Publications by authors named "Philippe Roch"

25 Publications

  • Page 1 of 1

Toll signal transduction pathway in bivalves: complete cds of intermediate elements and related gene transcription levels in hemocytes of immune stimulated Mytilus galloprovincialis.

Dev Comp Immunol 2014 Aug 4;45(2):300-12. Epub 2014 Apr 4.

Ecologie des Systèmes Marins Côtiers (EcoSym), CNRS-Université de Montpellier 2-IRD, cc 093, place E. Bataillon, 34095 Montpellier, France. Electronic address:

Based on protein domain structure and organization deduced from mRNA contigs, 15 transcripts of the Toll signaling pathway have been identified in the bivalve, Mytilus galloprovincialis. Identical searches performed on publicly available Mytilus edulis ESTs revealed 11 transcripts, whereas searches performed in genomic and new transcriptome sequences of the Pacific oyster, Crassostrea gigas, identified 21 Toll-related transcripts. The remarkable molecular diversity of TRAF and IKK coding sequences of C. gigas, suggests that the sequence data inferred from Mytilus cDNAs may not be exhaustive. Most of the Toll pathway genes were constitutively and ubiquitously expressed in M. galloprovincialis, although at different levels, and clearly induced after in vivo injection with bacteria. Such over-transcription was more rapid and intense with Gram-negative than with Gram-positive bacteria. Injection of a fungus modulated the transcription of few Toll pathway genes, with the induction levels of TLR/MyD88 complex being always less intense. Purified LPS and β-glucans had marginal effect whereas peptidoglycans were ineffective. At the moment, we found no evidence of an IMD transcript in bivalves. In conclusion, mussels possess a complete Toll pathway which can be triggered either by Gram-positive or Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.dci.2014.03.021DOI Listing
August 2014

Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels.

Dev Comp Immunol 2013 Jun 26;40(2):158-66. Epub 2013 Feb 26.

Ecologie des Systèmes Marins et Côtiers EcoSym, Université Montpellier 2-CNRS, cc 093, place E. Bataillon, F-34095 Montpellier cedex 05, France.

TLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signaling pathway in Lophotrochozoa.
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http://dx.doi.org/10.1016/j.dci.2013.02.006DOI Listing
June 2013

Individual variability of mytimycin gene expression in mussel.

Fish Shellfish Immunol 2012 Sep 29;33(3):641-4. Epub 2012 Jun 29.

Ecologie des Systèmes Marins et Côtiers-EcoSym UMR5119, Université Montpellier 2-CNRS, F-34095 Montpellier Cedex 05, France.

The antifungal peptide mytimycin (MytM) is synthesized by hemocytes of the Mediterranean mussel, Mytilus galloprovincialis. In addition to sequence and gene structure diversities previously reported from pooled hemocytes, the present report focused on the expression of mytm gene in individual M. galloprovincialis, before and after challenge. Within untreated mussel, MytM mRNA was observed by ISH in about 42% of circulating hemocytes, characterized by large, diffuse nucleus. Injection with Fusarium oxysporum increased such percentage, but in only some of the mussels. Similarly, MytM gene expression increased after injection in only some of the mussels, as measured by qPCR. Responders and not responders are common evidence in any given population of organisms. Nevertheless, even if the use of proper pool size selection has been practised to find out and evaluate the most common response trends, individual analyses must be regarded as optimal.
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http://dx.doi.org/10.1016/j.fsi.2012.06.020DOI Listing
September 2012

Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

PLoS One 2011 7;6(11):e26680. Epub 2011 Nov 7.

Department of Biology, University of Padua, Padova, Italy.

Background: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP) precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions.

Methodology/principal Findings: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France), Vigo (Spain) and Venice (Italy). To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore) and wild (lagoon) Venice mussels, both injected with 10(7) Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC) minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation.

Conclusions/significance: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related to geographical origin or Vibrio injection emerged. Whether or not the contact with potential pathogens can increase the amount of AMP transcript variants in mussels requires additional study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026680PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210125PMC
May 2012

MIF from mussel: coding sequence, phylogeny, polymorphism, 3D model and regulation of expression.

