing chick embryos in vivo without showing cytotoxicity. Notably, the angiostatic peptides blocked tumor cell-induced angiogenesis by suppressing the expression levels of hypoxia-inducible factor-1α and its target gene, vascular endothelial growth factor (VEGF). To our knowledge, our findings demonstrate for the first time that the antimicrobial peptides from EML-CAP3 possess antiangiogenic potential and may thus be used for the treatment of hypervascularized tumors.

Surface modification and properties of Bombyx mori silk fibroin films by antimicrobial peptide

NASA Astrophysics Data System (ADS)

Bai, Liqiang; Zhu, Liangjun; Min, Sijia; Liu, Lin; Cai, Yurong; Yao, Juming

2008-03-01

The Bombyx mori silk fibroin films (SFFs) were modified by a Cecropin B ( CB) antimicrobial peptide, (NH 2)-NGIVKAGPAIAVLGEAAL-CONH 2, using the carbodiimide chemistry method. In order to avoid the dissolution of films during the modification procedure, the SFFs were first treated with 60% (v/v) ethanol aqueous solution, resulting a structural transition from unstable silk I to silk II. The investigation of modification conditions showed that the surface-modified SFFs had the satisfied antimicrobial activity and durability when they were activated by EDC·HCl/NHS solution followed by a treatment in CB peptide/PBS buffer (pH 6.5 or 8) solution at ambient temperature for 2 h. Moreover, the surface-modified SFFs showed the smaller contact angle due to the hydrophilic antimicrobial peptides coupled on the film surface, which is essential for the cell adhesion and proliferation. AFM results indicated that the surface roughness of SFFs was considerably increased after the modification by the peptides. The elemental composition analysis results also suggested that the peptides were tightly coupled to the surface of SFFs. This approach may provide a new option to engineer the surface-modified implanted materials preventing the biomaterial-centered infection (BCI).

Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

PubMed

Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

2016-10-01

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds.

PubMed

Yokoyama, Seiya; Kato, Kouji; Koba, Atsuko; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio

2008-12-01

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

Synthesis and Antibacterial Activities of Antibacterial Peptides with a Spiropyran Fluorescence Probe

PubMed Central

Chen, Lei; Zhu, Yu; Yang, Danling; Zou, Rongfeng; Wu, Junchen; Tian, He

2014-01-01

In this report, antibacterial peptides1-3 were prepared with a spiropyran fluorescence probe. The probe exhibits a change in fluorescence when isomerized from a colorless spiro-form (spiropyran, Sp) to a colored open-form (merocyanine, Mc) under different chemical environments, which can be used to study the mechanism of antimicrobial activity. Peptides 1-3 exhibit a marked decrease in antimicrobial activity with increasing alkyl chain length. This is likely due to the Sp-Mc isomers in different polar environments forming different aggregate sizes in TBS, as demonstrated by time-dependent dynamic light scattering (DLS). Moreover, peptides 1-3 exhibited low cytotoxicity and hemolytic activity. These probe-modified peptides may provide a novel approach to study the effect of structural changes on antibacterial activity, thus facilitating the design of new antimicrobial agents to combat bacterial infection. PMID:25358905

Host Defense Antimicrobial Peptides as Antibiotics: Design and Application Strategies

PubMed Central

Mishra, Biswajit; Reiling, Scott; Zarena, D.; Wang, Guangshun

2017-01-01

This review deals with the design and application strategies of new antibiotics based on naturally occurring antimicrobial peptides (AMPs). The initial candidate can be designed based on three-dimensional structure or selected from a library of peptides from natural or laboratory sources followed by optimization via structure-activity relationship studies. There are also advanced application strategies such as induction of AMP expression from host cells by various factors (e.g., metals, amino acids, vitamin D and sunlight), the use of engineered probiotic bacteria to deliver peptides, the design of prodrug and peptide conjugates to improve specific targeting. In addition, combined uses of newly developed AMPs with existing antimicrobial agents may provide a practical avenue for effective management of antibiotic-resistant bacteria (superbugs, including biofilm). Finally, we highlight AMPs already in use or under clinical trials. PMID:28399505

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action.

PubMed

Scocchi, Marco; Tossi, Alessandro; Gennaro, Renato

2011-07-01

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells.

Structure and antimicrobial activity of platypus 'intermediate' defensin-like peptide.

PubMed

Torres, Allan M; Bansal, Paramjit; Koh, Jennifer M S; Pagès, Guilhem; Wu, Ming J; Kuchel, Philip W

2014-05-02

The three-dimensional structure of a chemically synthesized peptide that we have called 'intermediate' defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and β-defensins, however the presence of a third antiparallel β-strand makes its structure more similar to the β-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP.

PubMed

Jia, Fengjing; Wang, Jiayi; Peng, Jinxiu; Zhao, Ping; Kong, Ziqing; Wang, Kairong; Yan, Wenjin; Wang, Rui

2017-10-01

With the increasing emergence of resistant microbes toward conventional antimicrobial agents, there is an urgent need for the development of antimicrobial agents with novel action mode. Antimicrobial peptides (AMPs) are believed to be one kind of ideal alternatives. However, AMPs can be easily degraded by protease, which limited their therapeutic use. In the present study, D-amino acid substitution strategy was employed to enhance the stability of polybia-CP. We investigated the stability of peptides against the degradation of trypsin and chymotrypsin by determining the antimicrobial activity or determining the HPLC profile of peptides after incubation with proteases. Our results showed that both the all D-amino acid derivative (D-CP) and partial D-lysine substitution derivative (D-lys-CP) have an improved stability against trypsin and chymotrypsin. Although D-CP takes left-hand α-helical conformation and D-lys-CP loses some α-helical content, both of the D-amino acid-substituted derivatives maintain their parental peptides' membrane active action mode. In addition, D-lys-CP showed a slight weaker antimicrobial activity than polybia-CP, but the hemolytic activity decreased greatly. These results suggest that D-CP and D-lys-CP can offer strategy to improve the property of AMPs and may be leading compounds for the development of novel antimicrobial agents. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Lacrain: the first antimicrobial peptide from the body extract of the Brazilian centipede Scolopendra viridicornis.

PubMed

Chaparro, E; da Silva, P I

2016-09-01

Antimicrobial activities have previously been described by traditional Eastern medicine in Chilopoda body extracts, but until now no bioactive peptides have been described. In this study, a novel antimicrobial peptide, lacrain, was isolated from the body extract of the Brazilian Chilopoda Scolopendra viridicornis. The peptide was isolated by reverse-phase high-performance liquid chromatography (RP-HPLC). Its activity was tested using a liquid growth inhibition assay and the peptide was characterised using mass spectrometry. Lacrain has a sequence composed of eight amino acid residues and a molecular mass of 925.5 Da. A synthetic peptide of the native lacrain had identical characteristics to those of the isolated material, confirming its sequence. The synthetic peptide was active only against Gram-negative bacteria, showing strong bactericidal activity. Moreover, the peptide did not present haemolytic activity against human erythrocytes. Lacrain represents a novel molecule with powerful antibacterial activity that could be used as a new template for the development of drugs against clinically resistant bacterial strains. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits.

PubMed

Chakraborty, Saswata; Liu, Runhui; Hayouka, Zvi; Chen, Xinyu; Ehrhardt, Jeffrey; Lu, Qin; Burke, Eileen; Yang, Yiqing; Weisblum, Bernard; Wong, Gerard C L; Masters, Kristyn S; Gellman, Samuel H

2014-10-15

Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residues, and most HDP-mimetic polymers have therefore contained binary combinations of hydrophobic and cationic subunits. However, HDP-mimetic polymers rarely duplicate the hydrophobic surface and cationic charge density found among HDPs ( Hu , K. ; et al. Macromolecules 2013 , 46 , 1908 ); the charge and hydrophobicity are generally higher among the polymers. Statistical analysis of HDP sequences ( Wang , G. ; et al. Nucleic Acids Res. 2009 , 37 , D933 ) has revealed that serine (polar but uncharged) is a very common HDP constituent and that glycine is more prevalent among HDPs than among proteins in general. These observations prompted us to prepare and evaluate ternary nylon-3 copolymers that contain a modestly polar but uncharged subunit, either serine-like or glycine-like, along with a hydrophobic subunit and a cationic subunit. Starting from binary hydrophobic-cationic copolymers that were previously shown to be highly active against bacteria but also highly hemolytic, we found that replacing a small proportion of the hydrophobic subunit with either of the polar, uncharged subunits can diminish the hemolytic activity with minimal impact on the antibacterial activity. These results indicate that the incorporation of polar, uncharged subunits may be generally useful for optimizing the biological activity profiles of antimicrobial polymers. In the context of HDP evolution, our findings suggest that there is a selective advantage to retaining polar, uncharged residues in natural antimicrobial peptides.

Antimicrobial Peptides: A Promising Therapeutic Strategy in Tackling Antimicrobial Resistance.

PubMed

Nuti, Ramya; Goud, Nerella S; Saraswati, A Prasanth; Alvala, Ravi; Alvala, Mallika

2017-01-01

Antimicrobial resistance (AMR) has posed a serious threat to global public health and it requires immediate action, preferably long term. Current drug therapies have failed to curb this menace due to the ability of microbes to circumvent the mechanisms through which the drugs act. From the drug discovery point of view, the majority of drugs currently employed for antimicrobial therapy are small molecules. Recent trends reveal a surge in the use of peptides as drug candidates as they offer remarkable advantages over small molecules. Newer synthetic strategies like organometalic complexes, Peptide-polymer conjugates, solid phase, liquid phase and recombinant DNA technology encouraging the use of peptides as therapeutic agents with a host of chemical functions, and tailored for specific applications. In the last decade, many peptide based drugs have been successfully approved by the Food and Drug Administration (FDA). This success can be attributed to their high specificity, selectivity and efficacy, high penetrability into the tissues, less immunogenicity and less tissue accumulation. Considering the enormity of AMR, the use of Antimicrobial Peptides (AMPs) can be a viable alternative to current therapeutics strategies. AMPs are naturally abundant allowing synthetic chemists to develop semi-synthetics peptide molecules. AMPs have a broad spectrum of activity towards microbes and they possess the ability to bypass the resistance induction mechanisms of microbes. The present review focuses on the potential applications of AMPs against various microbial disorders and their future prospects. Several resistance mechanisms and their strategies have also been discussed to highlight the importance in the current scenario. Breakthroughs in AMP designing, peptide synthesis and biotechnology have shown promise in tackling this challenge and has revived the interest of using AMPs as an important weapon in fighting AMR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cathelicidin-Derived Antimicrobial Peptides Inhibit Zika Virus Through Direct Inactivation and Interferon Pathway.

PubMed

He, Miao; Zhang, Hainan; Li, Yuju; Wang, Guangshun; Tang, Beisha; Zhao, Jeffrey; Huang, Yunlong; Zheng, Jialin

2018-01-01

Zika virus (ZIKV) is a neurotrophic flavivirus that is able to infect pregnant women and cause fetal brain abnormalities. Although there is a significant effort in identifying anti-ZIKV strategies, currently no vaccines or specific therapies are available to treat ZIKV infection. Antimicrobial peptides, which are potent host defense molecules in nearly all forms of life, have been found to be effective against several types of viruses such as HIV-1 and influenza A. However, they have not been tested in ZIKV infection. To determine whether antimicrobial peptides have anti-ZIKV effects, we used nine peptides mostly derived from human and bovine cathelicidins. Two peptides, GF-17 and BMAP-18, were found to have strong anti-ZIKV activities and little toxicity at 10 µM in an African green monkey kidney cell line. We further tested GF-17 and BMAP-18 in human fetal astrocytes, a known susceptible cell type for ZIKV, and found that GF-17 and BMAP-18 effectively inhibited ZIKV regardless of whether peptides were added before or after ZIKV infection. Interestingly, inhibition of type-I interferon signaling resulted in higher levels of ZIKV infection as measured by viral RNA production and partially reversed GF-17-mediated viral inhibition. More importantly, pretreatment with GF-17 and BMAP-18 did not affect viral attachment but reduced viral RNA early in the infection course. Direct incubation with GF-17 for 1 to 4 h specifically reduced the number of infectious Zika virions in the inoculum. In conclusion, these findings suggest that cathelicidin-derived antimicrobial peptides inhibit ZIKV through direct inactivation of the virus and via the interferon pathway. Strategies that harness antimicrobial peptides might be useful in halting ZIKV infection.

