Characterization of a Defensin from the Oyster Crassostrea gigas

Abstract

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized α-β motif (CS-αβ) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization. In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized α-β motif (CS-αβ) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization. Antimicrobial peptides (AMPs) 5The abbreviations used are: AMPantimicrobial peptideCg-DefC. gigas defensinDGdistance geometryDQF-COSYtwo-dimensional double-quantum filter correlation spectroscopyMALDI-TOF MSmatrix-assisted laser desorption/ionization time of flight mass spectrometryESI-MSelectrospray ionization mass spectrometryNOEnuclear Overhauser effectNOESYtwo-dimensional nuclear Overhauser effect spectroscopyRP-HPLCreversed-phase high performance liquid chromatographyr.m.s.d.root mean square deviationTOCSYtotal correlation spectroscopyRTreverse transcriptaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseMICminimum inhibitory concentrationEFelongation factorIfremerInstitut Francais de Recherche pour l'Exploitation de la MerCFUcolony forming units.5The abbreviations used are: AMPantimicrobial peptideCg-DefC. gigas defensinDGdistance geometryDQF-COSYtwo-dimensional double-quantum filter correlation spectroscopyMALDI-TOF MSmatrix-assisted laser desorption/ionization time of flight mass spectrometryESI-MSelectrospray ionization mass spectrometryNOEnuclear Overhauser effectNOESYtwo-dimensional nuclear Overhauser effect spectroscopyRP-HPLCreversed-phase high performance liquid chromatographyr.m.s.d.root mean square deviationTOCSYtotal correlation spectroscopyRTreverse transcriptaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseMICminimum inhibitory concentrationEFelongation factorIfremerInstitut Francais de Recherche pour l'Exploitation de la MerCFUcolony forming units. are important components of the innate immune system that have been conserved during evolution (1Yang D. Biragyn A. Kwak L.W. Oppenheim J.J. Trends Immunol. 2002; 23: 291-296Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar). They constitute a first line of host defense against pathogens in plants and animals (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar, 3Boman H.G. J. Intern. Med. 2003; 254: 197-215Crossref PubMed Scopus (868) Google Scholar). We estimate that more than 1000 antimicrobial peptides have been described at the level of their primary structure (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar). They are gathered in the Antimicrobial Sequence Database (www.bbc-m.units.it/∼tossi/amsdb.html). AMPs can be classified into three major groups: (i) linear peptides that form amphipathic α-helices, (ii) cyclic peptides containing cysteine-residue engaged in disulfide bonds, and (iii) peptides with an overrepresentation in certain amino acids (proline, arginine, glycine, or histidine). Despite their great diversity in terms of size, primary structure, amino acid composition, and mode of action, most AMPs are characterized by the preponderance in cationic and hydrophobic amino acids (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar). In most of the cases, this amphipathic character is considered as crucial for the interaction of the effective peptide with the membrane of sensitive microorganisms. This first interaction seems to be essential whatever the exact mode of action: (i) through disruption of their negatively charged cytoplasmic membranes or (ii) through killing following translocation into the bacteria without membrane lyses and binding to a specific target protein (4Brogden K.A. Nat. Rev. Microbiol. 2005; 3: 238-250Crossref PubMed Scopus (4314) Google Scholar). Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against pathogens. antimicrobial peptide C. gigas defensin distance geometry two-dimensional double-quantum filter correlation spectroscopy matrix-assisted laser desorption/ionization time of flight mass spectrometry electrospray ionization mass spectrometry nuclear Overhauser effect two-dimensional nuclear Overhauser effect spectroscopy reversed-phase high performance liquid chromatography root mean square correlation spectroscopy inhibitory concentration Francais de Recherche pour l'Exploitation de la forming units. antimicrobial peptide C. gigas defensin distance geometry two-dimensional double-quantum filter correlation spectroscopy matrix-assisted laser desorption/ionization time of flight mass spectrometry electrospray ionization mass spectrometry nuclear Overhauser effect two-dimensional nuclear Overhauser effect spectroscopy reversed-phase high performance liquid chromatography root mean square correlation spectroscopy inhibitory concentration Francais de Recherche pour l'Exploitation de la forming units. the defensins an important peptide They are and in and tissues that are in host defense against are cationic in size, containing three or disulfide and are active against a of bacteria and C. P. R. L. L. R. P. 2004; PubMed Scopus Google Scholar). The defensins can be into three the and are on the of the of their and more the cyclic J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The are produced and in of animals and in have been in and were to be or expressed H.G. J. Intern. Med. 2003; 254: 197-215Crossref PubMed Scopus (868) Google Scholar, A. