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P Bulet

Publications and source records attributed to P Bulet.

At least 37 records · Page 2Linked to original sources
PubMed1999

Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects.

Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested.

Amino Acid Sequence↗
PubMed1999

Mosquito-Plasmodium interactions in response to immune activation of the vector.

During the development of Plasmodium sp. within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina. We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P. berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut. Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development. Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls. Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae. aegypti from 4 h to 10 days after ingesting P. gallinaceum, suggesting that parasite ingestion, passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin. However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae. aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal. Bacteria-challenged Ae. aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts. Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage.

Aedes↗
PubMed1999

Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti.

Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 microM at 24 h post inoculation. cDNAs encoding the preprodefensins of three previously described mature Ae. aegypti defensins were amplified by PCR, cloned and sequenced. Genomic clones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B. The defensin gene mapped to chromosome 3, between two known loci, blt and LF168.

Aedes↗
PubMed1999

The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare.

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C-peptide (50 amino acids). These results were confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex-determination. In invertebrates, there is no other example where sex-differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti-Müllerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects.

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within 1 h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Algorithms↗
PubMed1999

Androctonin, a novel antimicrobial peptide from scorpion Androctonus australis: solution structure and molecular dynamics simulations in the presence of a lipid monolayer.

Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.

Amino Acid Sequence↗
PubMed1998

Differential display of peptides induced during the immune response of Drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study.

We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5-11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.

Amino Acid Sequence↗
PubMed1998

Solution structure of thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data.

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221-1225], our structural study evidences the importance of the beta-sheet structure and also suggests that anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.

Amino Acid Sequence↗
PubMed1998

Cysteine-rich antimicrobial peptides in invertebrates.

Antimicrobial peptides are pivotal elements of the innate immune defense against bacterial and fungal infections. Within the impressive list of antimicrobial peptides available at present, more than half have been characterized in arthropods. Cysteine-rich antimicrobial peptides represent the most diverse and widely distributed family among arthropods and, to a larger extent, among invertebrates. Proeminent groups of cysteine-rich peptides are peptides with the CS alpha beta motif and peptides forming an hairpin-like beta-sheet structure. Although these substances exhibit a large structural diversity and a wide spectrum of activity, they have in common the ability to permeabilize microbial cytoplasmic membranes. Drosophila has proved a remarkable system for the analysis of the regulation of expression of gene encoding antimicrobial cysteine-rich peptides. These studies have unraveled the striking parallels that exist between insect immunity and innate immunity in mammals that point to a common ancestry of essential aspects of innate immunity.

Amino Acid Sequence↗
PubMed1998

Plasmodium gallinaceum: differential killing of some mosquito stages of the parasite by insect defensin.

We examined several insect antimicrobial peptides to study their effect on Plasmodium gallinaceum zygotes, ookinetes, oocysts, and sporozoites. Only two insect defensins-Aeschna cyanea (dragon fly) and Phormia terranovae (flesh fly)-had a profound toxic effect on the oocysts in Aedes aegypti and on isolated sporozoites. The defensins affected the oocysts in a time-dependent manner. Injecting the peptide into the hemolymph 1 or 2 days after an infectious blood meal had no significant effect on prevalence of infection or relative oocyst density per mosquito. When injected 3 days after parasite ingestion, the relative oocyst density was significantly reduced. Injection on day 4 or later damaged the developing oocysts, although the oocysts density per mosquito was not significantly different when examined on day 8. The oocysts were swollen or had extensive internal vacuolization. The peptides had no detectable effect on the early stages of the parasite: the zygotes and ookinetes tested in vitro. Both the defensins were highly toxic to isolated sporozoites in vitro as indicated by disruption of the membrane permeability barrier, a change in morphology, and loss of motility. In contrast to the toxicity of cecropin and magainin for mosquitoes, defensin, at concentrations that kill parasites, is not toxic to mosquitoes, suggesting that defensin should be studied further as a potential molecule to block sporogonic development of Plasmodium.

Aedes↗
PubMed1998

A novel insect defensin from the ant Formica rufa.

By combination of size exclusion and reversed-phase chromatography, we have isolated a novel member of insect defensin-type antimicrobial peptides from the entire bodies of bacteria-challenged Formica rufa (hymenoptera, formicidae). The molecular mass of the purified peptide was estimated to be 4120.42 by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Sequence analysis revealed that this peptide consisted of 40 amino acid residues with six cysteines engaged in the formation of three intramolecular disulfide bridges. This peptide is unique among the arthropod defensins in terms of the presence of asparatic acid and alanine at position 33 and as C-terminal residue, respectively. In addition, this novel defensin from Formica rufa has the particularity to have no C-terminal extension in contrast to those reported for other hymenoptera defensins.

Amino Acid Sequence↗
PubMed1997

Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda).

We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimps Penaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by a pyroglutamic acid at the first position. Comparison of the data obtained from the cDNA clones and mass spectrometry showed that two of these peptides are probably COOH-terminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

Amino Acid Sequence↗
PubMed1997

A matrix-assisted laser desorption ionization time-of-flight mass spectrometry approach to identify the origin of the glycan heterogeneity of diptericin, an O-glycosylated antibacterial peptide from insects.

In a previous study, electrospray ionization mass spectrometry was used to analyze the structure of the O-glycopeptide diptericin, an antibacterial peptide from the fleshfly Phormia terranovae. Several glycoforms of diptericin differing in the length of their oligosaccharide chains were present at the final stage of purification. In order to determine the origin of this glycan heterogeneity, we analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) the relative abundance of the different diptericin glycoforms in fractions obtained after each purification step, and directly in the hemolymph and in the fat body which produces diptericin. MALDI-MS clearly shows that the purification procedure had no effect on the O-linked oligosaccharide chains of diptericin, suggesting that diptericin is synthesized as a family of heterogeneous glycopeptides. In addition, in these experiments, differential mapping by MALDI-MS of the hemolymph and fat body tissue from bacteria-challenged and naive larvae allowed us to detect induced or repressed molecules which may be involved in the immune response of P. terranovae.

Amino Acid Sequence↗
PubMed1997

Novel antibacterial peptides isolated from a European bumblebee, Bombus pascuorum (Hymenoptera, Apoidea).

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.

Amino Acid Sequence↗
PubMed1996

Characterization of novel cysteine-rich antimicrobial peptides from scorpion blood.

We have isolated, from the hemolymph of unchallenged scorpions of the species Androctonus australis, three distinct antimicrobial peptides, which we have fully characterized by Edman degradation, electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. Two are novel molecules: (i) androctonin, a 25-residue peptide with two disulfide bridges, active against both bacteria (Gram-positive and Gram-negative) and fungi and showing marked sequence homology to tachyplesins and polyphemusins from horseshoe crabs; and (ii) buthinin, a 34-residue antibacterial (Gram-positive and Gram-negative) peptide with three disulfide bridges. The third peptide contains 37 residues and three disulfide bridges and clearly belongs to the family of anti-Gram-positive insect defensins. We have synthesized androctonin and explored its activity spectrum and mode of action.

Amino Acid Sequence↗