Animal Venom Peptides: Potential for New Antimicrobial Agents.

PubMed

Primon-Barros, Muriel; José Macedo, Alexandre

2017-01-01

Microbial infections affect people worldwide, causing diseases with significant impact on public health, indicating the need for research and development of new antimicrobial agents. Animal venoms represent a vast and largely unexploited source of biologically active molecules with attractive candidates for the development of novel therapeutics. Venoms consist of complex mixtures of molecules, including antimicrobial peptides (AMPs). Since the discovery of AMPs, they have been studied as promising new antimicrobial drugs. Amongst the remarkable sources of AMPs with known antimicrobial activities are ants, bees, centipedes, cone snails, scorpions, snakes, spiders, and wasps. The antimicrobial tests against bacteria, protozoans, fungi and viruses using 170 different peptides isolated directly from crude venoms or cDNA libraries of venom glands are listed and discussed in this review, as well as hemolytic ativity. The potential of venoms as source of new compounds, including AMPs, is extensively discussed. Currently, there are six FDA-approved drugs and many others are undergoing preclinical and clinical trials. The search for antimicrobial "weapons" makes the AMPs from venoms promising candidates. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat as preservative.

PubMed

Przybylski, Rémi; Firdaous, Loubna; Châtaigné, Gabrielle; Dhulster, Pascal; Nedjar, Naïma

2016-11-15

Bovine cruor, a slaughterhouse by-product, contains mainly hemoglobin, broadly described as a rich source of antimicrobial peptides. In the current context of food safety, bioactive peptides could be of interest as preservatives in the distribution of food products. The aim of this work was to study the α137-141 fragment of hemoglobin (Thr-Ser-Lys-Tyr-Arg), a small (653Da) and hydrophilic antimicrobial peptide. Its production was fast, with more 65% finally produced at 24h already produced after 30min of hydrolysis with pepsin. Moreover, increasing substrate concentration (from 1 to 8% (w/v)) resulted in a proportional augmentation of α137-141 production (to 807.95±41.03mgL(-1)). The α137-141 application on meat as preservative (0.5%, w/w) reduced the lipid oxidation about 60% to delay meat rancidity. The α137-141 peptide also inhibited the microbial growths under refrigeration during 14days. These antimicrobial effects were close to those of the butylated hydroxytoluene (BHT). Copyright © 2016 Elsevier Ltd. All rights reserved.

Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy.

PubMed

Mayo, Daniel J; Sahu, Indra D; Lorigan, Gary A

2018-07-01

Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66° ± 5°, 76° ± 5°, 70° ± 5°, and 72° ± 5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

Antimicrobial Peptide Simulations and the Influence of Force Field on the Free Energy for Pore Formation in Lipid Bilayers.

PubMed

Bennett, W F Drew; Hong, Chun Kit; Wang, Yi; Tieleman, D Peter

2016-09-13

Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations - including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100's of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a consistent manner, suggesting a common physical basis.

Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control.

PubMed

Datta, Aritreyee; Ghosh, Anirban; Airoldi, Cristina; Sperandeo, Paola; Mroue, Kamal H; Jiménez-Barbero, Jesús; Kundu, Pallob; Ramamoorthy, Ayyalusamy; Bhunia, Anirban

2015-07-06

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Novel antimicrobial peptide CPF-C1 analogs with superior stabilities and activities against multidrug-resistant bacteria.

PubMed

Xie, Junqiu; Zhao, Qian; Li, Sisi; Yan, Zhibin; Li, Jing; Li, Yao; Mou, Lingyun; Zhang, Bangzhi; Yang, Wenle; Miao, Xiaokang; Jiang, Xianxing; Wang, Rui

2017-11-01

As numerous clinical isolates are resistant to most conventional antibiotics, infections caused by multidrug-resistant bacteria are associated with a higher death rate. Antimicrobial peptides show great potential as new antibiotics. However, a major obstacle to the development of these peptides as useful drugs is their low stability. To overcome the problem of the natural antimicrobial peptide CPF-C1, we designed and synthesized a series of analogs. Our results indicated that by introducing lysine, which could increase the number of positive charges, and by introducing tryptophan, which could increase the hydrophobicity, we could improve the antimicrobial activity of the peptides against multidrug-resistant strains. The introduction of d-amino acids significantly improved stability. Certain analogs demonstrated antibiofilm activities. In mechanistic studies, the analogs eradicated bacteria not just by interrupting the bacterial membranes, but also by linking to DNA, which was not impacted by known mechanisms of resistance. In a mouse model, certain analogs were able to significantly reduce the bacterial load. Among the analogs, CPF-9 was notable due to its greater antimicrobial potency in vitro and in vivo and its superior stability, lower hemolytic activity, and higher antibiofilm activity. This analog is a potential antibiotic candidate for treating infections induced by multidrug-resistant bacteria. © 2017 John Wiley & Sons A/S.

Induction of the antimicrobial peptide CRAMP in the blood-brain barrier and meninges after meningococcal infection.

PubMed

Bergman, Peter; Johansson, Linda; Wan, Hong; Jones, Allison; Gallo, Richard L; Gudmundsson, Gudmundur H; Hökfelt, Tomas; Jonsson, Ann-Beth; Agerberth, Birgitta

2006-12-01

Antimicrobial peptides are present in most living species and constitute important effector molecules of innate immunity. Recently, we and others have detected antimicrobial peptides in the brain. This is an organ that is rarely infected, which has mainly been ascribed to the protective functions of the blood-brain barrier (BBB) and meninges. Since the bactericidal properties of the BBB and meninges are not known, we hypothesized that antimicrobial peptides could play a role in these barriers. We addressed this hypothesis by infecting mice with the neuropathogenic bacterium Neisseria meningitidis. Brains were analyzed for expression of the antimicrobial peptide CRAMP by immunohistochemistry in combination with confocal microscopy. After infection, we observed induction of CRAMP in endothelial cells of the BBB and in cells of the meninges. To explore the functional role of CRAMP in meningococcal disease, we infected mice deficient of the CRAMP gene. Even though CRAMP did not appear to protect the brain from invasion of meningococci, CRAMP knockout mice were more susceptible to meningococcal infection than wild-type mice and exhibited increased meningococcal growth in blood, liver, and spleen. Moreover, we could demonstrate that carbonate, a compound that accumulates in the circulation during metabolic acidosis, makes meningococci more susceptible to CRAMP.

Induction of the Antimicrobial Peptide CRAMP in the Blood-Brain Barrier and Meninges after Meningococcal Infection▿

PubMed Central

Bergman, Peter; Johansson, Linda; Wan, Hong; Jones, Allison; Gallo, Richard L.; Gudmundsson, Gudmundur H.; Hökfelt, Tomas; Jonsson, Ann-Beth; Agerberth, Birgitta

2006-01-01

Antimicrobial peptides are present in most living species and constitute important effector molecules of innate immunity. Recently, we and others have detected antimicrobial peptides in the brain. This is an organ that is rarely infected, which has mainly been ascribed to the protective functions of the blood-brain barrier (BBB) and meninges. Since the bactericidal properties of the BBB and meninges are not known, we hypothesized that antimicrobial peptides could play a role in these barriers. We addressed this hypothesis by infecting mice with the neuropathogenic bacterium Neisseria meningitidis. Brains were analyzed for expression of the antimicrobial peptide CRAMP by immunohistochemistry in combination with confocal microscopy. After infection, we observed induction of CRAMP in endothelial cells of the BBB and in cells of the meninges. To explore the functional role of CRAMP in meningococcal disease, we infected mice deficient of the CRAMP gene. Even though CRAMP did not appear to protect the brain from invasion of meningococci, CRAMP knockout mice were more susceptible to meningococcal infection than wild-type mice and exhibited increased meningococcal growth in blood, liver, and spleen. Moreover, we could demonstrate that carbonate, a compound that accumulates in the circulation during metabolic acidosis, makes meningococci more susceptible to CRAMP. PMID:17030578

A heterodimer comprised of two bovine lactoferrin antimicrobial peptides exhibits powerful bactericidal activity against Burkholderia pseudomallei.

PubMed

Puknun, Aekkalak; Bolscher, Jan G M; Nazmi, Kamran; Veerman, Enno C I; Tungpradabkul, Sumalee; Wongratanacheewin, Surasakdi; Kanthawong, Sakawrat; Taweechaisupapong, Suwimol

2013-07-01

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17-30 and LFampin268-284, a shorter fragment of LFampin265-284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10(5) CFU/ml, could be killed by 5-10 μM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.

Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria.

PubMed

Ovchinnikov, Kirill V; Chi, Hai; Mehmeti, Ibrahim; Holo, Helge; Nes, Ingolf F; Diep, Dzung B

2016-09-01

From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates (L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria, and Enterococcus Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria

PubMed Central

Chi, Hai; Mehmeti, Ibrahim; Holo, Helge; Nes, Ingolf F.

2016-01-01

ABSTRACT From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates (L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus. All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria, and Enterococcus. IMPORTANCE Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine. PMID:27316965

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles.

PubMed

Schibli, D J; Hwang, P M; Vogel, H J

1999-03-12

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.

Catesbeianin-1, a novel antimicrobial peptide isolated from the skin of Lithobates catesbeianus (American bullfrog).

PubMed

Xu, Huihui; Zhang, Yang; Feng, Xin; Tie, Kunyuan; Cao, Yuan; Han, Wenyu

2017-06-01

To identify and characterize a novel antimicrobial peptide, catesbeianin-1. Catesbeianin-1 is 25 amino acids long and is α-helical, cationic and amphipathic. It had antimicrobial activity against Gram-positive and Gram-negative bacteria. It was resistant against trypsin and pepsin. Catesbeianin-1 exhibited moderate hemolytic activity (approx 8%) at 100 μg/ml, and its HC 50 (50% hemolytic concentration) was 300 μg/ml. Its cytotoxicity was approx 10-20% at 100 μg/ml, and its CC 50 (50% cytotoxic concentration) was >100 μg/ml. The LD 50 of catesbeianin-1 in mice was 80 mg/kg. At 3.1 µg/ml, catesbeianin-1 significantly inhibited the growth of methicillin-resistant Staphylococcus aureus. A new antimicrobial peptide from the skin of Lithobates catesbeianus (American bullfrog) may represent a template for the development of novel antimicrobial agents.

Interaction of Mastoparan with Model Membranes

NASA Astrophysics Data System (ADS)

Haloot, Justin

2010-10-01

The use of antimicrobial agents began during the 20th century to reduce the effects of infectious diseases. Since the 1990s, antimicrobial resistance has become an ever-increasing global problem. Our laboratory recently found that small antimicrobial peptides (AMPs) have potent antimicrobial activity against a wide range of Gram-negative and Gram-positive organisms including antibiotic resistant organisms. These AMPs are potential therapeutic agents against the growing problem of antimicrobial resistance. AMPs are small peptides produced by plants, insects and animals. Several hypotheses concede that these peptides cause some type of structural perturbations and increased membrane permeability in bacteria however, how AMPs kill bacteria remains unclear. The goal of this study was to design an assay that would allow us to evaluate and monitor the pore forming ability of an AMP, Mastoparan, on model membrane structures called liposomes. Development of this model will facilitate the study of how mastoparan and related AMPs interact with the bacterial membrane.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

PubMed Central

Falkenberg, Shollie M.; Register, Karen B.; Samorodnitsky, Daniel; Nicholson, Eric M.; Reinhardt, Timothy A.