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Microbiol. 2002; PubMed Google Scholar). In from their antimicrobial activities, defensins play an important role in and of specific P. Microbiol. 2003; PubMed Scopus Google Scholar). In with the of the on their structure, the in of the defensins is on their (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar). The defensins differ from the defensins by their disulfide P. C. PubMed Scopus Google Scholar). are the most of and defensins have been isolated in arthropods and and (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar). of the defensins were isolated from the of in and defensins are in of animals (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Scholar). In AMPs have been in as in the C. P. J. Full Text Full Text PDF PubMed Scopus Google and P. PubMed Scopus Google Scholar). defensins from the have been found to sequence to defensins from to in antimicrobial activities have been detected in the of AMPs have been characterized despite to from and by using de J. J. Immunol. Rev. 2004; 198: PubMed Scopus Google Scholar). In the we report the characterization of the first in oyster. The Crassostrea gigas defensin (Cg-Def) mRNA been isolated from mantle edge using the expressed sequence tag approach. Cg-Def sequence with of the arthropod defensin and shares with the defensins and from the mussel a of To on the of defensins in and the of this peptide in the defense of the we expressed Cg-Def in Escherichia and using the its of activity against a of bacteria and fungi. To the structure of the Cg-Def was determined in solution by NMR spectroscopy and and with its from mussels. the Cg-Def gene expression was in to bacterial challenge of oysters. and of C. were from a local oyster in or of and in at of a defensin was by from a C. gigas mantle edge in of were used to the Cg-def from the the of the was using the C. gigas mantle edge as and using a and a as were by on of the size was from the using a into the using a and of C. gigas of C. gigas was in of was to recombinant were at to and at high with the Cg-def were and to and using the to that the of was determined by were determined by the sequence to the of Cg-def in was isolated from tissues using to the during at with of to of was using of and of at for in the The was by at for was using the was used for of of with the following and as and of the target was by a and a of oyster was to the expression of the Cg-def The level of Cg-def expression is for of the gene following the Recombinant of Cg-Def was expressed in as an protein using the system a was of the Cg-def and with the in the of The Cg-def sequence was by using with The was by the at for by at for for and for for using The in the a to a of the of the peptide to the described for recombinant J. R. 2003; PubMed Scopus Google Scholar). Recombinant Cg-Def was expressed in with the The were at to in of of and of with of was with at for bacterial were by and at The were by bacteria in in by at for using a The was by in a at for at to protein and of Recombinant Cg-Def protein was by chromatography by with acid at a of in for at were with two of against acid in membranes and The at the Cg-Def was to by the in of to and of the for in the at The was by of by and the peptide was in at in the presence of The was to using a high performance liquid chromatography and the of were The peptide was with a at a of on a The of the Cg-Def was at in at during the Cg-Def was to using an additional using the and as the peptide was at in a solution containing and and in the presence of and R. J. 2003; PubMed Scopus Google Scholar). the Cg-Def was to by using a as described was by and the peptide concentration was by amino acid and at on the of the of the peptides were determined using matrix-assisted laser ionization mode mass spectrometry and electrospray ionization mass spectrometry Antimicrobial activity of recombinant Cg-Def was against the Gram-positive the Gram-negative and The activity of the peptide was against the following and inhibitory were determined in by the liquid on the described by and C. P. in Scholar). was used for and was used for was in at with was at on a or effect was by following a at was with of at a concentration the was with of an of at a of in and at of were after and of on The of was after a at a the bacterial was with NMR of Cg-Def were either in a or in to a The was to the by of or and at with a They are for the were with to to the NMR were on a with a and were in the of In the was at the of the at the spectroscopy PubMed Scopus Google Scholar, J. spectroscopy A. J. Scholar, J. and nuclear Overhauser effect spectroscopy D. J. were in the mode using the D. R. A. J. Scholar). in the was by the J. PubMed Scopus Google for the where a was The were with a time of and with of and were by using the The was using the described by NMR of and Google Scholar). To the the was in at and first a and an was for their and were from two at and with a time of and into to their and were into and distance The were from the and the were from the of the and To the distance and were used as in the that with in P. C. J. PubMed Scopus Google Scholar). In the first of the an of structures was from a structure with and In disulfide was used as were considered as the distance was than and the was than to the Cg-Def structure, and the of the and the disulfide were used as of was and the structures with a of of were with The was with R. J. PubMed Scopus Google and the of the structure and the were determined with D. P. 23: PubMed Scopus Google Scholar). 