Biomedical subjects

Evelyne Bachère

Publications and source records attributed to Evelyne Bachère.

13 recordsLinked to original sources

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Evidence in oyster of a plasma extracellular superoxide dismutase which binds LPS.

We have characterized in the oyster Crassostrea gigas an extracellular superoxide dismutase (Cg-EcSOD) which appears to bind lipopolysaccharides (LPS). The protein has been purified from the oyster plasma and identified as a Cu/ZnSOD according to its N-terminal sequencing and biological activity. Cg-EcSOD expression and synthesis are restricted to hemocytes as revealed by in situ hybridization and immunocytochemistry. Cg-EcSOD-expressing hemocytes were seen in blood circulation, in connective tissues, and closely associated to endothelium blood vessels. Cg-EcSOD presents in its amino acid sequence a LPS-binding motif found in the endotoxin receptor CD14 and we show that the protein displays an affinity to Escherichia coli bacteria and with LPS and Lipid A. Additionally, an RGD motif known to be implicated in the association to membrane integrin receptor is present in the amino acid sequence. The purified Cg-EcSOD was shown to bind to oyster hemocytes and to be immunocolocalized with a beta-integrin-like receptor.

Amino Acid Sequence↗

PubMed2005

Molecular characterization of penaeidins from two Atlantic Brazilian shrimp species, Farfantepenaeus paulensis and Litopenaeus schmitti.

We report here the molecular cloning of new members of the penaeidin family from two Atlantic penaeids from Brazil, Litopenaeus schmitti and Farfantepenaeus paulensis. The presence of penaeidins in the granular hemocytes of both shrimps was first evidenced by immunofluorescence, using polyclonal antibodies raised against L. vannamei penaeidin Litvan PEN3-1. cDNAs from the hemocytes of both Brazilian species were obtained by reverse transcription and the sequences encoding penaeidins were amplified by PCR, using primers based on penaeidin consensus sequences. Five penaeidin clones were obtained. According to the international penaeidin classification (PenBase, http://www.penbase.immunaqua.com), the deduced amino acid sequences of two clones from L. schmitti and two from F. paulensis belong to the PEN2 subgroup and one clone from L. schmitti to the PEN4 subgroup of penaeidins. Surprisingly, no penaeidin from the PEN3 subgroup was obtained in both shrimp species, even though this subgroup appears to be the most commonly expressed in the hemocytes of penaeids.

Amino Acid Sequence↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Identification of genes that are differentially expressed in hemocytes of the Pacific blue shrimp (Litopenaeus stylirostris) surviving an infection with Vibrio penaeicida.

Considerable progress has been made in the field of invertebrate immunity through the characterization of genes involved in the response to infection and/or stress. However, the mechanisms by which commercially important marine invertebrates can successfully survive an infection remain largely unknown. For the first time in an invertebrate model, we have searched to discover genes involved in the survival capacity of shrimp using the highly pathogenic bacteria, Vibrio penaeicida. In the present study, we applied the technique of suppression subtractive hybridization (SSH) to hemocyte cDNAs from infected and uninfected shrimp, only using samples from individuals that had survived 96 h postinfection. The resulting library contains 260 expressed sequence tagged (EST) cDNA clones potentially representing highly expressed genes in surviving shrimp. Sequence similarity comparisons were made, and putative identities were assigned to clones that were at least 51% identical to known genes. This analysis showed two functional categories that were highly represented: those of genes involved in immune reactions (10.7% of the ESTs) and those involved in proliferation-hematopoiesis (10.3%). Expression pattern profile analyses of selected ESTs at different times postinfection confirmed the differential expression of the genes and efficiency of the SSH method. Differences in gene transcript abundance, for select ESTs encoding antimicrobial effectors, were evidenced by real-time PCR between shrimp that survived acute Vibrio infection and those individuals that did not survive acute Vibrio infection. These results suggest there are basic differences at the level of transcript abundance for genes directly involved in immune and hematopoietic processes from shrimp that survive and do not survive infection.

Animals↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2004

A new class (penaeidin class 4) of antimicrobial peptides from the Atlantic white shrimp (Litopenaeus setiferus) exhibits target specificity and an independent proline-rich-domain function.

