Molecular scaffold of a new pokeweed antifungal peptide deduced by H nuclear magnetic resonance

The antifungal peptide from seeds of Phytolacca Americana (Pokeweed), designated PAFP-S hereinafter, is a recently found cationic peptide which consists of 38 amino acid residues and exihibites a broad spectrum of antifungal activity, including inhibition of certain saprophytic fungi and some plant pathogens. The secondary structure and three cysteine pairings have been investigated by ¹H NMR analysis. The results show that the molecular scaffold of PAFP-S features a triple-stranded antiparallel β-sheet knotted by a typical disulfide bridge motif, which characterizes the knottin fold. CD spectroscopy indicates a high stability of the molecule in solution. Therefore, PAFP-S should be a new member of the knottin structural family and the first antifungal peptide that adopts the knottin-like fold.     

The activity of echinocandins, amphotericin B and voriconazole against fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates

The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.     

Peptides of the constant region of antibodies display fungicidal activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.     

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.

Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.     

The structure of a novel insect peptide explains its Ca2+ channel blocking and antifungal activities

Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.     

The active site of drosomycin, a small insect antifungal protein, delineated by comparison with the modeled structure of Rs-AFP2, a plant antifungal protein.

Drosomycin is the first strictly antifungal protein isolated from an insect (Drosophila melanogaster). The solution structure of this 44-residue protein has been reported previously. It involves a three-stranded beta-sheet and an alpha-helix, the protein global fold being maintained by four disulfide bridges. Rs-AFP2 is a plant antifungal protein exhibiting 41% sequence similarity with drosomycin. Mutational analysis of Rs-AFP2 showed the importance of some residues in the antifungal activity of the protein against the fungus target. In order to determine the structural features responsible for antifungal activity in both drosomycin and Rs-AFP2, we modeled the three-dimensional structure of Rs-AFP2, and of other antifungal proteins, using the solution structure of drosomycin as a template. Structure analysis of drosomycin and Rs-AFP2, and comparisons with the other modeled antifungal structures, revealed that the two proteins shared a hydrophobic cluster located at the protein surface in which a lysine residue is embedded. Based on these close structural similarities and the experimental data available for Rs-AFP2 mutants, an antifungal active site of the insect protein is proposed.     

Lead optimization of antifungal peptides with 3D NMR structures analysis

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.     

PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H-2-D NMR study and molecular modeling

PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys57-Arg77). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.     

In vitro antifungal activity of voriconazole: New data after the first years of clinical experience

Voriconazole has been developed to meet the increasing need for new and useful antifungal agents for the treatment of invasive mycoses. This review describes the spectrum of voriconazole antifungal activity based on data from in vitro studies published during the last three years. This survey demonstrates that voriconazole has a broad antifungal spectrum against the most common fungal pathogens being its action fungistatic for Candida and fungicidal for Aspergillus and other filamentous fungi. Overall, more than 95% of all Candida isolates tested are susceptible to voriconazole and less than 3% are resistant. Similar or even better activity rates have been described for Aspergillus, Cryptococcus and most of yeasts and moulds of medical importance. We also discuss the limitations related to the azole cross-resistance observed in some Candida glabrata isolates, the poor activity of voriconazole against Scedosporium prolificans, its activity against fungal biofilms and the great potential usefulness of combination of voriconazole with other antifungal drugs.     

NMR solution structure of ATTp,an Arabidopsis thaliana trypsin inhibitor

The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (approximately 7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degrees C by multinuclear magnetic resonance spectroscopy. The protein contains one alpha-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands. The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini. The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 A) and the antifungal protein Rs-Afp1 [a knottin protein from radish (Raphanus sativus), rmsd of 2.7 A]. The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by (15)N heteronuclear NOE analysis. The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold.     

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.     

Solution structure of PAFP-S: a new knottin-type antifungal peptide from the seeds of Phytolacca americana

The three-dimensional solution structure of PAFP-S, an antifungal peptide extracted from the seeds of Phytolacca americana, was determined using 1H NMR spectroscopy. This cationic peptide contains 38 amino acid residues. Its structure was determined from 302 distance restraints and 36 dihedral restraints derived from NOEs and coupling constants. The peptide has six cysteines involved in three disulfide bonds. The previously unassigned parings have now been determined from NMR data. The solution structure of PAFP-S is presented as a set of 20 structures using ab initio dynamic simulated annealing, with an average RMS deviation of 1.68 A for the backbone heavy atoms and 2.19 A for all heavy atoms, respectively. For the well-defined triple-stranded beta-sheet involving residues 8-10, 23-27, and 32-36, the corresponding values were 0.39 and 1.25 A. The global fold involves a cystine-knotted three-stranded antiparallel beta-sheet (residues 8-10, 23-27, 32-36), a flexible loop (residues 14-19), and four beta-reverse turns (residues 4-8, 11-14, 19-22, 28-32). This structure features all the characteristics of the knottin fold. It is the first structural model of an antifungal peptide that adopts a knottin-type structure. PAFP-S has an extended hydrophobic surface comprised of residues Tyr23, Phe25, Ile27, Tyr32, and Val34. The side chains of these residues are well-defined in the NMR structure. Several hydrophilic and positively charged residues (Arg9, Arg38, and Lys36) surround the hydrophobic surface, giving PAFP-S an amphiphilic character which would be the main structural basis of its biological function.     

