Molecular Characterization of Two Novel Antibacterial Peptides Inducible upon Bacterial Challenge in an Annelid, the Leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects. Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects. All of the multicellular organisms including plants, invertebrates, and vertebrates have developed an immediate response to defend themselves against infectious microorganisms (1Garcia-Olmedo F. Molina A. Alamillo J.M. Rodriguez-Palenzuela P. Biopolymers. 1998; 47: 479-491Google Scholar, 2Zasloff M. Nature. 2002; 415: 389-395Google Scholar). Recent studies on the components of the innate immune system have demonstrated the contribution of antimicrobial peptides to the host defense. Antibiotic peptides are small molecules. Based on their structural features, three classes were defined: 1) linear α-helical peptides without cysteines of which the prototype of this family is the cecropin; 2) open-ended cyclic cysteine-rich peptides among which defensins are the most widespread; and 3) linear peptides containing a high proportion of one or two amino acids like indolicidin, for example (3Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar). Despite great diversity in the primary structure, the majority of antimicrobial peptides documented are characterized by a preponderance of cationic and hydrophobic amino acids. This amphipathic structure allows them to interact with bacterial membrane. Brogden et al. (4Brogden K.A. De Lucca A.J. Bland J. Elliott S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 412-416Google Scholar) have determined in ovine that a novel class of peptides with anionic properties also has antimicrobial properties (4Brogden K.A. De Lucca A.J. Bland J. Elliott S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 412-416Google Scholar). In marked contrast to cationic antimicrobial peptides, no anionic antibacterial molecules have been described hitherto in invertebrates. Antimicrobial peptides appear to be essential anti-infectious factors that have been conserved during evolution. Meanwhile, their implications in immune processes are different according to species, cells, and tissues (5Salzet M. Trends Immunol. 2001; 22: 285-288Google Scholar). The involvement of antimicrobial peptides in natural resistance to infection is sustained by their strategic location in phagocytes, in body fluids, and at the epithelial level, i.e. at interfaces between the organisms and its environment. This action is strengthened by the rapid induction of such antibacterial peptide genes in bacteria-challenged plants or animals. Drosophila melanogaster, which is subjected to septic injury, massively induces and synthesizes a battery of potent antibiotic molecules from fat body, a functional equivalent of the mammalian liver (6Hoffmann J.A. Reichhart J.M. Hetru C. Curr. Opin. Immunol. 1996; 8: 8-13Google Scholar). The active peptides then are released into the hemolymph where they exert their antifungal and/or antibacterial activities. Adding to their systemic response, antibiotic molecules of the fruitfly are produced by cells of most of the barrier epithelia such as tracheal epithelium, gut lining, or salivary glands where they provide a local first defense against microorganisms (7Tzou P. Ohresser S. Ferrandon D. Capovilla M. Reichhart J.M. Lemaitre B. Hoffmann J.A. Imler J.L. Immunity. 2000; 13: 737-748Google Scholar, 8Tzou P. De Gregorio E. Lemaitre B. Curr. Opin. Microbiol. 2002; 5: 102-110Google Scholar). In worms, even if antimicrobial activities have been detected in the body fluids of several species, antimicrobial peptides have been characterized fully in nematodes and annelids only. A 71-amino acid peptide named ASABF 1The abbreviations used are: ASABF, Ascaris suum antibacterial factor; ACN, acetonitrile; RP-HPLC, reversed-phase high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; MIC, minimal inhibitory concentration; ISH, in situ hybridization; LFC, large fat cells. (for Ascaris suum antibacterial factor) possessing structural and functional similarities to the insect/arthropod defensins has been characterized in the parasitic nematode as A. suum (9Kato Y. Komatsu S. J. Biol. Chem. 1996; 271: 30493-30498Google Scholar). Recently, Pillai et al. (10Pillai A. Ueno S. Zhang H. Kato Y. Biochem. J. 2003; 371: 663-668Google Scholar) show an enhancement of the ASABF transcript in the body wall upon bacterial challenge. ASABF presents a significant sequence identity with peptides deduced from a cDNA sequence (yk150c7) and from a putative gene (T22H6.5) of the free-living nematode Caenorhabditis elegans. Although no variation of the C. elegans ASABF gene expression was detected using high density cDNA microarray, Mallo et al. (11Mallo G.V. Kurz C.L. Couillault C. Pujol N. Granjeaud S. Kohara Y. Ewbank J.J. Curr. Biol. 2002; 12: 1209-1214Google Scholar) demonstrate that Gram-negative bacteria infection of this worm provokes a marked up-regulation of genes encoding lysozyme and lectins that are known to be involved in the immune responses of other species. These authors (11Mallo G.V. Kurz C.L. Couillault C. Pujol N. Granjeaud S. Kohara Y. Ewbank J.J. Curr. Biol. 2002; 12: 1209-1214Google Scholar) also establish that certain infection-inducible genes are under the control of the DBL-transforming growth factor-β pathway. The