2018-01-01

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality. PMID:29771981

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin.

PubMed

Maky, Mohamed Abdelfattah; Ishibashi, Naoki; Zendo, Takeshi; Perez, Rodney Honrada; Doud, Jehan Ragab; Karmi, Mohamed; Sonomoto, Kenji

2015-07-01

Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A pocket guide to explorations of the defensin field.

PubMed

Selsted, Michael E

2007-01-01

Antimicrobial peptides are among the most ancient effectors of host defense. Intersecting lines of research demonstrate that life forms as diverse as plants, insects, and vertebrates employ antimicrobial peptides to kill or neutralize a wide variety of microbial species. Defensins, of which there are three structural sub-families, constitute a major category of host defense peptides in vertebrates. Presented here is a brief history of the emergence of the defensin field with an emphasis on the role of these peptides in mammalian innate immunity.

Towards the Improved Discovery and Design of Functional Peptides: Common Features of Diverse Classes Permit Generalized Prediction of Bioactivity

PubMed Central

Mooney, Catherine; Haslam, Niall J.; Pollastri, Gianluca; Shields, Denis C.

2012-01-01

The conventional wisdom is that certain classes of bioactive peptides have specific structural features that endow their particular functions. Accordingly, predictions of bioactivity have focused on particular subgroups, such as antimicrobial peptides. We hypothesized that bioactive peptides may share more general features, and assessed this by contrasting the predictive power of existing antimicrobial predictors as well as a novel general predictor, PeptideRanker, across different classes of peptides. We observed that existing antimicrobial predictors had reasonable predictive power to identify peptides of certain other classes i.e. toxin and venom peptides. We trained two general predictors of peptide bioactivity, one focused on short peptides (4–20 amino acids) and one focused on long peptides ( amino acids). These general predictors had performance that was typically as good as, or better than, that of specific predictors. We noted some striking differences in the features of short peptide and long peptide predictions, in particular, high scoring short peptides favour phenylalanine. This is consistent with the hypothesis that short and long peptides have different functional constraints, perhaps reflecting the difficulty for typical short peptides in supporting independent tertiary structure. We conclude that there are general shared features of bioactive peptides across different functional classes, indicating that computational prediction may accelerate the discovery of novel bioactive peptides and aid in the improved design of existing peptides, across many functional classes. An implementation of the predictive method, PeptideRanker, may be used to identify among a set of peptides those that may be more likely to be bioactive. PMID:23056189

A novel covalent approach to bio-conjugate silver coated single walled carbon nanotubes with antimicrobial peptide.

PubMed

Chaudhari, Atul A; Ashmore, D'andrea; Nath, Subrata Deb; Kate, Kunal; Dennis, Vida; Singh, Shree R; Owen, Don R; Palazzo, Chris; Arnold, Robert D; Miller, Michael E; Pillai, Shreekumar R

2016-07-13

Due to increasing antibiotic resistance, the use of silver coated single walled carbon nanotubes (SWCNTs-Ag) and antimicrobial peptides (APs) is becoming popular due to their antimicrobial properties against a wide range of pathogens. However, stability against various conditions and toxicity in human cells are some of the major drawbacks of APs and SWCNTs-Ag, respectively. Therefore, we hypothesized that APs-functionalized SWCNTs-Ag could act synergistically. Various covalent functionalization protocols described previously involve harsh treatment of carbon nanotubes for carboxylation (first step in covalent functionalization) and the non-covalently functionalized SWCNTs are not satisfactory. The present study is the first report wherein SWCNTs-Ag were first carboxylated using Tri sodium citrate (TSC) at 37 °C and then subsequently functionalized covalently with an effective antimicrobial peptide from Therapeutic Inc., TP359 (FSWCNTs-Ag). SWCNTs-Ag were also non covalently functionalized with TP359 by simple mixing (SWCNTs-Ag-M) and both, the FSWCNTs-Ag (covalent) and SWCNTs-Ag-M (non-covalent), were characterized by Fourier transform infrared spectroscopy (FT-IR), Ultraviolet visualization (UV-VIS) and transmission electron microscopy (TEM). Further the antibacterial activity of both and TP359 were investigated against two gram positive (Staphylococcus aureus and Streptococcus pyogenes) and two gram negative (Salmonella enterica serovar Typhimurium and Escherichia coli) pathogens and the cellular toxicity of TP359 and FSWCNTs-Ag was compared with plain SWCNTs-Ag using murine macrophages and lung carcinoma cells. FT-IR analysis revealed that treatment with TSC successfully resulted in carboxylation of SWCNTs-Ag and the peptide was indeed attached to the SWCNTs-Ag evidenced by TEM images. More importantly, the present study results further showed that the minimum inhibitory concentration (MIC) of FSWCNTs-Ag were much lower (~7.8-3.9 µg/ml with IC50: ~4-5 µg/ml) compared to SWCNTs-Ag-M and plain SWCNTs-Ag (both 62.6 µg/ml, IC50: ~31-35 µg/ml), suggesting that the covalent conjugation of TP359 with SWCNTs-Ag was very effective on their counterparts. Additionally, FSWCNTs-Ag are non-toxic to the eukaryotic cells at their MIC concentrations (5-2.5 µg/ml) compared to SWCNTs-Ag (62.5 µg/ml). In conclusion, we demonstrated that covalent functionalization of SWCNTs-Ag and TP359 exhibited an additive antibacterial activity. This study described a novel approach to prepare SWCNT-Ag bio-conjugates without loss of antimicrobial activity and reduced toxicity, and this strategy will aid in the development of novel and biologically important nanomaterials.

Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

PubMed

Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

2006-09-01

Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

Prediction of Antibacterial Activity from Physicochemical Properties of Antimicrobial Peptides

PubMed Central

Melo, Manuel N.; Ferre, Rafael; Feliu, Lídia; Bardají, Eduard; Planas, Marta; Castanho, Miguel A. R. B.

2011-01-01

Consensus is gathering that antimicrobial peptides that exert their antibacterial action at the membrane level must reach a local concentration threshold to become active. Studies of peptide interaction with model membranes do identify such disruptive thresholds but demonstrations of the possible correlation of these with the in vivo onset of activity have only recently been proposed. In addition, such thresholds observed in model membranes occur at local peptide concentrations close to full membrane coverage. In this work we fully develop an interaction model of antimicrobial peptides with biological membranes; by exploring the consequences of the underlying partition formalism we arrive at a relationship that provides antibacterial activity prediction from two biophysical parameters: the affinity of the peptide to the membrane and the critical bound peptide to lipid ratio. A straightforward and robust method to implement this relationship, with potential application to high-throughput screening approaches, is presented and tested. In addition, disruptive thresholds in model membranes and the onset of antibacterial peptide activity are shown to occur over the same range of locally bound peptide concentrations (10 to 100 mM), which conciliates the two types of observations. PMID:22194847

Current state of a dual behaviour of antimicrobial peptides-Therapeutic agents and promising delivery vectors.

PubMed

Piotrowska, Urszula; Sobczak, Marcin; Oledzka, Ewa

2017-12-01

Micro-organism resistance is an important challenge in modern medicine due to the global uncontrolled use of antibiotics. Natural and synthetic antimicrobial peptides (AMPs) symbolize a new family of antibiotics, which have stimulated research and clinical interest as new therapeutic options for infections. They represent one of the most promising antimicrobial substances, due to their broad spectrum of biological activity, against bacteria, fungi, protozoa, viruses, yeast and even tumour cells. Besides, being antimicrobial, AMPs have been shown to bind and neutralize bacterial endotoxins, as well as possess immunomodulatory, anti-inflammatory, wound-healing, angiogenic and antitumour properties. In contrast to conventional antibiotics, which have very defined and specific molecular targets, host cationic peptides show varying, complex and very rapid mechanisms of actions that make it difficult to form an effective antimicrobial defence. Importantly, AMPs display their antimicrobial activity at micromolar concentrations or less. To do this, many peptide-based drugs are commercially available for the treatment of numerous diseases, such as hepatitis C, myeloma, skin infections and diabetes. Herein, we present an overview of the general mechanism of AMPs action, along with recent developments regarding carriers of AMPs and their potential applications in medical fields. © 2017 John Wiley & Sons A/S.

Investigation of the role of interleukin-6 and hepcidin antimicrobial peptide in the development of anemia with age

PubMed Central

McCranor, Bryan J.; Langdon, Jacqueline M.; Prince, Olivier D.; Femnou, Laurette K.; Berger, Alan E.; Cheadle, Chris; Civin, Curt I.; Kim, Airie; Rivera, Seth; Ganz, Tomas; Vaulont, Sophie; Xue, Qian-Li; Walston, Jeremy D.; Roy, Cindy N.

2013-01-01

Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults. PMID:23996485

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages

PubMed Central

Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

2015-01-01

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation. PMID:26017270

Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

PubMed

Shim, Do-Wan; Heo, Kang-Hyuck; Kim, Young-Kyu; Sim, Eun-Jeong; Kang, Tae-Bong; Choi, Jae-Wan; Sim, Dae-Won; Cheong, Sun-Hee; Lee, Seung-Hong; Bang, Jeong-Kyu; Won, Hyung-Sik; Lee, Kwang-Ho

2015-01-01

Antimicrobial peptides (AMPs), also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4)- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

Antimicrobial activity of peptides derived from olive flounder lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI).

PubMed

Nam, Bo-Hye; Moon, Ji-Young; Park, Eun-Hee; Kim, Young-Ok; Kim, Dong-Gyun; Kong, Hee Jeong; Kim, Woo-Jin; Jee, Young Ju; An, Cheul Min; Park, Nam Gyu; Seo, Jung-Kil

2014-10-17

We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

Medicinal Chemistry of ATP Synthase: A Potential Drug Target of Dietary Polyphenols and Amphibian Antimicrobial Peptides

PubMed Central

Ahmad, Zulfiqar; Laughlin, Thomas F.

2015-01-01

In this review we discuss the inhibitory effects of dietary polyphenols and amphibian antimicrobial/antitumor peptides on ATP synthase. In the beginning general structural features highlighting catalytic and motor functions of ATP synthase will be described. Some details on the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it an interesting drug target with respect to dietary polyphenols and amphibian antimicrobial peptides will also be reviewed. ATP synthase is known to have distinct polyphenol and peptide binding sites at the interface of α/β subunits. Molecular interaction of polyphenols and peptides with ATP synthase at their respective binding sites will be discussed. Binding and inhibition of other proteins or enzymes will also be covered so as to understand the therapeutic roles of both types of molecules. Lastly, the effects of polyphenols and peptides on the inhibition of Escherichia coli cell growth through their action on ATP synthase will also be presented. PMID:20586714

Antimicrobial Activity of Lactoferrin-Related Peptides and Applications in Human and Veterinary Medicine.

PubMed

Bruni, Natascia; Capucchio, Maria Teresa; Biasibetti, Elena; Pessione, Enrica; Cirrincione, Simona; Giraudo, Leonardo; Corona, Antonio; Dosio, Franco

2016-06-11

Antimicrobial peptides (AMPs) represent a vast array of molecules produced by virtually all living organisms as natural barriers against infection. Among AMP sources, an interesting class regards the food-derived bioactive agents. The whey protein lactoferrin (Lf) is an iron-binding glycoprotein that plays a significant role in the innate immune system, and is considered as an important host defense molecule. In search for novel antimicrobial agents, Lf offers a new source with potential pharmaceutical applications. The Lf-derived peptides Lf(1-11), lactoferricin (Lfcin) and lactoferrampin exhibit interesting and more potent antimicrobial actions than intact protein. Particularly, Lfcin has demonstrated strong antibacterial, anti-fungal and antiparasitic activity with promising applications both in human and veterinary diseases (from ocular infections to osteo-articular, gastrointestinal and dermatological diseases).

Bacterial strategies of resistance to antimicrobial peptides.