The Solution Structure of CCL28 Reveals Structural Lability that Does Not Constrain Antifungal Activity

The chemokine CCL28 is constitutively expressed in mucosal tissues and is abundant in low-salt mucosal secretions. Beyond its traditional role as a chemoattractant, CCL28 has been shown to act as a potent and broad-spectrum antimicrobial agent with particular efficacy against the commensal fungus and opportunistic pathogen Candida albicans. However, the structural features that allow CCL28 to perform its chemotactic and antimicrobial functions remain unknown. Here, we report the structure of CCL28, solved using nuclear magnetic resonance spectroscopy. CCL28 adopts the canonical chemokine tertiary fold, but also has a disordered C-terminal domain that is partially tethered to the core by a non-conserved disulfide bond. Structure–function analysis reveals that removal of the C-terminal tail reduces the antifungal activity of CCL28 without disrupting its structural integrity. Conversely, removal of the non-conserved disulfide bond destabilizes the tertiary fold of CCL28 without altering its antifungal effects. Moreover, we report that CCL28 unfolds in response to low pH but is stabilized by the presence of salt. To explore the physiologic relevance of the observed structural lability of CCL28, we investigated the effects of pH and salt on the antifungal activity of CCL28 in vitro. We found that low pH enhances the antifungal potency of CCL28, but also that this pH effect is independent of CCL28's tertiary fold. Given its dual role as a chemoattractant and antimicrobial agent, our results suggest that changes in the salt concentration or pH at mucosal sites may fine-tune CCL28's functional repertoire by adjusting the thermostability of its structure.     

Oxime derivatives of sordaricin as potent antifungal agents

Oxime derivatives of the sordarin aglycone have been identified as potent antifungal agents. The in vitro spectrum of activity includes coverage against Candida albicans and Candida glabrata with MICs as low as 0.06 microg/mL. The antifungal activity was established to be exquisitely sensitive to the spatial orientation of the lipophilic side chains.     

Evaluation of the antimicrobial activity of the acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid, norstictic acid, and protocetraric acid constituents

The acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid constituent showed antimicrobial activity against Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, and Candida glabrata. Norstictic acid was active against Aeromonas hydrophila as well as the above microorganisms except Yersinia enterocolitica. Protocetraric acid showed activity only against the tested yeasts Candida albicans and Candida glabrata. The MIC values of the extract as well as of the three substances were determined. No antifungal activity of the acetone extract has been observed against ten filamentous fungi.     

Synthesis and evaluation of novel macrocyclic antifungal peptides

Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the β-(1,3)-D-glucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl groups present in these complex macrocyclic lipopeptides, most structure-activity relationship studies have relied upon semisynthetic derivatives. In order to probe the influence of the cyclic peptide backbone on the antifungal activity we developed a successful strategy for the synthesis of novel echinocandins analogues by on-resin ring closing metathesis or disulfide formation. The specific minimum inhibitory activity of each mimic was determined against Candida albicans. Our results indicate that ring size is an important factor for antifungal activity.     

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution

Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C¹X₃C²X_nC³X₃C⁴), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C¹–C⁴ and C²–C³. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.     

Growth inhibitory properties of chalcones to Candida

The growth inhibitory properties of derivatives of chalcone (1,3-diphenyl-2-propen-1-one) were studied against oral Candida species, including C. albicans. C. tropicalis and C. glabrata. The antifungal activities of 2-hydroxychalcone and 2,5-dihydroxychalcone were so high that they inhibited the growth of most strins at the concentration of 200 μg ml^-1, whereas chalcone was less active (to a limited number of strains) or inactive. 2-Hydroxychalcone was active only against C. glabrata. The other chalcones without a 2-hydroxyl group showed no significant antifungal activity. The minimum inhibitory concentration of 2-hydroxychalcone was 75 μ ml-1 to all strains of three Candida species sensitive to it. The antifungal property was based on a fungicidal action. The structure-activity relationship indicates that the presence of a hydroxyl group at the 2-position potentially improves the antifungal property.