Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.

Cabanillasin, a new antifungal metabolite, produced by entomopathogenic Xenorhabdus cabanillasii JM26.

Since the early 1980s, fungi have emerged as a major cause of human disease. Fungal infections are associated with high levels of morbidity and mortality, and are now recognized as an important public health problem. Gram-negative bacterial strains of genus Xenorhabdus are known to form symbiotic associations with soil-dwelling nematodes of the Steinernematidae family. We describe here the discovery of a new antifungal metabolite, cabanillasin, produced by Xenorhabdus cabanillasii. We purified this molecule by cation-exchange chromatography and reverse-phase chromatography. We then determined the chemical structure of cabanillasin by homo- and heteronuclear NMR and MS-MS. Cabanillasin was found to be active against yeasts and filamentous fungi involved in opportunistic infections.

Convergent evolution-guided design of antimicrobial peptides derived from influenza A virus hemagglutinin.

Antimicrobial activity and solution structures of four 13-amino acid peptides derived from the fusion domain of viral hemagglutinin proteins are presented. The results show that carboxyl-terminal amidation is a key factor to switch a viral fusion domain-derived sequence into an antimicrobial peptide. Optimization of amphiphilic balance on the amidated analogue largely improves efficacy and enlarges antimicrobial spectra of these peptides. Our work indicates that viral fusion domains have potential to be engineered into potent antimicrobial peptides.

Insight into invertebrate defensin mechanism of action: oyster defensins inhibit peptidoglycan biosynthesis by binding to lipid II.

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.

MeuTXKbeta1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity.

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.

Identification of a new antimicrobial lysine-rich cyclolipopeptide family from Xenorhabdus nematophila.

Entomopathogenic bacteria of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family. These bacteria are transported by their nematode hosts into the hemocoel of the insect larvae, where they proliferate and produce insecticidal proteins, inhibitors of the insect immune system and antimicrobial molecules. In this study, we describe the discovery of a new family (PAX) of five antimicrobial compounds produced by fermentation of the Xenorhabdus nematophila F1 strain and purified by cation exchange chromatography and reversed phase chromatography. The chemical structure of PAX 3, a lysine-rich cyclolipopetide, was obtained from the analysis of homo and heteronuclear 2D NMR and confirmed by MS-MS experiments. The five members of the PAX family showed significant activity against plants and human fungal pathogens and moderate activity against few bacteria and yeast. No cytotoxicity was observed on CHO or insect cells.

Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa.

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP.

NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs.

Complete hydrolysis of myo-inositol hexakisphosphate by a novel phytase from Debaryomyces castellii CBS 2923.

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60 degrees C and pH 4.0. The K (m) value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P(6)) to inositol and inorganic phosphate. The sequence of Ins P(6) hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P(5), Ins (1,2,5,6) P(4), Ins (1,2,6) P(3), Ins (1,2) P(2), Ins (1 or 2) P(1), and inositol (notation 3/4/5/6/1 or 2).

NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides.

Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II. Hydrophilic and hydrophobic areas of mytilin account for 63% and 37%, respectively, a ratio very close to that of MGD-1 (64% and 36%). One linear and three cyclic fragments were designed from the interstrand loop sequence known to retain the biological activities in MGD-1. Only the fragment of 10 amino acids (C10C) constrained by two disulfide bonds in a stable beta-hairpin structure was able to inhibit the mortality of Palaemon serratus shrimp injected with white spot syndrome virus (WSSV). Fifty percent inhibition was obtained by in vitro pre-incubation of WSSV with 45 microM of C10C compared with 7 microM for mytilin. Interaction between the fragment and the virus occurred very rapidly as 40% survival was recorded after only 1 min of pre-incubation. In addition, C10C was capable of inhibiting in vitro growth of Vibrio splendidus LGP32 (MIC 125 microM), Vibrio anguillarum (MIC 2mM), Micrococcus lysodeikticus and Escherichia coli (MIC 1mM). Destroying the cysteine-stabilized alphabeta structure or shortening the C10C fragment to the C6C fragment with only one disulfide bond resulted in loss of both antiviral and antibacterial activities. Increasing the positive net charge did not enforce the antibacterial activity and completely suppressed the antiviral one. The C10C-designed peptide from mytilin appeared comparable in composition and structure with protegrin, tachyplesin and polyphemusin.

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin.

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.

Laspartomycin, an acidic lipopeptide antibiotic with a unique peptide core.

