André Aumelas

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 α-β motif (CS-αβ) 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.

CHANGE IN MEMBRANE PERMEABILITY INDUCED BY PROTEGRIN 1 : IMPLICATION OF DISULPHIDE BRIDGES FOR PORE FORMATION

Protegrin 1 (PG‐1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG‐1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.

Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1).

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.

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.

Oligomerization of protegrin-1 in the presence of DPC micelles. A proton high-resolution NMR study

Protegrins are members of a family of five Cys‐rich naturally occurring cationic antimicrobial peptides. The NMR solution structure of protegrin‐1 (PG‐1) has been previously determined as a monomeric β‐hairpin both in water and in dimethylsulfoxide solution. Protegrins are bactericidal peptides but their mechanism of action is still unknown. In order to investigate the structural basis of their cytotoxicity, we studied the effect of lipid micelles on the structure of PG‐1. The NMR study reported in the present work indicates that PG‐1 adopts a dimeric structure when it binds to dodecylphosphocholine micelles. Moreover, the amide proton exchange study suggests the possibility of an association between several dimers.

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 15N uniformly labeled rALF‐Pm3 protein. Its 3D structure was established by NMR and consists of three α‐helices packed against a four‐stranded β‐sheet. The C34C55 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 β‐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 β‐hairpin synthetic peptides corresponding to ALF‐L β‐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.

Structure of the cathelicidin motif of protegrin-3 precursor: structural insights into the activation mechanism of an antimicrobial protein.

Cathelicidins are a family of antimicrobial proteins isolated from leucocytes and epithelia cells that contribute to the innate host defense mechanisms in mammalians. Located in the C-terminal part of the holoprotein, the cathelicidin-derived antimicrobial peptide is liberated by a specific protease cleavage. Here, we report the X-ray structure of the cathelicidin motif of protegrin-3 solved by MAD phasing using the selenocysteine-labeled protein. Its overall structure represents a fold homologous to the cystatin family and adopts two native states, a monomer, and a domain-swapped dimer. This crystal structure is the first example of a structural characterization of the highly conserved cathelicidin motif and thus provides insights into the possible mechanism of activation of the antimicrobial protegrin peptide.

MeuTXKβ1, 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.

Nouveaux cyclotétrapeptides isolés de l'ascidie cystodytes delle chiajei.

Resume Three cyclotetrapeptides cyclo (L- Pro - L - Leu) 2 , cyclo (L - Pro - L - Val) 2 and cyclo (L - Pro - L - Phe) 2 were isolated from the marine ascidian Cystodytes delle chiajei (Didemnidae). Their structures were determined by spectroscopic means and confirmed by synthesis.

ISOLATION AND STRUCTURE ELUCIDATION OF A HIGHLY HAEMOLYTIC SAPONIN FROM THE MERCK SAPONIN EXTRACT USING HIGH-FIELD GRADIENT-ENHANCED NMR TECHNIQUES

Saponins SAPO50 and SAPO30, of which SAPO50 is highly haemolytic, have been isolated from the commercial Merck Saponin. Their structures have been determined exclusively by high-field gradient-enhanced NMR methods. The 1H and 13C NMR spectra of these saponins in pyridine-deuterium oxide have been assigned by homonuclear and heteronuclear correlation experiments. Anomeric configurations were obtained by combined use of 1JCH, 3JH-1.H-2, and 1D-NOESY data. Sugar residues were identified by use of 3JHH values obtained from their subspectra recorded using an optimized 1D-zeta-TOCSY sequence. Linkage assignments were made using the ge-HMBC and 1D-NOESY spectra. This study shows that SAPO50 represents a hitherto undescribed saponin with the following structure: 3-O-beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl- (1-->2)]-beta-D-glucuronopyranosyl gypsogenin 28-O-(6-deoxy-beta-D-glucopyranosyl)-(1-->4)-[beta-D-xylopyranosyl-(1--> 3)- beta-D-xylopyranosyl-(1-->4)]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D- fucopyranoside. SAPO30, however, corresponds to a saponin previously described [D. Frechet, B. Christ, B. Monegier du Sorbier, H. Fischer, and M. Vuilhorgne, Phytochemistry, 30 (1991) 927-931].