Dev Comp Immunol 2012 Apr 4;36(4):688-96. Epub 2011 Nov 4.

Marine Immunobiology Laboratory, University of Palermo, Via Archirafi 18, 90123 Palermo, Italy.

Three macrophage migration inhibitory factor (MIF)-related sequences were identified from a Mytilus galloprovincialis EST library. The consensus sequence included a 5'-UTR of 32 nucleotides, the complete ORF of 345 nucleotides, and a 3'-UTR of 349 nucleotides. As for other MIFs, M. galloprovincialis ORF does not include any signal or C-terminus extensions. The translated sequence of 115 amino acids possesses a molecular mass of 12,681.4, a pI of 6.27 and a stability index of 21.48. Its 3D structure resembles human MIF except for one shorter α-helix. Although evolutionary separated from ticks and vertebrates, Mg-MIF appeared to be closely related to Pinctada fucata and Haliotis, but not to Chlamys farreri and Biomphalaria glabrata. Numerous mutation points were observed within the Mg-MIF ORF, defining 11 amino acid variants within the mussels from Palavas-France and 14 amino acid variants within the mussels from Palermo-Italy. The 2 major variants from Palavas were identical to 2 of the 4 major variants from Palermo. In all the 18 Mg-MIF variants, residues involved in tautomerase and in oxidoreductase activities were conserved. Generally, one mussel expressed 2 Mg-MIF amino acid sequences but with different frequencies of occurrence. Mg-MIF is constitutively expressed principally in hemocytes and in the mantle. In contrast to other animal models, Mg-MIF expression was always down regulated following challenge by bacteria and fungi, confirming previous data obtained with microarray. Down regulation started as soon as 1 h and Mg-MIF expression returned to background 9-48 h after the challenge. Exception was regarding the yeast, Candidaalbicans, down-regulation between 9 and 72 h, suggesting yeast and bacteria-filamentous fungi trigger different mechanisms of elimination.
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http://dx.doi.org/10.1016/j.dci.2011.10.014DOI Listing
April 2012

Gene expression specificity of the mussel antifungal mytimycin (MytM).

Fish Shellfish Immunol 2012 Jan 24;32(1):45-50. Epub 2011 Oct 24.

Ecologie des Systèmes Marins et Côtiers, Université Montpellier 2-CNRS, cc 093, place E. Bataillon, F-34095 Montpellier cedex 05, France.

We previously reported the nucleotide sequences and diversity of mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis. Using real-time PCR (q-PCR), we observed that the MytM gene was mainly expressed in circulating hemocytes and to a less extent in the mantle. In vivo challenge with bacteria or with the yeast, Candida albicans, did not increase the expression as measured by q-PCR in hemocytes. By contrast, injection of the filamentous fungus, Fusarium oxysporum, induced a sudden and strong increase of expression at 9h p.i. (stimulation index of 25.7 ± 2.1). Optimum stimulating dose was 10(4) spores of F. oxysporum per mussel. In the same samples, AMP mytilin and myticin showed no stimulation. Consequently, we hypothesized the existence of 2 different signal transduction pathways, one activated by bacteria and yeast, the other triggered by filamentous fungi. A second challenge performed with F. oxysporum 24 h after the first challenge induced an increase of MytM gene expression (stimulation index of 3.5 ± 1.7). However, this second increase was significantly lower than the first, suggesting less efficient response rather than significant protection.
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http://dx.doi.org/10.1016/j.fsi.2011.10.017DOI Listing
January 2012

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis.

BMC Genomics 2011 Jan 26;12:69. Epub 2011 Jan 26.

Department of Biology, University of Padova, Via U, Bassi, 58/B, 35121 Padova, Italy.

Background: Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.

Results: We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.

Conclusions: The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.
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http://dx.doi.org/10.1186/1471-2164-12-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039611PMC
January 2011

Diversity of coding sequences and gene structures of the antifungal peptide mytimycin (MytM) from the Mediterranean mussel, Mytilus galloprovincialis.

Mar Biotechnol (NY) 2011 Oct 19;13(5):857-67. Epub 2011 Jan 19.