PubMed

Joo, Hwang-Soo; Fu, Chih-Iung; Otto, Michael

2016-05-26

Antimicrobial peptides (AMPs) are a key component of the host's innate immune system, targeting invasive and colonizing bacteria. For successful survival and colonization of the host, bacteria have a series of mechanisms to interfere with AMP activity, and AMP resistance is intimately connected with the virulence potential of bacterial pathogens. In particular, because AMPs are considered as potential novel antimicrobial drugs, it is vital to understand bacterial AMP resistance mechanisms. This review gives a comparative overview of Gram-positive and Gram-negative bacterial strategies of resistance to various AMPs, such as repulsion or sequestration by bacterial surface structures, alteration of membrane charge or fluidity, degradation and removal by efflux pumps.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'. © 2016 The Author(s).

Skin secretion peptides: the molecular facet of the deimatic behavior of the four-eyed frog, Physalaemus nattereri (Anura, Leptodactylidae).

PubMed

Barbosa, Eder Alves; Iembo, Tatiane; Martins, Graciella Ribeiro; Silva, Luciano Paulino; Prates, Maura Vianna; Andrade, Alan Carvalho; Bloch, Carlos

2015-11-15

Amphibians can produce a large amount of bioactive peptides over the skin. In order to map the precise tissue localization of these compounds and evaluate their functions, mass spectrometry imaging (MSI) and gene expression studies were used to investigate a possible correlation between molecules involved in the antimicrobial defense mechanisms and anti-predatory behavior by Physalaemus nattereri. Total skin secretion of P. nattereri was analyzed by classical Protein Chemistry and proteomic techniques. Intact inguinal macroglands were dissected from the rest of the skin and both tissues were analyzed by MSI and real-time polymerase chain reaction (RT-PCR) experiments. Peptides were primarily identified by de novo sequencing, automatic Edman degradation and cDNA data. Fifteen bradykinin (BK)-related peptides and two antimicrobial peptides were sequenced and mapped by MSI on the inguinal macrogland and the rest of P. nattereri skin. RT-PCR results revealed that BK-related peptide levels of expression were about 30,000 times higher on the inguinal macroglands than on the any other region of the skin, whilst antimicrobial peptide ions appear to be evenly distributed in both investigated regions. The presence of antimicrobial peptides in all investigated tissue regions is in accordance with the defensive role against microorganisms thoroughly demonstrated in the literature, whereas BK-related molecules are largely found on the inguinal macroglands suggesting an intriguing link between their noxious activities against potential predators of P. nattereri and the frog's deimatic behavior. Copyright © 2015 John Wiley & Sons, Ltd.

Distinct antimicrobial peptide expression determines host species-specific bacterial associations

PubMed Central

Franzenburg, Sören; Walter, Jonas; Künzel, Sven; Wang, Jun; Baines, John F.; Bosch, Thomas C. G.; Fraune, Sebastian

2013-01-01

Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations. PMID:24003149

Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus.

PubMed

Gonzalez-Curiel, Irma; Castañeda-Delgado, Julio; Lopez-Lopez, Nallely; Araujo, Zaida; Hernandez-Pando, Rogelio; Gandara-Jasso, Benjamin; Macias-Segura, Noe; Enciso-Moreno, Antonio; Rivas-Santiago, Bruno

2011-08-01

Tuberculosis (TB) is one of the most important infectious diseases, causing 1.8 million deaths annually worldwide. This problem has increased because of the association with human immmunodeficiency virus and diabetes mellitus type 2, mainly in developing countries. In the past few years it has been highlighted the significance of antimicrobial peptides in the immunopathogenesis of TB ex vivo and in experimental models studies. In this study we analyzed the expression of CAMP, DEFA1, DEFB4, and DEFB103A in patients with latent TB and progressive TB with and without comorbidity with diabetes mellitus type 2. Antimicrobial peptide gene expression increased during progressive TB, which could be used as a biomarker for reactivation. By contrast, patients with diabetes mellitus type 2 have lower antimicrobial peptides gene expression, suggesting that the lack of its proper production in these patients contribute to enhance the risk for TB reactivation. Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Protein-only, antimicrobial peptide-containing recombinant nanoparticles with inherent built-in antibacterial activity.

PubMed

Serna, Naroa; Sánchez-García, Laura; Sánchez-Chardi, Alejandro; Unzueta, Ugutz; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio

2017-09-15

The emergence of bacterial antibiotic resistances is a serious concern in human and animal health. In this context, naturally occurring cationic antimicrobial peptides (AMPs) might play a main role in a next generation of drugs against bacterial infections. Taking an innovative approach to design self-organizing functional proteins, we have generated here protein-only nanoparticles with intrinsic AMP microbicide activity. Using a recombinant version of the GWH1 antimicrobial peptide as building block, these materials show a wide antibacterial activity spectrum in absence of detectable toxicity on mammalian cells. The GWH1-based nanoparticles combine clinically appealing properties of nanoscale materials with full biocompatibility, structural and functional plasticity and biological efficacy exhibited by proteins. Because of the largely implemented biological fabrication of recombinant protein drugs, the protein-based platform presented here represents a novel and scalable strategy in antimicrobial drug design, that by solving some of the limitations of AMPs offers a promising alternative to conventional antibiotics. The low molecular weight antimicrobial peptide GWH1 has been engineered to oligomerize as self-assembling protein-only nanoparticles of around 50nm. In this form, the peptide exhibits potent and broad antibacterial activities against both Gram-positive and Gram-negative bacteria, without any harmful effect over mammalian cells. As a solid proof-of-concept, this finding strongly supports the design and biofabrication of nanoscale antimicrobial materials with in-built functionalities. The protein-based homogeneous composition offer advantages over alternative materials explored as antimicrobial agents, regarding biocompatibility, biodegradability and environmental suitability. Beyond the described prototype, this transversal engineering concept has wide applicability in the design of novel nanomedicines for advanced treatments of bacterial infections. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

PubMed Central

2010-01-01

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID:20122244

Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens.

PubMed

Tanhaeian, Abbas; Shahriari Ahmadi, Farajollah; Sekhavati, Mohammad Hadi; Mamarabadi, Mojtaba

2018-04-04

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 μg/ml for different bacterial isolates.

Engineered chimeric peptides with antimicrobial and titanium-binding functions to inhibit biofilm formation on Ti implants.

PubMed

Geng, Hongjuan; Yuan, Yang; Adayi, Aidina; Zhang, Xu; Song, Xin; Gong, Lei; Zhang, Xi; Gao, Ping

2018-01-01

Titanium (Ti) implants have been commonly used in oral medicine. However, despite their widespread clinical application, these implants are susceptible to failure induced by microbial infection due to bacterial biofilm formation. Immobilization of chimeric peptides with antibacterial properties on the Ti surface may be a promising antimicrobial approach to inhibit biofilm formation. Here, chimeric peptides were designed by connecting three sequences (hBD-3-1/2/3) derived from human β-defensin-3 (hBD-3) with Ti-binding peptide-l (TBP-l: RKLPDAGPMHTW) via a triple glycine (G) linker to modify Ti surfaces. Using X-ray photoelectron spectroscopy (XPS), the properties of individual domains of the chimeric peptides were evaluated for their binding activity toward the Ti surface. The antimicrobial and anti-biofilm efficacy of the peptides against initial settlers, Streptococcus oralis (S. oralis), Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis), was evaluated with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Transmission electron microscopy (TEM) and real-time quantitative PCR (qRT-PCR) were used to study cell membrane changes and the underlying antimicrobial mechanism. Compared with the other two peptides, TBP-1-GGG-hBD3-3 presented stronger antibacterial activity and remained stable in saliva and serum. Therefore, it was chosen as the best candidate to modify Ti surfaces in this study. This peptide inhibited the growth of initial streptococci and biofilm formation on Ti surfaces with no cytotoxicity to MC3T3-E1 cells. Disruption of the integrity of bacterial membranes and decreased expression of adhesion protein genes from S. gordonii revealed aspects of the antibacterial mechanism of TBP-1-GGG-hBD3-3. We conclude that engineered chimeric peptides with antimicrobial activity provide a potential solution for inhibiting biofilm formation on Ti surfaces to reduce or prevent the occurrence of peri-implant diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Antimicrobial activity of buttermilk and lactoferrin peptide extracts on poultry pathogens.

PubMed

Jean, Catherine; Boulianne, Martine; Britten, Michel; Robitaille, Gilles

2016-11-01

Antibiotics are commonly used in poultry feed as growth promoters. This practice is questioned given the arising importance of antibiotic resistance. Antimicrobial peptides can be used as food additives for a potent alternative to synthetic or semi-synthetic antibiotics. The objective of this study was to develop a peptide production method based on membrane adsorption chromatography in order to produce extracts with antimicrobial activity against avian pathogens (Salmonella enterica var. Enteritidis, Salmonella enterica var. Typhimurium, and two Escherichia coli strains, O78:H80 and TK3 O1:K1) as well as Staphylococcus aureus. To achieve this, buttermilk powder and purified lactoferrin were digested with pepsin. The peptide extracts (<10 kDa) were fractionated depending on their charges through high-capacity cation-exchange and anion-exchange adsorptive membranes. The yields of cationic peptide extracts were 6·3 and 15·4% from buttermilk and lactoferrin total peptide extracts, respectively. Antimicrobial activity was assessed using the microdilution technique on microplates. Our results indicate that the buttermilk cationic peptide extracts were bactericidal at less than 5 mg/ml against the selected avian strains, with losses of 1·7 log CFU/ml (Salm. Typhimurium) to 3 log CFU/ml (E. coli O78:H80); viability decreased by 1·5 log CFU/ml for Staph. aureus, a Gram-positive bacterium. Anionic and non-adsorbed peptide extracts were inactive at 5 mg/ml. These results demonstrate that membrane adsorption chromatography is an effective way to prepare a cationic peptide extract from buttermilk that is active against avian pathogens.

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) by antimicrobial peptides (AMPs) and plant essential oils.

PubMed

Zouhir, Abdelmajid; Jridi, Taoufik; Nefzi, Adel; Ben Hamida, Jeannette; Sebei, Khaled

2016-12-01

Drug-resistant bacterial infections cause considerable patient mortality and morbidity. The annual frequency of deaths from methicillin-resistant Staphylococcus aureus (MRSA) has surpassed those caused by human immunodeficiency virus/acquired immune deficiency syndrome. The antimicrobial peptides (AMPs), plant essential oils (EOs) and their combinations have proven to be quite effective in killing a wide selection of bacterial pathogens including MRSA. This review summarizes the studies in the use of AMPs, plant EOs and their combinations for coping with MRSA bacteria, and to formulate new prospects for future studies on this topic. The sources of scientific literature such as PubMed, library search, Google Scholar, Science Direct and electronic databases such as 'The Antimicrobial Peptide Database', 'Collection of Anti-Microbial Peptides' and 'YADAMP'. Physicochemical data of anti-MRSA peptides were determined by Scientific DataBase Maker software. Of the 118 peptides, 88 exhibited an activity against MRSA with the highest activity of minimum inhibitory concentration values. Various plant EOs have been effective against MRSA. Remarkably, lemongrass EOs completely inhibited all MRSA growth on the plate. Lemon myrtle, Mountain savory, Cinnamon bark and Melissa EOs showed a significant inhibition. Several of these AMPs, EOs and their combinations were effective against MRSA. Their activities have implications for the development of new drugs for medical use.

