Antimicrobial peptides

Scientific publications - Antimicrobial peptides

 

​Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, Pag U, Jansen A, Nielsen AK, Mygind PH, Raventós DS, Neve S, Ravn B, Bonvin AM, De Maria L, Andersen AS, Gammelgaard LK, Sahl HG, Kristensen HH.

"Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II"

 

June 2010

Pharmaceutical Microbiology Section, Institute for Medical Microbiology, Immunology, and Parasitology, University of Bonn, D-53115 Bonn, Germany.

 
Abstract
Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.
 
PMID: 20508130 [PubMed - in process]
 

Thomas Kruse Bjarke Christensen Dorotea Ravento´s, Allan K. Nielsen Jesper D. Nielsen, Natasa Vukmirovic, Hans-Henrik Kristensen.

"Transcriptional Profile of Escherichia coli in Response to Novispirin G10"

26 September 2008

 
Abstract
Using a novel methodology, we have investigated the transcriptional response of Escherichia coli to novispirin G10, an α-helical cationic antimicrobial peptide. We show that novispirin G10 induces an exceptionally coherent transcriptional response in E. coli, resulting in upregulation of genes involved in response to osmotic stress, acid shock, phage shock, and antimicrobial peptides, and own-regulation of the heat shock response genes, e.g., dnaJK, GroES, and GroEL. This transcriptional pattern indicates that novispirin G10 acts by compromising the bacterial membrane and possibly also by targeting the heat shock response. The impact of novispirin G10 on E. coli cells was monitored directly using the fluorescent LIVE/DEAD assay verifying that the peptide, indeed, targets bacterial membranes. Furthermore, in agreement with the observed heat shock transcriptional response, we show that overexpression of the heat shock transcription factor in E. coli, σ 32, leads to a significant decrease in sensitivity towards novispirin G10.
 
 

Thevissen, K., Kristensen, H.-H., Thomma, B.P.H.J., Cammue, B.P.A., François, I.E.J.A.

"Therapeutic potential of antifungal plant and insect defensins"

Drug Discovery Today, 12 (21-22), pp. 966-971. (2007)

 
Abstract
To defend themselves against invading fungal pathogens, plants and insects largely depend on the production of a wide array of antifungal molecules, including antimicrobial peptides such as defensins. Interestingly, plant and insect defensins display antimicrobial activity not only against plant and insect pathogens but also against human fungal pathogens, including Candida spp. and Aspergillus spp. This review focuses on these defensins as novel leads for antifungal therapeutics. Their mode of action, involving interaction with fungus-specific sphingolipids, and heterologous expression, required for cost-effective production, are major assets for development of plant and insect defensins as antifungal leads. Studies evaluating their in vivo antifungal efficacy demonstrate their therapeutic potential. © 2007 Elsevier Ltd. All rights reserved.
 
 

Jacobsen, F., Mohammadi-Tabrisi, A., Hirsch, T., Mittler, D., Mygind, P.H., Sonksen, C.P., Raventos, D., Kristensen, H.H., Gatermann, S., Lehnhardt, M., Daigeler, A., Steinau, H.U., Steinstraesser, L.

"Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin"

Journal of Antimicrobial Chemotherapy, 59 (3), pp. 493-498. (2007)

 
Abstract
Objectives: The growing number of patients with impaired wound healing and the development of multidrug-resistant bacteria demand the investigation of alternatives in wound care. The antimicrobial activity of naturally occurring host defence peptides and their derivatives could be one alternative to the existing therapy options for topical treatment of wound infection. Therefore, the aim of this study was to investigate the antimicrobial activity of proline-novispirin G10 (P-novispirin G10) in vitro and in the infected porcine titanium wound chamber model. Methods: The new derived designer host defence peptide P-novispirin G10 was tested in vitro against Gram-positive and Gram-negative bacterial strains. Additionally, cytotoxicity and haemolytic activities of P-novispirin G10 and protegrin-1 were measured. For in vivo studies, six wound chambers were implanted on each flank of Göttinger minipigs (n = 2, female, 6 months old, 15-20 kg). Eleven wound chambers were inoculated 8 days post-operatively with 5 × 108 of Staphylococcus aureus; one wound chamber remained uninfected as a system control. After wound infection had been established (4 days after inoculation), each wound chamber was topically treated with P-novispirin G10, protegrin-1 or carrier control. Wound fluid was harvested every hour for a total follow up of 3 h. Results: P-novispirin G10 demonstrated broad-spectrum antimicrobial activity with moderate haemolytic and cytotoxic activities compared with protegrin-1. In the infected wound chamber model P-novispirin G10 demonstrated a 4 log10 reduction in bacterial counts. Conclusions: This implicates the potential of P-novispirin G10 as an alternative in future antimicrobial wound care. However, more studies are necessary to further define clinical applications and potential side effects in greater detail. © The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
 
 

Taboureau, O., Olsen, O.H., Nielsen, J.D., Raventos, D., Mygind, P.H., Kristensen, H.-H.