Ecosystèmes Lagunaires, CNRS-Université Montpellier 2, cc093, place E. Bataillon, 34095, Montpellier, Cedex 05, France.

Knowledge on antifungal biomolecules is limited compared to antibacterial peptides. A strictly antifungal peptide from the blue mussel, Mytilus edulis named mytimycin (MytM) was reported in 1996 as partial NH(2) 33 amino acid sequence. Using back-translations of the previous sequence, MytM-related nucleotide sequences were identified from a normalized Mytilus galloprovincialis expressed sequence tag library. Primers designed from a consensus sequence have been used to obtain a fragment of 560 nucleotides, including the complete coding sequence of 456 nucleotides. Precursor is constituted by a signal peptide of 23 amino acids, followed by MytM of 54 amino acids (6.2-6.3 kDa, 12 cysteines) and C-terminal extension of 75 amino acids. Only two major amino acid precursor sequences emerged, one shared by M. galloprovincialis from Venice and Vigo, the other belonging to M. galloprovincialis from Palavas, with nine amino acid differences between the two MytM. Predicted disulfide bonds suggested the presence of two constrained domains joined by amino acidic NIFG track. Intriguing was the presence of conserved canonical EF hand-motif located in the C-terminus extension of the precursor. The MytM gene was found interrupted by two introns. Intron 2 existed in two forms, a long (1,112 nucleotides) and a short (716 nucleotides) one resulting from the removal of the central part of the long one. Both the short (GenBank FJ804479) and the long (GenBank FJ804478) genes are simultaneously present in the mussel genome.
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http://dx.doi.org/10.1007/s10126-010-9345-4DOI Listing
October 2011

Effects of vibrio challenge on digestive gland biomarkers and antioxidant gene expression in Mytilus galloprovincialis.

Comp Biochem Physiol C Toxicol Pharmacol 2010 Sep 28;152(3):399-406. Epub 2010 Jun 28.

Dipartimento di Biologia, Università di Genova, Italy.

In bivalve molluscs, responses to bacterial infection have been largely characterized in terms of both functional responses and gene expression in the immune cells, the hemocytes. The effects of bacterial challenge at the tissue level, where bacterial infection may cause stressful conditions, have not been so far specifically investigated. Biomarkers are widely utilised to evaluate the health status of bivalves, from the molecular to the organism level, in response to both natural and anthropogenic stressors. In this work, the effects of in vivo challenge with heat-killed vibrio species, Vibrio splendidus LGP32 and Vibrio anguillarum (ATCC19264), on different biomarkers in the digestive gland of the marine bivalve Mytilus galloprovincialis were investigated. Mussels were injected with either vibrio and tissues sampled at 3, 6 and 24 h post injection (p.i.). Lysosomal biomarkers, such as lysosomal membrane stability (LMS) and lipofuscin accumulation, as well as specific activities of antioxidant enzymes (catalase and glutathione transferase-GST) were evaluated. Moreover, the expression of antioxidant molecules (catalase, GST-pi and metallothioneins MT10 and MT20) was determined by quantitative RT-PCR. Both V. splendidus and V. anguillarum significantly affected all parameters measured, to a different extent and at different times p.i. Interestingly, whereas both vibrios induced lysosomal membrane destabilisation and increases in the activities of antioxidant enzymes, distinct responses were observed in terms of lysosomal lipofuscin accumulation and expression of antioxidant molecules. In particular, V. splendidus induced a general increase in the transcription of antioxidant genes, indicating that Mytilus digestive gland can mount an efficient antioxidant response towards this vibrio species. On the other hand, a general down-regulation or no effect was observed with V. anguillarum. The lack of this response was reflected in stronger oxidative stress conditions in the digestive gland of mussels challenged with V. anguillarum, as indicated by higher levels of lysosomal lipofuscin observed at longer times p.i. Overall, these data indicate that lysosomal and oxidative stress biomarkers could be usefully applied in order to monitor early changes in the health status of bivalves induced by bacteria. Moreover, the results support the hypothesis that host responses to bacteria may be taken into account when interpreting biomarker data in ecotoxicological studies.
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http://dx.doi.org/10.1016/j.cbpc.2010.06.008DOI Listing
September 2010

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis.