A highly pure, chemically defined representative of a new class of antimicrobial peptide from the Atlantic white shrimp (Litopenaeus setiferus), penaeidin class 4 [Pen4-1 (penaeidin class 4 isoform 1)], was produced synthetically. Chemical synthesis was achieved by native ligation from two separate domains yielding a bioactive peptide that reflected the characteristics of native penaeidin. Synthetic Pen4-1 proved to be an effective antimicrobial peptide, particularly against the broad-spectrum pathogen Fusarium oxysporum, exhibiting a complex effect on reproductive growth at inhibitory concentrations resulting in the suppression of spore formation. Pen4-1 exhibits unique features [not previously observed for penaeidins from the Pacific white shrimp (L. vannamei)], including target-species specificity against Gram-positive bacteria, indicating a potential partitioning of antimicrobial function among this family of peptides. The proline-rich domain of penaeidin class 4 alone was an active antimicrobial peptide, having the same target range as the full-length Pen4-1. These findings indicate that the proline-rich domain of penaeidin is sufficient to confer target specificity and that divergence in this domain between classes can result in a gain in antimicrobial function as observed for the proline-rich domain of Pen4-1.

Amino Acid Sequence↗

PubMed2004

Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas.

Research on innate immunity of the penaeid shrimps and the oyster Crassostrea gigas is motivated greatly by economical necessities. Indeed, the aquaculture of these organisms is now limited by the development of infectious diseases. Studying anti-microbial peptides/proteins (AMPs), which are effector molecules of the host defense, is particularly attractive not only for progressing basic knowledge on immunity but also because they offer various possible applications for disease management in aquaculture. AMPs are explored with a global approach,considering their structure, properties, function, gene expression, and tissue distribution during the response to infections. In shrimp, investigations of the penaeidins, which are constitutively expressed peptides, have highlighted the importance of hemocytes and hematopoiesis as major elements of the immune response, providing both local and systemic reactions. The activation of hematopoiesis must be regarded as a regulatory way for the expression and distribution of constitutively expressed immune effectors. As complementary approaches, genomics and gene profiling are promising to deepen our understanding of the anti-microbial defense of the oyster and the shrimp. However, real progress will depend also on the characterization of hemocyte lineages and hematopoiesis of these marine invertebrates as well as on the ontogenesis of their immune systems.

Animals↗

PubMed2003

Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide.

Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.

Amino Acid Sequence↗

PubMed2003

Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas.

An expressed sequence tag program was undertaken to isolate genes involved in defense mechanisms of the Pacific oyster, Crassostrea gigas. Putative function could be assigned to 54% of the 1142 sequenced cDNAs. We built a public database where all EST information are accessible through numerous search profiles (http://www.ifremer.fr/GigasBase). Based on sequence similarities we identified 20 genes that may be implicated in immune function. We investigated the expression of four of these genes during bacterial challenge of oysters. Three of them were induced in response to challenge lending support to their involvement in oyster immunity. Moreover, four other genes were highly homologous to components of the NF-kappa B signaling pathway which is involved in innate immune response in Drosophila and mammals. Altogether, our results open a new way to investigate the immune response in mollusks.

Animals↗

PubMed2003

Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei.

Penaeidins are a family of antimicrobial peptides constitutively produced and stored in the hemocytes of penaeid shrimps. We have determined the expression and the localization of penaeidins in the first early larval stages (Nauplius V, Zoea I, II, III and Mysis II) and in post-larvae (1 and 8) of the shrimp Penaeus vannamei. Using in situ hybridization and immunohistochemical analyses, we localized penaeidin transcripts and peptides in a few hemocytes of larvae from Mysis II stage. However, RT-PCR analyses showed that penaeidin mRNAs are already present in the early stage of Nauplius V. In addition, penaeidin expression could not be detected in other cells than hemocytes. Our observations highlight the potential involvement of penaeidins during larval development and the ontogeny of immune system through hemocytes during this period of life, where shrimps are particularly susceptible to infectious diseases.

Animals↗

PubMed2002

Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp.

Penaeidins are a family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp. In response to microbial stimulation, they are released into the blood circulation and they further attach to shrimp cuticle surfaces through a chitin-binding property. In the present paper, we have analysed their expression, regulation and distribution in shrimp tissues in response to experimental microbial challenge. We have shown that penaeidin mRNA and protein are restricted to granular haemocytes and that their expression and distribution are regulated through dramatic changes in haemocyte populations, both circulating and infiltrating shrimp tissues. Two distinct phases in the immune reactions were evidenced: (a) a migration of haemocytes towards the infection site within the first 12 h following microbial injection, with a local and massive release of peptides; (b) the appearance into the blood circulation and tissues of a haemocyte population displaying increased penaeidin-transcriptional activity, which may correspond to a systemic reaction involving haemocyte proliferation process. Finally, in vitro confrontation of haemocytes and bacteria revealed that penaeidins are released from granular haemocytes by a novel phenomenon of intracellular degranulation, probably followed by the lysis of the cells. Furthermore, penaeidins were shown covering bacterial surfaces suggesting that the peptides could be involved in opsonic activity. Penaeidin-positive bacteria were observed to be phagocytosed mainly by hyaline cells, a population that does not express penaeidins.

Animals↗