Antimicrobial Peptides from Marine Proteobacteria

PubMed Central

Desriac, Florie; Jégou, Camille; Balnois, Eric; Brillet, Benjamin; Le Chevalier, Patrick; Fleury, Yannick

2013-01-01

After years of inadequate use and the emergence of multidrug resistant (MDR) strains, the efficiency of “classical” antibiotics has decreased significantly. New drugs to fight MDR strains are urgently needed. Bacteria hold much promise as a source of unusual bioactive metabolites. However, the potential of marine bacteria, except for Actinomycetes and Cyanobacteria, has been largely underexplored. In the past two decades, the structures of several antimicrobial compounds have been elucidated in marine Proteobacteria. Of these compounds, polyketides (PKs), synthesised by condensation of malonyl-coenzyme A and/or acetyl-coenzyme A, and non-ribosomal peptides (NRPs), obtained through the linkage of (unusual) amino acids, have recently generated particular interest. NRPs are good examples of naturally modified peptides. Here, we review and compile the data on the antimicrobial peptides isolated from marine Proteobacteria, especially NRPs. PMID:24084784

A review of fish-derived antioxidant and antimicrobial peptides: their production, assessment, and applications.

PubMed

Najafian, L; Babji, A S

2012-01-01

Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals. Copyright © 2011 Elsevier Inc. All rights reserved.

Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial.

PubMed

Mily, Akhirunnesa; Rekha, Rokeya Sultana; Kamal, S M Mostafa; Arifuzzaman, Abu Saleh Mohammad; Rahim, Zeaur; Khan, Lamia; Haq, Md Ahsanul; Zaman, Khaliqu; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H; Agerberth, Birgitta; Raqib, Rubhana

2015-01-01

Development of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects. To examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients. Adult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and ex vivo bactericidal function of monocyte-derived macrophages (MDM) were determined at week 0, 4, 8, 12 and additionally at week 24 for plasma 25(OH)D3. At week 4, 71% (46/65) of the patients in the PBA+vitD3-group (p = 0.001) and 61.3% (38/62) in the vitD3-group (p = 0.032) were culture negative compared to 42.2% (27/64) in the placebo-group. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (p = 0.001) and 2.2 times higher in vitD3-group (p = 0.032) compared to placebo. The concentration of LL-37 in MDM was significantly higher in the PBA-group compared to placebo at week 12 (p = 0.034). Decline in intracellular Mtb growth in MDM was earlier in the PBA-group compared to placebo (log rank 11.38, p = 0.01). Adjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB. clinicaltrials.gov NCT01580007.

Significant Effects of Oral Phenylbutyrate and Vitamin D3 Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial

PubMed Central

Kamal, S. M. Mostafa; Arifuzzaman, Abu Saleh Mohammad; Rahim, Zeaur; Khan, Lamia; Haq, Md. Ahsanul; Zaman, Khaliqu; Bergman, Peter; Brighenti, Susanna; Gudmundsson, Gudmundur H.; Agerberth, Birgitta; Raqib, Rubhana

2015-01-01

Background Development of new tuberculosis (TB) drugs and alternative treatment strategies are urgently required to control the global spread of TB. Previous results have shown that vitamin D3 (vitD3) and 4-phenyl butyrate (PBA) are potent inducers of the host defense peptide LL-37 that possess anti-mycobacterial effects. Objective To examine if oral adjunctive therapy with 5,000IU vitD3 or 2x500 mg PBA or PBA+vitD3 to standard chemotherapy would lead to enhanced recovery in sputum smear-positive pulmonary TB patients. Methods Adult TB patients (n = 288) were enrolled in a randomized, double-blind, placebo-controlled trial conducted in Bangladesh. Primary endpoints included proportions of patients with a negative sputum culture at week 4 and reduction in clinical symptoms at week 8. Clinical assessments and sputum smear microscopy were performed weekly up to week 4, fortnightly up to week 12 and at week 24; TB culture was performed at week 0, 4 and 8; concentrations of LL-37 in cells, 25-hydroxyvitamin D3 (25(OH)D3) in plasma and ex vivo bactericidal function of monocyte-derived macrophages (MDM) were determined at week 0, 4, 8, 12 and additionally at week 24 for plasma 25(OH)D3. Results At week 4, 71% (46/65) of the patients in the PBA+vitD3-group (p = 0.001) and 61.3% (38/62) in the vitD3-group (p = 0.032) were culture negative compared to 42.2% (27/64) in the placebo-group. The odds of sputum culture being negative at week 4 was 3.42 times higher in the PBA+vitD3-group (p = 0.001) and 2.2 times higher in vitD3-group (p = 0.032) compared to placebo. The concentration of LL-37 in MDM was significantly higher in the PBA-group compared to placebo at week 12 (p = 0.034). Decline in intracellular Mtb growth in MDM was earlier in the PBA-group compared to placebo (log rank 11.38, p = 0.01). Conclusion Adjunct therapy with PBA+vitD3 or vitD3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery and holds potential for host-directed-therapy in the treatment of TB. Trial Registration clinicaltrials.gov NCT01580007 PMID:26394045

Recombinant probiotics with antimicrobial peptides: a dual strategy to improve immune response in immunocompromised patients.

PubMed

Mandal, Santi M; Silva, Osmar N; Franco, Octavio L

2014-08-01

Bacterial infectious diseases are currently a serious health problem, especially in patients compromised by illness or those receiving immune-suppressant drugs. In this context, it is not only essential to improve the understanding of infectious mechanisms and host response but also to discover novel therapies with extreme urgency. Probiotics and antimicrobial peptides are also favorably viewed as novel strategies in the control of resistant bacteria. The present review will shed some light on the use of probiotic microorganisms expressing antimicrobial peptides as a dual therapy to control bacterial infectious diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties

PubMed Central

2008-01-01

Polyguanidinium oxanorbornene (PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents. PMID:18850741

Transcriptome analysis of the responses of Staphylococcus aureus to antimicrobial peptides and characterization of the roles of vraDE and vraSR in antimicrobial resistance

PubMed Central

Pietiäinen, Milla; François, Patrice; Hyyryläinen, Hanne-Leena; Tangomo, Manuela; Sass, Vera; Sahl, Hans-Georg; Schrenzel, Jacques; Kontinen, Vesa P

2009-01-01

Background Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial for optimizing antimicrobial therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). Gene expression changes in S. aureus strain Newman exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two are derived from frog, temporin L and dermaseptin K4-S4(1-16), and the ovispirin-1 is obtained from sheep. Results The peptides induced the VraSR cell-wall regulon and several other genes that are also up-regulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, appeared to be strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 is dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps (GraRS) sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, revealed a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA. Conclusion Defense against different CAMPs involves not only general signaling pathways but also CAMP-specific ones. These results suggest that CAMPs or a mixture of CAMPs could constitute a potential additive to standard antibiotic treatment. PMID:19751498

High-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells.

PubMed

Rai, Akhilesh; Pinto, Sandra; Evangelista, Marta B; Gil, Helena; Kallip, Silvar; Ferreira, Mario G S; Ferreira, Lino

2016-03-01

Medical device-associated infections are a multi-billion dollar burden for the worldwide healthcare systems. The modification of medical devices with non-leaching coatings capable of killing microorganisms on contact is one of the strategies being investigated to prevent microorganism colonization. Here we developed a robust antimicrobial coating based on the chemical immobilization of the antimicrobial peptide (AMP), cecropin-melittin (CM), on gold nanoparticles coated surfaces. The concentration of AMP immobilized (110 μg/cm(2)) was higher than most of the studies reported so far (<10 μg/cm(2)). This translated onto a coating with high antimicrobial activity against Gram positive and negative bacteria sp., as well as multi-drug resistant bacteria. Studies with E. coli reporter bacteria showed that these coatings induced the permeability of the outer membrane of bacteria in less than 5 min and the inner membrane in approximately 20 min. Importantly, the antimicrobial properties of the coating are maintained in the presence of 20% (v/v) human serum, and have low probability to induce bacteria resistance. We further show that coatings have low toxicity against human endothelial and fibroblast cells and is hemocompatible since it does not induce platelet and complement activation. The antimicrobial coating described here may be promising to prevent medical device-associated infections. In recent years, antimicrobial peptides (AMPs) have been chemically immobilized on surfaces of medical devices to render them with antimicrobial properties. Surfaces having immobilized cationic peptides are susceptible to be adsorbed by plasma proteins with the subsequent loss of antimicrobial activity. Furthermore, with the exception of very few studies that have determined the cytotoxicity of surfaces in mammalian cells, the effect of the immobilized AMP on human cells is relatively unknown. Here we report a coating based on cecropin-melittin peptide (CM) that maintains its antimicrobial activity against Gram-positive and negative bacteria including multi-drugs resistance bacteria in the presence of serum and has relatively low cytotoxicity against human cells. The reported coatings may be translated on to variety of substrates (glass and titanium) and medical devices to prevent device-associated microbial infection. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Investigation of antimicrobial peptide genes associated with fungus and insect resistance in maize

USDA-ARS?s Scientific Manuscript database

Antimicrobial peptides (AMPs) are small defense proteins present in various organisms. Major groups of AMPs include beta-barrelin, hevein, knottin, lipid transfer protein (LTP), thionin, defensin, snakin, and cyclotide. Most plant AMPs involve host plant resistance to pathogens such as fungi, viruse...

Multitasking antimicrobial peptides, plant development, and host defense against biotic/abiotic stress

USDA-ARS?s Scientific Manuscript database

Crop losses due to pathogens are a major threat to global food security. Plants employ a multilayer defense system against pathogens including use of physical barriers (cell wall), induction of hypersensitive defense response (HR), resistance (R) proteins, and synthesis of antimicrobial peptides (AM...

Therapeutic peptides: new arsenal against drug resistant pathogens.

PubMed

Mok, Wendy W K; Li, Yingfu

2014-01-01

Our incessant tug-of-war with multidrug resistant pathogenic bacteria has prompted researchers to explore novel methods of designing therapeutics in order to defend ourselves against infectious diseases. Combined advances in whole genome analysis, bioinformatics algorithms, and biochemical techniques have led to the discovery and subsequent characterization of an abundant array of functional small peptides in microorganisms and multicellular organisms. Typically classified as having 10 to 100 amino acids, many of these peptides have been found to have dual activities, executing important defensive and regulatory functions in their hosts. In higher organisms, such as mammals, plants, and fungi, host defense peptides have been shown to have immunomodulatory and antimicrobial properties. In microbes, certain growth-inhibiting peptides have been linked to the regulation of diverse cellular processes. Examples of these processes include quorum sensing, stress response, cell differentiation, biofilm formation, pathogenesis, and multidrug tolerance. In this review, we will present a comprehensive overview of the discovery, characteristics, and functions of host- and bacteria-derived peptides with antimicrobial activities. The advantages and possible shortcomings of using these peptides as antimicrobial agents and targets will also be discussed. We will further examine current efforts in engineering synthetic peptides to be used as therapeutics and/or drug delivery vehicles.

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Efficacy of a novel antimicrobial peptide against periodontal pathogens in both planktonic and polymicrobial biofilm states.

PubMed

Wang, Hong-Yan; Cheng, Jya-Wei; Yu, Hui-Yuan; Lin, Li; Chih, Ya-Han; Pan, Ya-Ping

2015-10-01

Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis represent the early, middle and late colonizers of the bacterial accretion in dental plaque biofilms. These sessile communities constitute a protected mode of growth that promotes survival in a hostile environment. This study describes a novel and unrecognized role for a synthetic cationic antimicrobial peptide, Nal-P-113, which inhibits and kills periodontal bacteria in planktonic state, inhibits the formation of biofilms and eradicates polymicrobial biofilms. Nal-P-113 is also stable in saliva, serum and saline solution. At a concentration less than 320 μg/mL which is harmless to normal oral cells, Nal-P-113 can kill bacteria in planktonic state. At a concentration of antimicrobial peptide Nal-P-113 (1280 μg/mL) which only causes slight damages to normal oral cells is needed to kill bacteria in biofilm state. It is worth mentioning that this concentration of Nal-P-113 is harmless to rat oral mucosa compared to chlorhexidine. The mechanism of Nal-P-113 inhibiting and killing periodontal bacteria might rely on the abilities to permeabilize and/or to form pores within the cytoplasmic membranes, thus causes the death of bacteria. Here, we provided a novel and stable antimicrobial peptide with very low mammalian cytotoxicity, which can inhibit and kill periodontal bacteria in both planktonic and polymicrobial biofilm states. Nal-P-113 is a potent antimicrobial peptide with strong antimicrobial ability, improved deficiency compared with other antibacterial peptides, and remains stable in phosphate buffered saline, saliva, brain-heart infusion medium and bovine calf serum. Nal-P-113 exhibits a broad spectrum of bacteriocidal activity with excellent eradicating capability on oral pathogens and the respective biofilms. In this study, we used propidium iodide staining, scanning electron microscopy and transmission electron microscopy to confirm that Nal-P-113 can perforate plasmalemma thereby resulting in the death of oral pathogens and disintegrate the respective biofilms. Nal-P-113 also showed effective anti-plaque biofilms and cytotoxicity in the rat periodontitis model. No adverse effects can be observed on the gingivomucosa tissue. In short, the antimicrobial peptide Nal-P-113 presented to be an effective yet have low mammalian cytotoxicity agent with potential application in the clinic. This study provides a proof of concept in applying antimicrobial peptides in the clinical perspective. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Cationic Antimicrobial Peptides Derived from Crocodylus siamensis Leukocyte Extract, Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells.