J Invertebr Pathol 2010 Sep 20;105(1):49-55. Epub 2010 May 20.

DISUAN, Dipartimento di Scienze dell'Uomo, dell'Ambiente e della Natura, Italy.

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components.
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http://dx.doi.org/10.1016/j.jip.2010.05.010DOI Listing
September 2010

Expression of Mytilus immune genes in response to experimental challenges varied according to the site of collection.

Fish Shellfish Immunol 2010 Apr 1;28(4):640-8. Epub 2010 Jan 1.

Ecosystèmes Lagunaires, UMR 5119, Université de Montpellier 2-CNRS, cc093, place E. Bataillon, F-34095 Montpellier cedex 05, France.

Mussels live in diverse coastal environments experience various physical, chemical and biological conditions, which they counteract with functional adjustments and heritable adaptive changes. In order to investigate possible differences in immune system capabilities, we analyzed by qPCR the expression levels of 4 immune genes (defensin, mytilin B, myticin B, lysozyme) and HSP70 in the Mediterranean mussel, Mytilus galloprovincialis collected in 3 European farming areas {Atlantic Ocean-Ría de Vigo-Spain (RV), French Mediterranean Gulf of Lion-Palavas-Prévost lagoon (PP) and Northern Adriatic Sea-Venice-Italy (VI)} in response to one injection of one of the 3 bacterial species (Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus), and to heat shock or cold stress. We confirmed that the 5 genes are constitutively expressed in hemocytes, defensin being the less expressed, myticin B the highest. As suspected, the same gene resulted differently expressed according to mussel group, with the biggest difference being for HSP70 and lysozyme and lowest expression of all the 5 genes in mussels from RV. In addition, gene expression levels varied according to the challenge. Most frequent effect of bacterial injections was down-regulation, especially for mytilin B and myticin B. Heat shock enhanced transcript levels, particularly in mussels from RV, whereas cold stress had no effect. In situ hybridization of labelled probes on mussel hemocytes indicated that bacterial injections did not change the mRNA patterns of defensin and myticin B whereas mytilin B mRNA almost disappeared. In conclusion, these results demonstrated that constitutive level, nature and intensity of immune gene expression regulations strongly depended from mussel group, and support the concept of gene-environment interactions.
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http://dx.doi.org/10.1016/j.fsi.2009.12.022DOI Listing
April 2010

Influence of temperature, salinity and E. coli tissue content on immune gene expression in mussel: results from a 2005-2008 survey.

Dev Comp Immunol 2009 Sep 13;33(9):974-9. Epub 2009 May 13.

Ecosystèmes Lagunaires, JRU CNRS-IFREMER-Université Montpellier 2, Montpellier, France.

Several bivalves, including mussels, suffered from mortalities particularly in summer. To look for the possible effect of environmental parameters on immune capacities, Mytilus galloprovincialis were collected monthly from August 2005 to July 2008 from the Palavas Laguna, French Mediterranean coast. Q-PCR was used to quantify the expression of three antimicrobial peptide genes (defensin, mytilin B and myticin B), in addition to lysozyme and HSP70. House keeping gene was 28S rRNA. Defensin, myticin B and lysozyme appeared more expressed in spring-summer than in winter. In contrast, HSP70 expression was higher in winter. Statistical studies using principal component analysis (PCA) and multiple regression models revealed positive influence of temperature on 28S rRNA, defensin, myticin B and lysozyme expressions, but not on mytilin B and HSP70. The positive influence was significant for defensin and lysozyme expression, but relationships cannot be quantified. Similarly, salinity appeared to influence defensin expression, but this relationship cannot be quantified neither. E. coli tissue content appeared without influence. Consequently, there was no clear relationship between environmental parameters and immune-related gene expressions, demonstrating anti-infectious capabilities cannot be evaluated using only the expression of such genes as markers.
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http://dx.doi.org/10.1016/j.dci.2009.04.002DOI Listing
September 2009

Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge.

Comp Biochem Physiol B Biochem Mol Biol 2009 Aug 22;153(4):365-71. Epub 2009 Apr 22.

