Biomedical subjects

Yannick Gueguen

Publications and source records attributed to Yannick Gueguen.

14 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↗

PubMed2004

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans.

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.

Adenosine Triphosphate↗

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

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

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.

Mass Spectrometry↗

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

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi.

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).

DNA Polymerase I↗

PubMed2002

Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

Adenosine Triphosphatases↗

PubMed2002

Comment on "The first description of an archaeal hemicellulase: the xylanase from Thermococcus zilligii strain AN1": evidence that the unique N-terminal sequence proposed comes from a maltodextrin phosphorylase.

Uhl and Daniel reported in this journal in 1999 (Extremophiles 3:263-267) the characterization of the first archaeal hemicellulase with a unique N-terminal sequence showing no homology with any xylanase or other protein from the databases. A genomic library of the chromosomal DNA of Thermococcus zilligii strain AN1 was screened by using a degenerate probe deduced from the N-terminal sequence. A positive clone was identified, and an amino acid sequence analysis revealed that the N-terminal sequence from this protein and the N-terminal sequence from the putative xylanase of T. zilligii were identical. However, the comparison of the amino acid sequence of the protein with sequences in the main protein databases revealed significant similarities with maltodextrin phosphorylases. In conclusion, it is likely that the N-terminal sequence proposed by Uhl and Daniel is not that of the T. zilligii xylanase, but corresponds to an archaeal T. zilligii maltodextrin phosphorylase.

Amino Acid Sequence↗