Laspartomycin was originally isolated and characterized in 1968 as a lipopeptide antibiotic related to amphomycin. The molecular weight and structure remained unknown until now. In the present study, laspartomycin was purified by a novel calcium chelate procedure, and the structure of the major component (1) was determined. The structure of laspartomycin C (1) differs from that of amphomycin and all related antibiotics as a result of its peptide region being acidic rather than amphoteric and the amino acid branching into the side chain being diaminopropionic rather than diaminobutyric. In addition, the fatty acid side chain is 2,3-unsaturated compared to 3,4-unsaturated for amphomycin and other related antibiotics. Calcium ion addition to stabilize a particular conformer was found to be important for an enzymatic deacylation of the antibiotic. A peptide resulting from the deacylation was critical for chemical structure determination by NMR studies, which also involved addition of calcium ions to stabilize a conformer.

Structure and mechanism of action of the antimicrobial peptide piscidin.

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.

PenBase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature.

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.

Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression.

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon.

Anti-lipopolysaccharide factors (ALFs), originally characterized from horseshoe crabs, have been recently identified from hemocytes of the black tiger shrimp, Penaeus monodon, by a genomic approach. In order to characterize the properties and biological activities of this immune effector in shrimp, ALFPm3, the most abundant isoform found in P. monodon, was expressed in the yeast Pichia pastoris. Large-scale production in fermentor provided 262 mg/l of recombinant ALFPm3 which was purified to homogeneity by single chromatography step on expanded-bed Streamline SP6XL. The rALFPm3 was further characterized in terms of N-terminal sequencing and mass spectrometry. Anti-microbial assays demonstrated that rALFPm3 has a broad spectrum of anti-fungal properties against filamentous fungi, and anti-bacterial activities against both Gram-positive and Gram-negative bacteria, associated with a bactericidal effect. Interestingly, rALFPm3 is highly efficient against various Vibrio species including strains pathogenic for shrimp. Finally, a synthetic peptide corresponding to a part of the putative LPS-binding site of ALFPm3 was shown to display activities mainly directed against Gram-positive bacteria indicating the involvement of the full molecule to the anti-microbial activity for Gram-negative bacteria.

Solution structure of synthetic penaeidin-4 with structural and functional comparisons with penaeidin-3.

Antimicrobial peptide structure has direct implications for the complexity of functions and mechanisms of action. The penaeidin antimicrobial peptide family from shrimp is divided into multiple class designations based on primary structure. The penaeidin classes are not only characterized by variability in primary sequence but also by variation in target specificity and effectiveness. Whereas class 4 exhibits low isoform diversity within species and is highly conserved between species, the primary sequence of penaeidin class 3 is less conserved between species and exhibits considerable isoform diversity within species. All penaeidins, regardless of class or species, are composed of two dramatically different domains: an unconstrained proline-rich domain and a disulfide bond-stabilized cysteine-rich domain. The proline-rich domain varies in length and is generally less conserved, whereas the spacing and specific residue content of the cysteine-rich domain is more conserved. The structure of the synthetic penaeidin class 4 (PEN4-1) from Litopenaeus setiferus was analyzed using several approaches, including chemical mapping of disulfide bonds, circular dichroism analysis of secondary structural characteristics, and complete characterization of the solution structure of the peptide by proton NMR. L. setiferus PEN4-1 was then compared with the previously characterized structure of penaeidin class 3 from Litopenaeus vannamei. Moreover, the specificity of these antimicrobial peptides was examined through direct comparison of activity against a panel of microbes. The penaeidin classes differ in microbial target specificity, which correlates to variability in specific domain sequence. However, the tertiary structure of the cysteine-rich domain and indeed the overall structure of penaeidins are conserved across classes.

High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide.

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.

Cytotoxic effects of mammea type coumarins from Calophyllum brasiliense.

Calophyllum brasiliense (Clusiaceae) is a big tree from the Tropical Rain Forests of the American continent. The organic extracts from the leaves yielded coumarins of the mammea type: mammea A/BA, A/BB, B/BA, B/BB, C/OA, C/OB, B/BA cyclo F, B/BB cyclo F, and isomammeigin. The triterpenoids friedelin and canophyllol, as well as the biflavonoid amentoflavone, protocatechuic and shikimic acids, were also obtained. Most of the isolated compounds were tested in vitro against K562, U251, and PC3 human tumor cell lines. The coumarins were cytotoxic against the three cell lines, the highest activity was shown by mammea A/BA (IC50 = 0.04 to 0.59 microM). The mixtures of mammea A/BA + A/BB, mammea B/BA + B/BB and mammea C/OA + C/OB were also highly active (IC50 < 4.05 microM). Friedelin was cytotoxic only against PC3, and U251 lines. Inhibition of HIV-1 reverse transcriptase was also assayed in vitro; however, none of the tested compounds (250 microM) prevented the activity of this enzyme. Most of the isolated compounds were also inactive against fourteen bacterial strains; however mammea A/BA + A/BB, and mammea C/OA + C/OB inhibited the growth of Staphylococcus aureus, S. epidermidis and Bacillus subtilis.