DISUAN, Dipartimento di Scienze dell'Uomo, dell'Ambiente e della Natura, Università Carlo Bo di Urbino, Italy.

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(-) Vibrio anguillarum and V. splendidus, Gram+ Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus >V. anguillarum >M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus >V. anguillarum >M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.
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http://dx.doi.org/10.1016/j.cbpb.2009.04.007DOI Listing
August 2009

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences.

BMC Genomics 2009 Feb 9;10:72. Epub 2009 Feb 9.

Department of Biology, University of Padova, Via U Bassi 58/B, Padova, Italy.

Background: Although bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.

Results: We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database http://mussel.cribi.unipd.it.

Conclusion: We defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.
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http://dx.doi.org/10.1186/1471-2164-10-72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657158PMC
February 2009

Polymorphism of mytilin B mRNA is not translated into mature peptide.

Mol Immunol 2009 Jan 2;46(3):384-92. Epub 2008 Dec 2.

Ecosystèmes Lagunaires UMR 5119, Université Montpellier 2, Montpellier, France.

Diversity of mRNAs from mytilin B, one of the five mytilins identified in the Mediterranean mussel, Mytilus galloprovincialis, has been investigated from circulating hemocytes. One mussel expressed simultaneously two to ten different mytilin B mRNAs as observed in denaturing gradient gel electrophoresis (DGGE), defining 10 individual DGGE patterns (named A to J) within the mussels from Messina, Sicily (Italy). Three patterns accounted for 79% of the individuals whereas other patterns were found in only 2-7% of the 57 analyzed mussels. Base mutations were observed at specific locations, mainly within COOH-terminus and 3'UTR, leading to 36 nucleotide sequence variants and 21 different coding sequences (cds) segregating in two different clusters. Most of the base mutations were silent, and the number of pro-peptide variants was restricted to four. Finally, as the two amino acid replacements occurred within COOH-terminus, mature peptide from mytilin B appeared unique. Multiple sequencing of partial mytilin B gene from one mussel revealed that one to four randomly distributed mutation points occurred within intron-3. Only one sequence out of the 91 analyzed contained 16 mutation points. In addition, this sequence was the only one containing four out of the six mutation points occurring within exon-4, that code for most of the COOH-terminus domain, including the unique amino acid replacement. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure for COOH-terminus domain.
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http://dx.doi.org/10.1016/j.molimm.2008.10.009DOI Listing
January 2009

Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria.

Fish Shellfish Immunol 2008 Dec 23;25(6):834-40. Epub 2008 Sep 23.

Ecosystèmes Lagunaires UMR5119, Université Montpellier 2, cc093, place E. Bataillon, F-34095 Montpellier cedex 05, France.

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.
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http://dx.doi.org/10.1016/j.fsi.2008.09.005DOI Listing
December 2008

Lysozyme gene expression and hemocyte behaviour in the Mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses.

Fish Shellfish Immunol 2008 Jul 13;25(1-2):143-52. Epub 2008 Apr 13.

Ecosystèmes Lagunaires UMR5119, Université Montpellier 2, CNRS, IFREMER, cc093, Place E. Bataillon, F-34095 Montpellier Cedex 05, France.

The aim of the present study was to evaluate the expression of the Mytilus galloprovincialis lysozyme gene in different in vivo stress situations, including injection of bacteria Vibrio splendidus LGP32, Vibrio anguillarum or Micrococcus lysodeikticus, as well as heat shock at 30 degrees C and cold stress at 5 degrees C. Injection of V. splendidus LGP32 resulted in: (i) a general down-regulation of lysozyme gene expression, as quantified by Q-PCR; (ii) reduction in the number of circulating hemocytes; (iii) decrease in the percentage of circulating hemocytes expressing lysozyme mRNA which was now restricted to only small cells, as observed by ISH; and (iv) accumulation of hemocytes expressing lysozyme in the muscle sinus where injection took place. Injection of V. anguillarum or M. lysodeikticus induced significant up-regulation of lysozyme gene expression, but only 2-3days post-injection, with no change in the total hemocyte counts but an increased percentage of hemocytes expressing lysozyme mRNA. Neither the control injection of PBS-NaCl nor temperature stress modified the lysozyme expression pattern. Consequently, the hemocyte population appears to be capable of discriminating between stress factors, and even between 2 Vibrio species.
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http://dx.doi.org/10.1016/j.fsi.2008.04.001DOI Listing
July 2008

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

Dev Comp Immunol 2008 26;32(3):227-38. Epub 2007 Jun 26.