PubMed

Theansungnoen, Tinnakorn; Maijaroen, Surachai; Jangpromma, Nisachon; Yaraksa, Nualyai; Daduang, Sakda; Temsiripong, Theeranan; Daduang, Jureerut; Klaynongsruang, Sompong

2016-06-01

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC50 values of KT2 and RT2 for HeLa and CaSki cells showed 28.7-53.4 and 17.3-30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.

Genomic and functional characterization of three new venom peptides from the scorpion Heterometrus spinifer.

PubMed

Wu, Shifen; Nie, Yao; Zeng, Xian-Chun; Cao, Hanjun; Zhang, Lei; Zhou, Lingli; Yang, Ye; Luo, Xuesong; Liu, Yichen

2014-03-01

Three new cysteine-free venom peptides, which are referred to as Heterin-1, Heterin-2 and Spiniferin, respectively, were identified from the scorpion Heterometrus spinifer. Heterin-1, Heterin-2 and Spiniferin contain 43, 24 and 13 amino acid residues, respectively. Genomic analysis showed that the genomic organizations of the three peptides are consistent with those of the known Na(+), K(+) or Cl(-)-channel specific toxins from scorpions; this suggests that the genes of the cysteine-free and cysteine-rich peptides from scorpions were derived from a common ancestor. Antimicrobial assay demonstrated that Heterin-1 possesses potent activities against both Gram-positive and Gram-negative bacteria. Among the tested bacterial species, Heterin-1 is the most active against Bacillus megaterium and Micrococcus luteus with MICs of 4.0 μM and 4.0 μM, respectively. Heterin-2 is able to potently inhibit the growth of Gram-positive bacteria with MICs from 5.6 μM to 30.0 μM; however, it has weaker activities against the tested Gram-negative bacteria. It is interesting to see that deletion of the C-terminal random coiled tail (KKD) in Heterin-2 markedly changed the antimicrobial specificity and activity of the peptide. Spiniferin has very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. We found that introducing three net charges into the polar face of Spiniferin significantly increased its antimicrobial activity against the majority of the tested bacteria; however, in some instances, net charge on the polar face is not important for the antimicrobial activity of the peptide. These studies have expanded our understanding of the diversity, evolution and structure/function relationships of the cysteine-free peptides from scorpions. Copyright © 2013 Elsevier Inc. All rights reserved.

NLF20: an antimicrobial peptide with therapeutic potential against invasive Pseudomonas aeruginosa infection.

PubMed

Papareddy, Praveen; Kasetty, Gopinath; Kalle, Martina; Bhongir, Ravi K V; Mörgelin, Matthias; Schmidtchen, Artur; Malmsten, Martin

2016-01-01

Increasing resistance to antibiotics makes antimicrobial peptides interesting as novel therapeutics. Here, we report on studies of the peptide NLF20 (NLFRKLTHRLFRRNFGYTLR), corresponding to an epitope of the D helix of heparin cofactor II (HCII), a plasma protein mediating bacterial clearance. Peptide effects were evaluated by a combination of in vitro and in vivo methods, including antibacterial, anti-inflammatory and cytotoxicity assays, fluorescence and electron microscopy, and experimental models of endotoxin shock and Pseudomonas aeruginosa sepsis. The results showed that NLF20 displayed potent antimicrobial effects against the Gram-negative bacteria Escherichia coli and P. aeruginosa, the Gram-positive Bacillus subtilis and Staphylococcus aureus and the fungi Candida albicans and Candida parapsilosis. Importantly, this antimicrobial effect was retained in human blood, particularly for P. aeruginosa. Fluorescence and electron microscopy studies showed that the peptide exerted membrane-breaking effects. In an animal model of P. aeruginosa sepsis, NLF20 reduced bacterial levels, resulting in improved survival. Reduced mortality was also observed in experimental animal models of endotoxin shock, which was paralleled with modulated IFN-γ, IL-10 and coagulation responses. Together, these results indicate that functional epitopes of HCII may have therapeutic potential against bacterial infection. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Using genomics to identify novel antimicrobials.

PubMed

Kim, W H; Lillehoj, H S; Gay, C G

2016-04-01

There is a critical need in animal agriculture to develop novel antimicrobials and alternative strategies that will help to reduce the use of antibiotics and address the challenges of antimicrobial resistance. High-throughput gene expression analysis is providing new tools that are enabling the discovery of host-derived antimicrobial peptides. Examples of gene-encoded natural antibiotics that have gained attention include antimicrobial peptides such as human granulysin and its multi-species homolog, namely NK-lysin, which provide a protective response against a broad range of microbes and are a principal component of innate immunity in vertebrates. Both granulysin and NK-lysin are localised in cytolytic granules in natural killer and cytotoxic T lymphocytes. Host-derived NK-lysins that were first described in mammals are also found in avian species, and they have been shown to have antimicrobial activities that could potentially be used to control important poultry pathogens. Morphological alterations observed following chicken NK-lysin binding to Eimeria sporozoites and Escherichia coli membranes indicate damage and disruption of cell membranes, suggesting that NK-lysin kills pathogenic protozoans and bacteria by direct interaction. Genotype analysis revealed that chicken NK-lysin peptides derived from certain alleles were more effective at killing pathogens than those derived from others, which could potentially affect susceptibility to diseases. Although the host-derived antimicrobial peptides described in this paper may not, by themselves, be able to replace the antibiotics currently used in animal production, their use as specific treatments based on their known mechanisms of action is showing promising results.

The use of antimicrobial peptides in ophthalmology: an experimental study in corneal preservation and the management of bacterial keratitis.

PubMed Central

Mannis, Mark J

2002-01-01

PURPOSE: Bacterial keratitis is an ocular infection with the potential to cause significant visual impairment. Increasing patterns of antibiotic resistance have necessitated the development of new antimicrobial agents for use in bacterial keratitis and other serious ocular infections. With a view to exploring the use of novel antimicrobial peptides in the management of ocular infection, we performed a series of experiments using synthetic antimicrobial peptides designed for the eradication of common and serious ophthalmic pathogens. METHODS: Experiments were performed with three clinical ocular isolates--Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis--in three experimental settings: (1) in vitro in a controlled system of 10 mM sodium phosphate buffer, (2) in vitro in modified chondroitin sulfate-based corneal preservation media (Optisol), and (3) in an in vivo animal model (rabbit) simulating bacterial keratitis. In all cases, outcomes were measured by quantitative microbiological techniques. RESULTS: The candidate peptides (CCI A, B, and C and COL-1) produced a total reduction of the test pathogens in phosphate buffered saline. In modified Optisol, the peptides were effective against S epidermidis at all temperatures, demonstrated augmented activity at 23 degrees C against the gram-positive organisms, but were ineffective against P aeruginosa. The addition of EDTA to the medium augmented the killing of P aeruginosa but made no difference in the reduction of gram-positive organisms. In an in vivo rabbit model of Pseudomonas keratitis, COL-1 demonstrated neither clinical nor microbicidal efficacy and appeared to have a very narrow dosage range, outside of which it appeared to be toxic to the ocular surface. CONCLUSION: Our data indicate that the antimicrobial peptides we tested were effective in vitro but not in vivo. In an age of increasing antibiotic resistance, antimicrobial peptides, developed over millions of years as innate defense mechanisms by plants and animals, may have significant potential for development as topical agents for the management of severe bacterial keratitis. However, modifications of the peptides, the drug delivery systems, or both, will be necessary for effective clinical application. PMID:12545697

Design of novel antimicrobial peptide dimer analogues with enhanced antimicrobial activity in vitro and in vivo by intermolecular triazole bridge strategy.

PubMed

Liu, Beijun; Huang, Haifeng; Yang, Zhibin; Liu, Beiyin; Gou, Sanhu; Zhong, Chao; Han, Xiufeng; Zhang, Yun; Ni, Jingman; Wang, Rui

2017-02-01

Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4-16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2×MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2-6h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120mg/kg. The 50% lethal dose (LD 50 ) of J-AA was 53.6mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option. Copyright © 2016 Elsevier Inc. All rights reserved.

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression

USDA-ARS?s Scientific Manuscript database

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. I...

Novel mode of action of plant defense peptides: hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases

USDA-ARS?s Scientific Manuscript database

The multilayered plant immune system relies on rapid recognition of pathogen-associated molecular patterns followed by activation of defense-related genes that results in the reinforcement of plant cell walls and production of antimicrobial compounds. To suppress plant defense, fungi secrete effecto...

Enzymatic fractionation of the antimicrobial peptides casocidin and isracidin by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

USDA-ARS?s Scientific Manuscript database

The cumulative effect of peptidase and protease activities associated with cells of Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LB) was evaluated on the milk-protein based antimicrobial peptides casocidin and isracidin. Reaction mixtures of casocidin or isracidin...

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

USDA-ARS?s Scientific Manuscript database

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response in various organisms, including cattle. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Collective results...

In vitro growth of growth of campylobacter spp. inhibited by selected antimicrobial peptides

USDA-ARS?s Scientific Manuscript database

Background: Novel alternatives to traditional antibiotics are urgently needed for food-animal production. A goal of our laboratory is to develop and evaluate antimicrobial peptides (AMP) to control and reduce foodborne pathogens in poultry. AMP have been found in most every class of living organism...

The next generation of antimicrobial peptides (AMPs) as molecular therapeutic tools for the treatment of diseases with social and economic impacts.

PubMed

da Cunha, Nicolau B; Cobacho, Nicole B; Viana, Juliane F C; Lima, Loiane A; Sampaio, Kamila B O; Dohms, Stephan S M; Ferreira, Arthur C R; de la Fuente-Núñez, César; Costa, Fabrício F; Franco, Octávio L; Dias, Simoni C

2017-02-01

Anti-infective drugs have had a key role in the contemporary world, contributing to dramatically decrease mortality rates caused by infectious diseases worldwide. Antimicrobial peptides (AMPs) are multifunctional effectors of the innate immune system of mucosal surfaces and present antimicrobial activity against a range of pathogenic viruses, bacteria, and fungi. However, the discovery and development of new antibacterial drugs is a crucial step to overcome the great challenge posed by the emergence of antibiotic resistance. In this review, we outline recent advances in the development of novel AMPs with improved antimicrobial activities that were achieved through characteristic structural design. In addition, we describe recent progress made to overcome some of the major limitations that have hindered peptide biosynthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro antimicrobial activity of a gel containing antimicrobial peptide AMP2041, chlorhexidine digluconate and Tris-EDTA on clinical isolates of Pseudomonas aeruginosa from canine otitis.