"Design of novispirin antimicrobial peptides by quantitative structure-activity relationship"

Chemical Biology and Drug Design, 68 (1), pp. 48-57. (2006)

 
Abstract
Novispirin G10 is an α-helical antimicrobial peptide designed in an effort to develop alternative treatments against multidrug-resistant micro-organisms. To further optimize the antimicrobial activity, 58 novispirin analogs were constructed and used to establish a quantitative structure-activity relationship model. A statistically significant model (r2 = 0.73, q2 = 0.61) was obtained using a set of 69 selected molecular descriptors. Among these, VolSurf and charged partial surface area descriptors played a dominant role. Analysis of the model indicated that hydrophobicity, amphipathicity and charge were the most important features influencing activity for this set of peptides. Furthermore, the ability of the quantitative structure-activity relationship model to predict bioactivity was evaluated by analyzing a set of 400 novispirin analogs designed by molecular modeling. Out of these 400, 16 new novispirins with a higher predicted antimicrobial activity were tested in the suicide expression system, and about three out of four appeared more potent than the parent novispirin G10. Combination of VolSurf and charged partial surface area descriptors seems relevant to depict the interaction between novispirin and its target(s), presumably the microbial cell membrane. The presented findings show that modeling and quantitative structure-activity relationship methods can be useful in the construction of and/or optimization of the bioactivity of antimicrobial peptides for further development as effective antibiotic therapeutics. © 2006 The Authors.
 
 

Wimmer, R., Andersen, K.K., Vad, B., Davidsen, M., Mølgaard, S., Nesgaard, L.W., Kristensen, H.H., Otzen, D.E.

"Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids"

Biochemistry, 45 (2), pp. 481-497. (2006)

 
Abstract
Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce α-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces α-helical structures above the cmc. Both types of aggregates contain significant amounts of β-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce α-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form α-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles. © 2006 American Chemical Society.
 
 

Mygind, P.H., Fischer, R.L., Schnorr, K.M., Hansen, M.T., Sönksen, C.P., Ludvigsen, S., Raventós, D., Buskov, S., Christensen, B., De Maria, L., Taboureau, O., Yaver, D., Elvig-Jørgensen, S.G., Sørensen, M.V., Christensen, B.E., Kjærulff, S., Frimodt-Moller, N., Lehrer, R.I., Zasloff, M., Kristensen, H.-H.

"Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus"

Nature, 437 (7061), pp. 975-980. (2005)

 
Abstract
Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene. © 2005 Nature Publishing Group.
 
 

Song, Z., Wu, H., Mygind, P., Raventos, D., Sonksen, G., Kristensen, H.-H., Høiby, N.

"Effects of intratracheal administration of novispirin G10 on a rat model of mucoid Pseudomonas aeruginosa lung infection"

Antimicrobial Agents and Chemotherapy, 49 (9), pp. 3868-3874. (2005)

 
Abstract
Chronic Pseudomonas aeruginosa lung infection is a major problem for patients with cystic fibrosis (CF). The biofilm mode of growth of the pathogen makes it highly resistant to antibiotic treatment, and this is especially pronounced with mucoid strains. In this study, novispirin G10, a synthetic antimicrobial peptide patterned loosely on sheep myeloid antimicrobial peptide 29, was tested in a rat model of mucoid P. aeruginosa lung infection. P. aeruginosa NH57388A, a mucoid strain isolated from a CF patient, was mixed with the alginate produced by the bacterium itself and adjusted to a concentration of 1010 CFU/ml. Each rat received 109 CFU of bacteria intratracheally in the left lung to establish lung infection. At 0 and 3 h post P. aeruginosa infection, the treated group of rats received novispirin G10 (0.1 mg/ml, 0.1 ml/rat) intratracheally, whereas the control group received vehicle treatment only. The animals were sacrificed on days 3, 5, 7, and 10 after challenge for evaluation of various parameters. On day 5, 50% of the rats in the treated group had cleared the bacteria from the lungs, whereas in the control group, none of the rats cleared the pathogen (P < 0.03). The average bacterial loads remaining in the lungs of treated rats on days 3 and 5 were more than 170- and 330-fold lower than in the control groups (P < 0.0005 and P < 0.0003). In accordance, the macroscopic and microscopic lung pathology was also significantly milder in the treated group compared to the control group (P < 0.0002). Lung cytokine responses in the treated group were significantly lower than in the control group. The results suggest that novispirin G10 might be useful in treating antibiotic-resistant P. aeruginosa lung infections. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
 
 

Raventós, D., Taboureau, O., Mygind, P.H., Nielsen, J.D., Sonksen, C.P., Kristensen, H.-H.