CNRS UMR5119-IFREMER-Université Montpellier 2, Ecosystèmes Lagunaires, cc093, place E. Bataillon, F-34095 Montpellier, France.

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.
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http://dx.doi.org/10.1016/j.dci.2007.05.006DOI Listing
April 2008

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax.

Comp Biochem Physiol B Biochem Mol Biol 2007 Apr 28;146(4):521-9. Epub 2006 Dec 28.

Marine Immunobiology Laboratory, University of Palermo, Via Archirafi 18, 90123 Palermo, Italy.

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68-71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.
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http://dx.doi.org/10.1016/j.cbpb.2006.12.007DOI Listing
April 2007

Deep sea immunity: unveiling immune constituents from the hydrothermal vent mussel Bathymodiolus azoricus.

Mar Environ Res 2007 Aug 5;64(2):108-27. Epub 2007 Jan 5.

IMAR/Department of Oceanography and Fisheries, Genetics and Molecular Laboratory, University of the Azores, Rua Comendador Fernando da Costa, 9901-862 Horta, Portugal.

Marine molluscs are subjected to constant microbial threats in their natural habitats. As a result, they represent suitable models for the study of the molecular mechanisms that govern defense reactions in marine organisms. To understand humoral and cellular defense reactions in animals defying extreme physical and chemical conditions we set out to investigate the deep sea hydrothermal vent mussel Bathymodiolus azoricus found in abundance at the Mid-Atlantic Ridge. In the present study, hemocytes were stimulated with compounds of microbial origin and cellular morphological alterations as well as the production of superoxide assessed. Consequently, zymosan, glucan and peptidoglycan were considered as potent inducers of cellular reactions for inducing drastic cell morphology changes and high levels of superoxide production. Furthermore, we have presented for the first time in a deep sea hydrothermal vent animal, molecular evidence of the Rel-homology domain, a conserved motif present in all members of the Rel/nuclear-factor NF-kappaB family. Additionally we have demonstrated the occurrence of the antibacterial gene mytilin in Bathymodiolus azoricus gill tissues. Our results support the premise of an evolutionary conserved innate immune system in Bathymodiolus. Such system is seemingly homologous to that of Insects and other Bivalves and may involve the participation of NF-kappaB transcription factors and antibacterial genes.
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http://dx.doi.org/10.1016/j.marenvres.2006.12.010DOI Listing
August 2007

Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and several bacterial challenges in mussels.

Fish Shellfish Immunol 2007 Apr 20;22(4):340-50. Epub 2006 Jun 20.

Pathogens and Immunity, UMR CNRS EcoLag, University of Montpellier 2, cc 093, Place E. Bataillon, 34095 Montpellier cedex 05, France.

Defensin, mytilin and myticin are antimicrobial peptides (AMP) involved in mussel innate immunity. Their in vitro antibacterial activity is different according to the targeted bacterial species. To determine if this specificity is correlated to different regulations of gene expressions, adult mussels were challenged in vivo with either Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus or by heat shock. RNAs were isolated from circulating hemocytes and AMP mRNAs were quantified by Q-PCR using 28S rRNA as housekeeping gene. In addition, HSP70 gene expression was also quantified as representing non-specific response to stress. In naïve mussels, the three AMP mRNAs were present in dramatically different quantities. Compared to defensin, myticin was expressed 300-fold more and mytilin 30-fold more. HSP70 was found expressed 80-fold more than defensin. AMP genes were differentially regulated according to the challenging bacteria, M. lysodeikticus being the only one inducing down-regulation. Such variations in mRNA quantities were observed immediately after challenging, lasting less than 24h. Only V. anguillarum effect was observed later, between 12h and 3 days post-challenge. Compared to their background expression in naïve mussels, the major effect of V. splendidus was the decrease of mytilin and myticin mRNAs, V. anguillarum mainly increased both mytilin and HSP70 mRNAs, whereas M. lysodeikticus almost suppressed defensin mRNA. As expected, heat shock increased HSP70 mRNA, but also myticin mRNA. Consequently, AMP genes responded specifically to the challenges, confirming that at least some of the innate immune mechanisms are specifically orientated.
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http://dx.doi.org/10.1016/j.fsi.2006.06.007DOI Listing
April 2007

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

Dev Comp Immunol 2006 31;30(11):984-97. Epub 2006 Jan 31.