PubMed

Ghibaudo, Giovanni; Santospirito, Davide; Sala, Andrea; Flisi, Sara; Taddei, Simone; Cavirani, Sandro; Cabassi, Clotilde Silvia

2016-10-01

Pseudomonas aeruginosa (PA) may cause suppurative otitis externa with severe inflammation and ulceration in dogs. Multidrug resistance is commonly reported for this organism, creating a difficult therapeutic challenge. The aim of this study was to evaluate the in vitro antimicrobial activity of a gel containing 0.5 μg/mL of antimicrobial peptide AMP2041, 0.07% chlorhexidine digluconate (CLX), 0.4% Tris and 0.1% EDTA on 30 clinical isolates of PA from canine otitis externa. Antimicrobial activity was evaluated through minimal bactericidal concentration (MBC). Standardized bacterial suspensions were incubated with different concentrations of the gel at 37°C for 30 min and plated for colony forming unit (CFU) counts. Time-to-kill kinetics were evaluated with the undiluted product and at MBC for each PA strain at 30 s, 1, 5, 10, 15, 30 min, 24 and 48 h. The MBC was 1:64 for two of 30 strains, 1:128 for 15 of 30 strains and 1:256 for 13 of 30 strains. The geometric mean was 1:165, equivalent to a concentration of 0.003 μg/mL AMP2041 + 0.0004% CLX + 0.0024%Tris + 0.0006% EDTA. Time-to-kill assays with the undiluted product showed complete bactericidal effect within 30 s for all isolates, whereas at the MBC this effect was reached within 5 min for 20 of 30 isolates and within 30 min for all isolates. Bactericidal activity was maintained after 48 h for all isolates. This gel has shown rapid, complete and long-lasting activity against a panel of 30 PA isolates from cases of canine otitis externa. © 2016 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the ESVD and ACVD.

Ab Initio Design of Potent Anti-MRSA Peptides based on Database Filtering Technology

PubMed Central

Mishra, Biswajit; Wang, Guangshun

2012-01-01

To meet the challenge of antibiotic resistance worldwide, a new generation of antimicrobials must be developed.1 This communication demonstrates ab initio design of potent peptides against methicillin-resistant Staphylococcus aureus (MRSA). Our idea is that the peptide is very likely to be active when most probable parameters are utilized in each step of the design. We derived the most probable parameters (e.g. amino acid composition, peptide hydrophobic content, and net charge) from the antimicrobial peptide database2 by developing a database filtering technology (DFT). Different from classic cationic antimicrobial peptides usually with high cationicity, DFTamP1, the first anti-MRSA peptide designed using this technology, is a short peptide with high hydrophobicity but low cationicity. Such a molecular design made the peptide highly potent. Indeed, the peptide caused bacterial surface damage and killed community-associated MRSA USA300 in 60 minutes. Structural determination of DFTamP1 by NMR spectroscopy revealed a broad hydrophobic surface, providing a basis for its potency against MRSA known to deploy positively charged moieties on the surface as a mechanism for resistance. A combination of our ab initio design with database screening3 led to yet another peptide with enhanced potency. Because of simple composition, short length, stability to proteases, and membrane targeting, the designed peptides are attractive leads for developing novel anti-MRSA therapeutics. Our database-derived design concept can be applied to the design of peptide mimicries to combat MRSA as well. PMID:22803960

Ab initio design of potent anti-MRSA peptides based on database filtering technology.

PubMed

Mishra, Biswajit; Wang, Guangshun

2012-08-01

To meet the challenge of antibiotic resistance worldwide, a new generation of antimicrobials must be developed. This communication demonstrates ab initio design of potent peptides against methicillin-resistant Staphylococcus aureus (MRSA). Our idea is that the peptide is very likely to be active when the most probable parameters are utilized in each step of the design. We derived the most probable parameters (e.g., amino acid composition, peptide hydrophobic content, and net charge) from the antimicrobial peptide database by developing a database filtering technology (DFT). Different from classic cationic antimicrobial peptides usually with high cationicity, DFTamP1, the first anti-MRSA peptide designed using this technology, is a short peptide with high hydrophobicity but low cationicity. Such a molecular design made the peptide highly potent. Indeed, the peptide caused bacterial surface damage and killed community-associated MRSA USA300 in 60 min. Structural determination of DFTamP1 by NMR spectroscopy revealed a broad hydrophobic surface, providing a basis for its potency against MRSA known to deploy positively charged moieties on the surface as a mechanism for resistance. Our ab initio design combined with database screening led to yet another peptide with enhanced potency. Because of the simple composition, short length, stability to proteases, and membrane targeting, the designed peptides are attractive leads for developing novel anti-MRSA therapeutics. Our database-derived design concept can be applied to the design of peptide mimicries to combat MRSA as well.

Periodontal disease in patients from the original Kostmann family with severe congenital neutropenia.

PubMed

Carlsson, Göran; Wahlin, Ylva-Britt; Johansson, Anders; Olsson, Anders; Eriksson, Torbjörn; Claesson, Rolf; Hänström, Lennart; Henter, Jan-Inge

2006-04-01

Patients with Kostmann syndrome (severe congenital neutropenia [SCN]) typically normalize their absolute neutrophil count (ANC) upon granulocyte colony-stimulating factor (G-CSF) therapy. However, although they no longer experience life-threatening bacterial infections, they frequently still have recurrent gingivitis and even severe periodontitis, often starting in early childhood. We studied the periodontal disease in the four surviving patients belonging to the family originally described by Kostmann. Their odontological records, x-rays, color photos, bacterial cultures, serum antibodies to oral bacteria, and histopathological examinations were reviewed. The data were also correlated to previous investigations on their antibacterial peptides and molecular biology. Three patients had periodontal disease, despite normal ANC and professional dental care, and had neutrophils deficient in antibacterial peptides. One of these patients also had a heterozygous mutation in the neutrophil elastase gene, had severe periodontal disease and overgrowth of the periodontal pathogen Actinobacillus actinomycetemcomitans in the dental flora, and 15 permanent teeth had been extracted by the age of 27. One bone marrow-transplanted patient had no periodontal disease. Normalized ANC levels are not sufficient to maintain normal oral health in SCN patients, and because neutrophils are important for first-line defense and innate immunity, the deficiency of the antibacterial peptide LL-37 probably explains their chronic periodontal disease. Professional dental care is still important for SCN patients, despite treatment with G-CSF and normal ANC levels. Whether antibacterial peptides play a role in the pathogenesis of periodontitis in other patients remains to be elucidated.

Effect of physicochemical properties of peptides from soy protein on their antimicrobial activity.

PubMed

Xiang, Ning; Lyu, Yuan; Zhu, Xiao; Bhunia, Arun K; Narsimhan, Ganesan

2017-08-01

Antimicrobial peptides (AMPs) kill microbial cells through insertion and damage/permeabilization of the cytoplasmic cell membranes and has applications in food safety and antibiotic replacement. Soy protein is an attractive, abundant natural source for commercial production of AMPs. In this research, explicit solvent molecular dynamics (MD) simulation was employed to investigate the effects of (i) number of total and net charges, (ii) hydrophobicity (iii) hydrophobic moment and (iv) helicity of peptides from soy protein on their ability to bind to lipid bilayer and their transmembrane aggregates to form pores. Interaction of possible AMP segments from soy protein with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPC/POPG) bilayers, a mimic of bacterial cell membrane, was investigated. Pore formation was insensitive to helicity and occurred for hydrophobicity threshold in the range of -0.3-0kcal/mol, hydrophobic moment threshold of 0.3kcal/mol, net charge threshold of 2. Though low hydrophobicity and high number of charges help in the formation of water channel for transmembrane aggregates, insertion of peptides with these properties requires overcome of energy barrier, as shown by potential of mean force calculations, thereby resulting in low antimicrobial activity. Experimental evaluation of antimicrobial activity of these peptides against Gram positive L. monocytogenes and Gram negative E. coli as obtained by spot-on-lawn assay was consistent with simulation results. These results should help in the development of guidelines for selection of peptides with antimicrobial activity based on their physicochemical properties. Copyright © 2017 Elsevier Inc. All rights reserved.

Antimicrobial Peptides as Potential Alternatives to Antibiotics in Food Animal Industry.

PubMed

Wang, Shuai; Zeng, Xiangfang; Yang, Qing; Qiao, Shiyan

2016-05-03

Over the last decade, the rapid emergence of multidrug-resistant pathogens has become a global concern, which has prompted the search for alternative antibacterial agents for use in food animals. Antimicrobial peptides (AMPs), produced by bacteria, insects, amphibians and mammals, as well as by chemical synthesis, are possible candidates for the design of new antimicrobial agents because of their natural antimicrobial properties and a low propensity for development of resistance by microorganisms. This manuscript reviews the current knowledge of the basic biology of AMPs and their applications in non-ruminant nutrition. Antimicrobial peptides not only have broad-spectrum activity against bacteria, fungi, and viruses but also have the ability to bypass the common resistance mechanisms that are placing standard antibiotics in jeopardy. In addition, AMPs have beneficial effects on growth performance, nutrient digestibility, intestinal morphology and gut microbiota in pigs and broilers. Therefore, AMPs have good potential as suitable alternatives to conventional antibiotics used in swine and poultry industries.

Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens.

PubMed

Hu, Fengjiao; Wu, Qiaoxing; Song, Shuang; She, Ruiping; Zhao, Yue; Yang, Yifei; Zhang, Meikun; Du, Fang; Soomro, Majid Hussain; Shi, Ruihan

2016-12-05

Hemoglobin is a rich source of biological peptides. As a byproduct and even wastewater of poultry-slaughtering facilities, chicken blood is one of the most abundant source of hemoglobin. In this study, the chicken hemoglobin antimicrobial peptides (CHAP) were isolated and the antimicrobial and bactericidal activities were tested by the agarose diffusion assay, minimum inhibitory concentration (MIC) analysis, minimal bactericidal concentration (MBC) analysis, and time-dependent inhibitory and bactericidal assays. The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains, including 9 multidrug-resistant bacterial strains. Bacterial biofilm and NaCl permeability assays, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were further performed to detect the mechanism of its antimicrobial effect. Additionally, CHAP showed low hemolytic activity, embryo toxicity, and high stability in different temperatures and animal plasma. CHAP may have great potential for expanding production and development value in animal medication, the breeding industry and environment protection.

Increased survival of experimentally evolved antimicrobial peptide-resistant Staphylococcus aureus in an animal host

PubMed Central

Dobson, Adam J; Purves, Joanne; Rolff, Jens

2014-01-01

Antimicrobial peptides (AMPs) have been proposed as new class of antimicrobial drugs, following the increasing prevalence of bacteria resistant to antibiotics. Synthetic AMPs are functional analogues of highly evolutionarily conserved immune effectors in animals and plants, produced in response to microbial infection. Therefore, the proposed therapeutic use of AMPs bears the risk of ‘arming the enemy’: bacteria that evolve resistance to AMPs may be cross-resistant to immune effectors (AMPs) in their hosts. We used a panel of populations of Staphylococcus aureus that were experimentally selected for resistance to a suite of individual AMPs and antibiotics to investigate the ‘arming the enemy’ hypothesis. We tested whether the selected strains showed higher survival in an insect model (Tenebrio molitor) and cross-resistance against other antimicrobials in vitro. A population selected for resistance to the antimicrobial peptide iseganan showed increased in vivo survival, but was not more virulent. We suggest that increased survival of AMP-resistant bacteria almost certainly poses problems to immune-compromised hosts. PMID:25469169

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

PubMed

Ghanmi, Fadoua; Carré-Mlouka, Alyssa; Vandervennet, Manon; Boujelben, Ines; Frikha, Doniez; Ayadi, Habib; Peduzzi, Jean; Rebuffat, Sylvie; Maalej, Sami

2016-05-01

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Sequence characterization of cDNA sequence of encoding of an antimicrobial Peptide with no disulfide bridge from the Iranian mesobuthus eupeus venomous glands.

