Fungal Defensin Research Articles

Eurocin, a New Fungal Defensin: STRUCTURE, LIPID BINDING, AND ITS MODE OF ACTION

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity.

BackgroundPlant defensins represent a major innate immune protein superfamily that displays strong inhibitory effects on filamentous fungi. The total number of plant defensins in a conifer species is unknown since there are no sequenced conifer genomes published, however the genomes of several angiosperm species provide an insight on the diversity of plant defensins. Here we report the identification of five new defensin-encoding genes from the Picea glauca genome and the characterization of two of their gene products, named PgD5 and endopiceasin.ResultsScreening of a P. glauca EST database with sequences of known plant defensins identified four genes with homology to the known P. glauca defensin PgD1, which were designated PgD2-5. Whereas in the mature PgD2-4 only 7–9 amino acids differed from PgD1, PgD5 had only 64% sequence identity. PgD5 was amplified from P. glauca genomic DNA by PCR. It codes for a precursor of 77-amino acid that is fully conserved within the Picea genus and has similarity to plant defensins. Recombinant PgD5, produced in Escherichia coli, had a molecular mass of 5.721 kDa, as determined by mass spectrometry. The PgD5 peptide exhibited strong antifungal activity against several phytopathogens without any effect on the morphology of the treated fungal hyphae, but strongly inhibited hyphal elongation. A SYTOX uptake assay suggested that the inhibitory activity of PgD5 could be associated with altering the permeability of the fungal membranes. Another completely unrelated defensin gene was identified in the EST library and named endopiceasin. Its gene codes for a 6-cysteine peptide that shares high similarity with the fungal defensin plectasin.ConclusionsScreening of a P. glauca EST database resulted in the identification of five new defensin-encoding genes. PgD5 codes for a plant defensin that displays non-morphogenic antifungal activity against the phytopathogens tested, probably by altering membrane permeability. PgD5 has potential for application in the plant biotechnology sector. Endopiceasin appears to derive from an endo- or epiphytic fungal strain rather than from the plant itself.

Characterization of recombinant plectasin: Solubility, antimicrobial activity and factors that affect its activity

Plectasin is the first known fungal defensin with potent activity against Gram-positive bacteria. To evaluate the potential therapeutic application of plectasin, we produced plectasin and investigated its solubility, activity and factors that affect its antimicrobial activity. Recombinant plectasin was produced from Escherichia coli in high yield by integration of fusion expression and on-column cleavage. Including 0.5M arginine significantly increased the solubility of plectasin in acetic acid buffer with 10% glycerol from 89μg/ml to 408μg/ml. Plectasin was soluble at 846μg/ml in Tris–glycerol–EDTA buffer. Plectasin was active against Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus with minimum inhibitory concentrations of 2 and 0.5μg/ml. Much lower or no activity was observed toward Gram-negative bacteria and fungi. Plectasin (128μg/ml) did not exhibit hemolytic activity toward rabbit erythrocytes. The activity of plectasin toward S. aureus was decreased by reduction with dithiothreitol, indicating that the disulfide-bond is essential for maximal activity. Plectasin was bactericidal under physiological concentrations of mono- and divalent cations. This activity was markedly attenuated by divalent cations in a concentration-dependent manner, however, with complete inhibition occurring at Ca2+ concentrations greater than 25mM. These results suggested that the presence of the disulfide-bond and the absence of divalent cations play key roles in the antimicrobial activity of plectasin.

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.

Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

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.

Defining Defensins' Mode of Action

Defensins are antimicrobial host defense peptides that play a role in innate immunity. Many such peptides act by disrupting the bacterial membrane; however, Schneider et al. (p. [1168][1]) now show that the fungal defensin, plecstasin, targets cell wall biosynthesis. Biochemical studies identified Lipid II as the cellular target of plecstasin and the residues involved in complex formation were identified using NMR spectroscopy and computational modeling. Initial studies identified two defensins from invertebrates that also target Lipid II. Plecstasin is active against some drug-resistant Gram-positive bacteria, and its action against a validated target makes it a promising lead for further drug development. [1]: /lookup/doi/10.1126/science.1185723

Biological Roles and Therapeutical Potential of Cationic Host Defence Peptides

Cationic antimicrobial peptides (AMPs; synonyms: host defence peptides, HDPs; defensins) are most frequently occurring antibiotics and are produced by virtually every life form as a first line of host defence. They do have both direct antibiotic activities which lead to rapid killing of microbes as well a immunomodulatory actvities which lead to upregulation of defence pathways and recruitment of immune cells (1). Both actvities are highly interesting for the development of innovative antiinfective strategies. However, in spite of considerable development activities, AMP-based antibiotics did not reach the market so far, and it may be necessary to better understand their molecular activities for AMP based rational drug design. Unlike conventional antibiotics which act via defined target molecules, antimicrobial host defence peptides are assumed to act unspecifically by permeabilising cell membranes. We used various naturally occurring HDPs and synthetic AMPs to study their antimicrobial activity in more detail. We found that the antibiotic activity of cationic amphiphilic peptides is based on multiple inhibitory activities and that the perturbation of the membrane lipid bilayer may be just one of the multiple activities. In general terms, AMPs appear to disturb the coordinated function of highly dynamic, membrane bound multi-enzyme machineries such as the cell wall biosynthesis machinery and respiratory chains. Defensins sensu strictu, as defined by the presence of a disulfide-bridged γ-core motif such as the human ß-defensins 2 and 3 (hBD2, hBD3) may, in addition, gain specificity for defined molecular targets. In particular, the fungal defensin plectasin, which binds with high affinity to the bacterial cell wall precursor Lipid II and thus blocks cell wall biosynthesis is taken forward through preclinical antibiotic development. 1) Hancock, R.E.W., Sahl H.G.: Antimicrobial and host defence peptides as novel anti-infective therapeutic strategies. Nature Biotechnology, 24, 1551-1557 (2006)