Pathogens and Immunity, UMR CNRS EcoLag, University of Montpellier 2, cc 093, Place E. Bataillon, 34095 Montpellier Cedex 05, France.

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.
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http://dx.doi.org/10.1016/j.dci.2005.12.009DOI Listing
October 2006

What can we learn from marine invertebrates to be used as complementary antibiotics?

Authors:
Philippe Roch

Adv Exp Med Biol 2004 ;546:391-403

Laboratoire DRIM, Université de Montpellier 2, case courrier 080, Place Eugène Bataillon, 34095 Montpellier cedex 5, France.

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http://dx.doi.org/10.1007/978-1-4757-4820-8_26DOI Listing
March 2005

Antiprotozoan and Antiviral Activities of Non-cytotoxic Truncated and Variant Analogues of Mussel Defensin.

Evid Based Complement Alternat Med 2004 09;1(2):167-174

Pathogènes et Immunité, UMR Ecosystèmes Lagunaires, Université de Montpellier 2, France.

We previously reported the crucial role displayed by loop 3 of defensin isolated from the Mediterranean mussel, Mytilus galloprovincialis, in antibacterial and antifungal activities. We now investigated antiprotozoan and antiviral activities of some previously reported fragments B, D, E, P and Q. Two fragments (D and P) efficiently killed Trypanosoma brucei (ID(50) 4-12 μM) and Leishmania major (ID(50) 12-45 μM) in a time/dose-dependent manner. Killing of T. brucei started as early as 1 h after initiation of contact with fragment D and reached 55% mortality after 6 h. Killing was temperature dependent and a temperature of 4 degrees C efficiently impaired the ability to kill T. brucei. Fragments bound to the entire external epithelium of T. brucei. Prevention of HIV-1 infestation was obtained only with fragments P and Q at 20 μM. Even if fragment P was active on both targets, the specificity of fragments D and Q suggest that antiprotozoan and antiviral activities are mediated by different mechanisms. Truncated sequences of mussel defensin, including amino acid replacement to maintain 3D structure and increased positive net charge, also possess antiprotozoan and antiviral capabilities. New alternative and/or complementary antibiotics can be derived from the vast reservoir of natural antimicrobial peptides (AMPs) contained in marine invertebrates.
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http://dx.doi.org/10.1093/ecam/neh033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516463PMC
September 2004

Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity.

Eur J Biochem 2003 Jul;270(13):2805-13

DRIM, Université Montpellier 2, France.

To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram-positive and Gram-negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25-33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non-naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the alpha-helical part or to the beta-strands of MGD1 had no detectable activity. The bacteriostatic activity of the sequence 25-33 was strictly dependent on the bridging of Cys25 and Cys33 and was proportional to the theoretical isoelectric point of the peptide, as deduced from the variation of activity in a set of peptide analogues of the 25-33 sequence with different numbers of cationic charges. By using confocal fluorescence microscopy, we found that the cyclic peptides bound to Gram-positive bacteria without apparent lysis. However, by using a fluorescent dye, we observed that dead bacteria had been permeated by the cyclic peptide 25-33. Sequence comparisons in the family of arthopod defensins indicate that MGD1 belongs to a subfamily of the insect defensins, characterized by the constant occurrence of both positively charged and hydrophobic amino acids in the loop. Modelling studies showed that in the MGD1 structure, positively charged and hydrophobic residues are organized in two layered clusters, which might have a functional significance in the docking of MGD1 to the bacterial membrane.
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http://dx.doi.org/10.1046/j.1432-1033.2003.03657.xDOI Listing
July 2003