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In this and for the first time in an we the characterization of an isolated from mantle a of the defensin is in and was three disulfide this defensin The amino acid sequence of the C. gigas defensin with defensins from the the as observed with the defensins and from Cg-Def disulfide This additional was to the peptide more in high as in A. P. A. PubMed Scopus Google Scholar). this is to the mussel defensins this additional disulfide C. P. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Cg-Def an to be a sequence for translocation to the of the but a an as observed in the mussel or the P. P. Immunol. PubMed Scopus Google Scholar). This would suggest a of for the oyster To its Cg-Def was produced in and in This recombinant was of peptide of for and for the of Cg-Def in was the presence of residues in the the of with disulfide is a by the of in a that the of and in a bacterial host system J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). described the of and in J. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). We report for the first time the in a bacterial host of an with that can be in The activity of the recombinant Cg-Def was against a of bacteria and fungi. with on defensins (2Bulet P. Stocklin R. Menin L. Immunol. Rev. 2004; 198: 169-184Crossref PubMed Scopus (849) Google Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. Cg-Def its in the presence of high to This the that Cg-Def its activity in in vivo and would play a role in the antimicrobial defense of the oyster C. Cg-Def a of for to a to of bacteria in a C. PubMed Google Scholar). This is for for it been that the primary the salt of the antimicrobial Full Text Full Text PDF PubMed Scopus Google Scholar, J. Med. 2002; PubMed Scopus Google Scholar). from the disulfide the global of Cg-Def the motif observed in the defensins isolated from A. P. A. PubMed Scopus Google C. 3: Full Text Full Text PDF PubMed Scopus Google and plants P. L. P. A. 2003; PubMed Scopus Google Scholar). The Cg-Def and MGD-1 were and their structures were and This revealed of residues with three P. J. PubMed Google Scholar). Cg-Def and MGD-1 the disulfide that to the and to the of their The residues were mainly gathered in and their sequence the Cg-Def structure was with that of MGD-1 A. P. A. PubMed Scopus Google Scholar). showed that the and were conserved and the was for the of the residues and of Cg-Def with residues and of MGD-1 of was for the the two structures the and a similar disulfide Nevertheless, were observed for the disulfide and for the of the three the three disulfide and for Cg-Def and and for the for Cg-Def and for is mainly to the of by the of the This is to the the and the first The is a of this disulfide that on the of the Cg-Def and in MGD-1 as in the and loops of Cg-Def additional the of the the are conserved the of the loops are This is more hydrophobic for than that of MGD-1 that two charged Nevertheless, to the in the sequence the for Cg-Def and at the for it that have with to the In the were it been that the of MGD-1 is for a of the activity P. C. J. 2003; PubMed Scopus Google this is is that the of hydrophobic and in AMPs is essential for their antimicrobial The showed that of and and were conserved and the two antimicrobial mainly differ by the presence of two aspartic residues and and by the of a peptide and at the the Cg-Def and MGD-1 mainly to the two aspartic the hydrophobic by the of the disulfide bonds, hydrophobic and charged residues and are at the to a of hydrophobic and Our results showed that in the mantle as the tissue Cg-Def. we were to by of defensin from the we detected its presence by mass spectrometry of mantle tissue The results are in with for the In the AMPs are produced in where are and following bacterial P. J. PubMed Google Scholar). In the AMPs are produced in the of and into the in to a the systemic in the expression of peptides is in the of tissues P. D. Full Text Full Text PDF PubMed Scopus Google as observed for the expression of peptides in defensin is expressed in the and in the and a and P. Immunol. 2005; PubMed Scopus Google in AMPs are produced by Immunol. PubMed Scopus Google Scholar). from most of the AMPs in the of have been isolated from de J. J. Immunol. Rev. 2004; 198: PubMed Scopus Google Scholar). Our results would suggest that Cg-Def is continuously expressed in the oyster at that this an important in host protection against in the mantle is a of to that a for bacterial and potential pathogens. more of Cg-def gene expression in to various be to gain insight into the role of Cg-Def in oyster In AMPs and characterization be from oyster on AMPs are of great to the oyster immune system with the and it to pathogens. We are to A. J. for during defensin recombinant We J. de and for and