"Improving on nature's defenses: Optimization & high throughput screening of antimicrobial peptides"

Combinatorial Chemistry and High Throughput Screening, 8 (3), pp. 219-233. (2005)

 
Abstract
Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The exacting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The trans-acting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems. © 2005 Bentham Science Publishers Ltd.
 
 

Ezra, D., Castillo, U.F., Strobel, G.A., Hess, W.M., Porter, H., Jensen, J.B., Condron, M.A.M., Teplow, D.B., Sears, J., Maranta, M., Hunter, M., Weber, B., Yaver, D.

"Coronamycins, peptide antibiotics produced by a verticillate Streptomyces sp. (MSU-2110) endophytic on Monstera sp"

Microbiology, 150 (4), pp. 785-793. (2004)

 
Abstract
Coronamycin is a complex of novel peptide antibiotics with activity against pythiaceous fungi and the human fungal pathogen Cryptococcus neoformans. It is also active against the malarial parasite, Plasmodium falciparum, with an IC50 of 9.0 ng ml-1. Coronamycin is produced by a verticillate Streptomyces sp. isolated as an endophyte from an epiphytic vine, Monstera sp., found in the Manu region of the upper Amazon of Peru. Bioassay-guided fractionation of the fermentation broths of this endophyte on silica gel and HPLC chromatography yielded two principal, inseparable, peptides with masses of 1217.9 and 1203.8 Da. Three other minor, but related components, are also present in the preparation. Amino acid analysis of coronamycin revealed residues of component 1, component 2, methionine, tyrosine and leucine at a ratio of 2: 2: 1: 1: 3. Other compounds with antifungal activities are also produced by this endophytic streptomycete. © 2004 SGM.
 
 

Per H. Mygind, Rikke L. Fischer, Kirk M. Schnorr, Mogens T. Hansen, Carsten P. S"nksen, Svend Ludvigsen, Dorotea Raventøs, Steen Buskov, Bjarke Christensen, Leonardo De Maria, Olivier Taboureau, Debbie Yaver, Signe G. Elvig-Jørgensen, Marianne V. Sørensen, Bjørn E. Christensen, Søren Kjærulff, Niels Frimodt-Moller, Robert I. Lehrer, Michael Zasloff and Hans-Henrik Kristensen.

"Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus."

Nature 437, 975-980 (13 October 2005)

 
Abstract
Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin - the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.
 
 

Song ZJ, Wu H, Mygind P, Raventos D, Sonksen C, Kristensen HH, Hoiby N.

"Effects of intratracheal administration of novispirin G10 on a rat model of mucoid Pseudomonas aeruginosa lung infection."

Antimicrobial Agents and Chemotherapy, Vol. 49 (9) pp. 3868-3874 (2005)

 
Abstract
Chronic Pseudomonas aeruginosa lung infection is a major problem for patients with cystic fibrosis (CF). The biofilm mode of growth of the pathogen makes it highly resistant to antibiotic treatment, and this is especially pronounced with mucoid strains. In this study, novispirin G10, a synthetic antimicrobial peptide patterned loosely on sheep myeloid antimicrobial peptide 29, was tested in a rat model of mucoid P. aeruginosa lung infection. P. aeruginosa NH57388A, a mucoid strain isolated from a CF patient, was mixed with the alginate produced by the bacterium itself and adjusted to a concentration of 10(10) CFU/ml. Each rat received 10(9) CFU of bacteria intratracheally in the left lung to establish lung infection. At 0 and 3 h post P. aeruginosa infection, the treated group of rats received novispirin G10 (0.1 mg/ml, 0.1 ml/rat) intratracheally, whereas the control group received vehicle treatment only. The animals were sacrificed on days 3, 5, 7, and 10 after challenge for evaluation of various parameters. On day 5, 50% of the rats in the treated group had cleared the bacteria from the lungs, whereas in the control group, none of the rats cleared the pathogen (P < 0.03). The average bacterial loads remaining in the lungs of treated rats on days 3 and 5 were more than 170- and 330-fold lower than in the control groups (P < 0.0005 and P < 0.0003) . In accordance, the macroscopic and microscopic lung pathology was also significantly milder in the treated group compared to the control group (P < 0.0002). Lung cytokine responses in the treated group were significantly lower than in the control group. The results suggest that novispirin G10 might be useful in treating antibiotic-resistant P. aeruginosa lung infections.
 
 

Raventos D, Taboureau O, Mygind PH, Nielsen JD, Sonksen CP, Kristensen HH.

"Improving on nature's defenses: Optimization & high throughput screening of antimicrobial peptides."

Combinatorial Chemistry and High Throughput Screening, Vol. 8 (3) pp. 219-233 (2005)

 
Abstract:
Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cis- acting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screen ing of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The transacting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.