PubMed

Farajzadeh-Sheikh, Ahmad; Jolodar, Abbas; Ghaemmaghami, Shamsedin

2013-01-01

Scorpion venom glands produce some antimicrobial peptides (AMP) that can rapidly kill a broad range of microbes and have additional activities that impact on the quality and effectiveness of innate responses and inflammation. In this study, we reported the identification of a cDNA sequence encoding cysteine-free antimicrobial peptides isolated from venomous glands of this species. Total RNA was extracted from the Iranian mesobuthus eupeus venom glands, and cDNA was synthesized by using the modified oligo (dT). The cDNA was used as the template for applying Semi-nested RT- PCR technique. PCR Products were used for direct nucleotide sequencing and the results were compared with Gen Bank database. A 213 BP cDNA fragment encoding the entire coding region of an antimicrobial toxin from the Iranian scorpion M. Eupeus venom glands were isolated. The full-length sequence of the coding region was 210 BP contained an open reading frame of 70 amino with a predicted molecular mass of 7970.48 Da and theoretical Pi of 9.10. The open reading frame consists of 210 BP encoding a precursor of 70 amino acid residues, including a signal peptide of 23 residues a propertied of 7 residues, and a mature peptide of 34 residues with no disulfide bridge. The peptide has detectable sequence identity to the Lesser Asian mesobuthus eupeus MeVAMP-2 (98%), MeVAMP-9 (60%) and several previously described AMPs from other scorpion venoms including mesobuthus martensii (94%) and buthus occitanus Israelis (82%). The secondary structure of the peptide mainly consisted of α-helical structure which was generally conserved by previously reported scorpion counterparts. The phylogenetic analysis showed that the Iranian MeAMP-like toxin was similar but not identical with that of venom antimicrobial peptides from lesser Asian scorpion mesobuthus eupeus.

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin.

PubMed

Falcao, Loeni L; Silva-Werneck, Joseilde O; Ramos, Alessandra de R; Martins, Natalia F; Bresso, Emmanuel; Rodrigues, Magali A; Bemquerer, Marcelo P; Marcellino, Lucilia H

2016-05-01

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH2), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH2), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC=40μM and MIC=127μM, respectively), as well as for P. pastoris (MIC=20μM and MIC=127μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi. Copyright © 2016 Elsevier Inc. All rights reserved.

Antimicrobial activity of synthetic cationic peptides and lipopeptides derived from human lactoferricin against Pseudomonas aeruginosa planktonic cultures and biofilms.

PubMed

Sánchez-Gómez, Susana; Ferrer-Espada, Raquel; Stewart, Philip S; Pitts, Betsey; Lohner, Karl; Martínez de Tejada, Guillermo

2015-07-07

Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen -particularly when it forms biofilms - can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least hydrophobic lipopeptides, DI-MB-LF11-322 (2,2-dimethylbutanoyl-PFWRIRIRR) and DI-MB-LF11-215, penetrated deep into the biofilm structure and homogenously killed biofilm-forming bacteria. We identified peptides derived from human lactoferricin with potent antimicrobial activity against P. aeruginosa growing either in planktonic or in biofilm mode. Although further structure-activity relationship analyses are necessary to optimize the anti-biofilm activity of these compounds, the results indicate that lactoferricin derived peptides are promising anti-biofilm agents.

DBAASP v.2: an enhanced database of structure and antimicrobial/cytotoxic activity of natural and synthetic peptides

PubMed Central

Pirtskhalava, Malak; Gabrielian, Andrei; Cruz, Phillip; Griggs, Hannah L.; Squires, R. Burke; Hurt, Darrell E.; Grigolava, Maia; Chubinidze, Mindia; Gogoladze, George; Vishnepolsky, Boris; Alekseev, Vsevolod; Rosenthal, Alex; Tartakovsky, Michael

2016-01-01

Antimicrobial peptides (AMPs) are anti-infectives that may represent a novel and untapped class of biotherapeutics. Increasing interest in AMPs means that new peptides (natural and synthetic) are discovered faster than ever before. We describe herein a new version of the Database of Antimicrobial Activity and Structure of Peptides (DBAASPv.2, which is freely accessible at http://dbaasp.org). This iteration of the database reports chemical structures and empirically-determined activities (MICs, IC50, etc.) against more than 4200 specific target microbes for more than 2000 ribosomal, 80 non-ribosomal and 5700 synthetic peptides. Of these, the vast majority are monomeric, but nearly 200 of these peptides are found as homo- or heterodimers. More than 6100 of the peptides are linear, but about 515 are cyclic and more than 1300 have other intra-chain covalent bonds. More than half of the entries in the database were added after the resource was initially described, which reflects the recent sharp uptick of interest in AMPs. New features of DBAASPv.2 include: (i) user-friendly utilities and reporting functions, (ii) a ‘Ranking Search’ function to query the database by target species and return a ranked list of peptides with activity against that target and (iii) structural descriptions of the peptides derived from empirical data or calculated by molecular dynamics (MD) simulations. The three-dimensional structural data are critical components for understanding structure–activity relationships and for design of new antimicrobial drugs. We created more than 300 high-throughput MD simulations specifically for inclusion in DBAASP. The resulting structures are described in the database by novel trajectory analysis plots and movies. Another 200+ DBAASP entries have links to the Protein DataBank. All of the structures are easily visualized directly in the web browser. PMID:26578581

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: biochemical and functional characterization of natural peptides and a single site-substituted analog.

PubMed

Almaaytah, Ammar; Zhou, Mei; Wang, Lei; Chen, Tianbao; Walker, Brian; Shaw, Chris

2012-06-01

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu-->Pro at position 2 and Phe-->Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20 μM and 150 μM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His-->Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5 μM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His-->Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40 μm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means. Copyright © 2012 Elsevier Inc. All rights reserved.

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium

PubMed Central

Silva, Tânia; Moreira, Ana C.; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G. M.; Rodrigues, Pedro N.; Bastos, Margarida

2017-01-01

ABSTRACT Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17–30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17–30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17–30 did not localize to M. avium-harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17–30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis, M. leprae, M. avium, etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17–30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17–30 on macrophages, which may be useful under other conditions in which macrophage activation is needed. PMID:28875176

Lactoferricin Peptides Increase Macrophages' Capacity To Kill Mycobacterium avium.

PubMed

Silva, Tânia; Moreira, Ana C; Nazmi, Kamran; Moniz, Tânia; Vale, Nuno; Rangel, Maria; Gomes, Paula; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

2017-01-01

Mycobacterial infections cause a significant burden of disease and death worldwide. Their treatment is long, toxic, costly, and increasingly prone to failure due to bacterial resistance to currently available antibiotics. New therapeutic options are thus clearly needed. Antimicrobial peptides represent an important source of new antimicrobial molecules, both for their direct activity and for their immunomodulatory potential. We have previously reported that a short version of the bovine antimicrobial peptide lactoferricin with amino acids 17 to 30 (LFcin17-30), along with its variants obtained by specific amino acid substitutions, killed Mycobacterium avium in broth culture. In the present work, those peptides were tested against M. avium living inside its natural host cell, the macrophage. We found that the peptides increased the antimicrobial action of the conventional antibiotic ethambutol inside macrophages. Moreover, the d-enantiomer of the lactoferricin peptide (d-LFcin17-30) was more stable and induced significant killing of intracellular mycobacteria by itself. Interestingly, d-LFcin17-30 did not localize to M. avium -harboring phagosomes but induced the production of proinflammatory cytokines and increased the formation of lysosomes and autophagosome-like vesicles. These results lead us to conclude that d-LFcin17-30 primes macrophages for intracellular microbial digestion through phagosomal maturation and/or autophagy, culminating in mycobacterial killing. IMPORTANCE The genus Mycobacterium comprises several pathogenic species, including M. tuberculosis , M. leprae , M. avium , etc. Infections caused by these bacteria are particularly difficult to treat due to their intrinsic impermeability, low growth rate, and intracellular localization. Antimicrobial peptides are increasingly acknowledged as potential treatment tools, as they have a high spectrum of activity, low tendency to induce bacterial resistance, and immunomodulatory properties. In this study, we show that peptides derived from bovine lactoferricin (LFcin) improve the antimicrobial activity of ethambutol against Mycobacterium avium growing inside macrophages. Moreover, the d-enantiomer of a short version of lactoferricin containing amino acids 17 to 30 (d-LFcin17-30) causes intramacrophagic death of M. avium by increasing the formation of lysosomes and autophagosomes. This work opens the way to the use of lactoferricin-derived peptides to treat infections caused by mycobacteria and highlights important modulatory effects of d-FLcin17-30 on macrophages, which may be useful under other conditions in which macrophage activation is needed.

[Research progress in fusion expression of antimicrobial peptides].

PubMed

Ma, Qingshan; Yu, Zhanqiao; Han, Bing; Zhang, Rijun

2011-10-01

Antimicrobial peptides (AMPs) are of great significance in the field of food, feed and medicine due to their wide spectrum of antimicrobial activity and new mechanism of action different from conventional antibiotics. AMPs production from natural sources is usually limited, and chemical synthesis is not economically practical, especially for the production of long peptides. Therefore, heterologous expression of AMPs has been widely studied as an alternative, and fusion expression plays an important role in increasing production. The present review mainly focuses on the types and bioactivities of AMPs. In addition, the recent strategies to the most commonly used carrier proteins for fusion expression of AMPs and prospects for future research were also discussed.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

PubMed

Polyansky, Anton A; Vassilevski, Alexander A; Volynsky, Pavel E; Vorontsova, Olga V; Samsonova, Olga V; Egorova, Natalya S; Krylov, Nicolay A; Feofanov, Alexei V; Arseniev, Alexander S; Grishin, Eugene V; Efremov, Roman G

2009-07-21

In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.).

PubMed

Fujimura, Masatoshi; Ideguchi, Mineo; Minami, Yuji; Watanabe, Keiichi; Tadera, Kenjiro

2004-03-01

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

Opposing effects of cationic antimicrobial peptides and divalent cations on bacterial lipopolysaccharides

NASA Astrophysics Data System (ADS)

Smart, Matthew; Rajagopal, Aruna; Liu, Wing-Ki; Ha, Bae-Yeun

2017-10-01

The permeability of the bacterial outer membrane, enclosing Gram-negative bacteria, depends on the interactions of the outer, lipopolysaccharide (LPS) layer, with surrounding ions and molecules. We present a coarse-grained model for describing how cationic amphiphilic molecules (e.g., antimicrobial peptides) interact with and perturb the LPS layer in a biologically relevant medium, containing monovalent and divalent salt ions (e.g., Mg2+). In our approach, peptide binding is driven by electrostatic and hydrophobic interactions and is assumed to expand the LPS layer, eventually priming it for disruption. Our results suggest that in parameter ranges of biological relevance (e.g., at micromolar concentrations) the antimicrobial peptide magainin 2 effectively disrupts the LPS layer, even though it has to compete with Mg2+ for the layer. They also show how the integrity of LPS is restored with an increasing concentration of Mg2+. Using the approach, we make a number of predictions relevant for optimizing peptide parameters against Gram-negative bacteria and for understanding bacterial strategies to develop resistance against cationic peptides.

Antimicrobial peptides: a new class of antimalarial drugs?

PubMed Central

Vale, Nuno; Aguiar, Luísa; Gomes, Paula

2014-01-01

A range of antimicrobial peptides (AMP) exhibit activity on malaria parasites, Plasmodium spp., in their blood or mosquito stages, or both. These peptides include a diverse array of both natural and synthetic molecules varying greatly in size, charge, hydrophobicity, and secondary structure features. Along with an overview of relevant literature reports regarding AMP that display antiplasmodial activity, this review makes a few considerations about those molecules as a potential new class of antimalarial drugs. PMID:25566072

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus

PubMed Central

Hieggelke, Lena; Schneiker, Bianca; Lysson, Mariola; Stoffels, Burkhard; Nuding, Sabine; Wehkamp, Jan; Kikhney, Judith; Moter, Annette; Kalff, Joerg C.

2018-01-01

Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery. PMID:29652914