existence of an inducible immune system in both nematodes and insects is consistent with the new molecule-based phylogeny, which considers that protostomes can be divided into two large groups: 1) ecdysozoans comprising nematodes, arthropods, and other phyla that have emerged and 2) lophotrochozoans regrouping annelids, flat worms, and mollusks (12Adoutte A. Balavoine G. Lartillot N. Lespinet O. Prud'homme B. de Rosa R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4453-4456Google Scholar). In lophotrochozoans, no inducible antimicrobial peptides have been described so far in the animals studied. The gene encoding lumbricin I, a proline-rich antibacterial peptide from the earthworm Lumbricus rubellus, is expressed constitutively (13Cho J.H. Park C.B. Yoon Y.G. Kim S.C. Biochim. Biophys. Acta. 1998; 1408: 67-76Google Scholar). The constitutive expression also has been reported in the mussel Mytilus galloprovincialis in which antibiotic peptides stored in hemocyte granules are released after bacterial challenge (14Mitta G. Vandenbulcke F. Roch P. FEBS Lett. 2000; 486: 185-190Google Scholar). In this study, we report the primary and secondary structures of two new antibacterial peptides, theromacin and theromyzin, isolated from the body fluid of an annelid, the leech Theromyzon tessulatum. Interestingly, both molecules do not present significant similarities with other known molecules. Theromacin is a novel cysteine-rich antimicrobial peptide, and theromyzin constitutes a new anionic antibiotic peptide. In an experimental model of we evidence for an enhancement of levels of both genes in specific tissues that be to the fat body of D. have that peptides are released massively into hemolymph where they exert their antimicrobial activity by a systemic These of the Drosophila antimicrobial are the first of antimicrobial peptide induction by a specific tissue in a leech is an of is in by as three blood from the first blood and after the and blood The to the animals were with or with 10 of a containing fluid and the animals were with a at and after challenge. were at for at and were in containing of The was to with at then was used to the which were according to et al. P. S. J.L. J. Reichhart J.M. Hoffmann D. Hetru C. Hoffmann J.A. J. Biol. Chem. Scholar). were with and in The then were in and for antimicrobial activity as described the were active and to by reversed-phase high pressure liquid All of the were on a of the were subjected to on a model was with a linear of in at a of The to were in in and for antimicrobial active were a model with a of in for 10 and for at a of The were and as was on a reversed-phase at a of using the described in The and the of theromacin and theromyzin were by mass in an using and by the matrix-assisted laser desorption ionization acids of the peptides was by on a liquid peptide were used to the mass of the theromacin and the mass of the from and of peptide were in amino containing and The was with and at for of was and the was with and at for of theromacin were in 10 of containing of was The was at of peptide were in 10 of of was This peptide at acid and with a at the were used to the mass of from the and as as the mass of the peptide from The were in acid containing at a of of the was directly the with of the acid in bacteria E. and the were used for the antimicrobial Antimicrobial antibacterial activity was by a liquid growth as described by et al. P. S. J.L. J. Reichhart J.M. Hoffmann D. Hetru C. Hoffmann J.A. J. Biol. Chem. Scholar). The minimal inhibitory and the minimal were determined according to the of R. E. activity was as described by et al. P. P. S. Hetru C. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: Scholar) by a liquid growth The was as described G. F. Roch P. J. Biochem. Scholar). cDNA for theromacin and cDNA for theromyzin were using from leech was using was into cDNA using the the of the of the was by using the and the sequence was deduced from with a for theromacin cDNA and from with a for theromyzin cDNA was for using one of in The were as for for and for of was using and from the theromacin and theromyzin cDNA first cDNA and the of a at its using a the of the was with an and the deduced from the cDNA in for theromacin and for were to described in All of the products were into and several different cDNA were In were in a containing and animals were in and were on and stored at The containing the of theromacin and theromyzin were used as for the of the and and were from cDNA by in using an and or were as described G. Vandenbulcke F. B. M. Roch P. J. Sci. 2000; Scholar). The were observed under a The control in with the control were by a with 10 A to were in using a was according to the 10 of and were on a containing and and to Theromacin and theromyzin were from and in the first was also as A and an were from the of and used in into The containing theromacin cDNA or cDNA were with and were from using the system of then were by using the and were as described In of the at the was using an and a system as C. D. S. S. 1998; Scholar). of theromacin and theromyzin were used for the of and according to the of were and as 1) 10 at in 2) containing and for at 3) with or in containing and at 10 in or in the for at 10 in in with the according to the for at and in then were in and observed using a on a was were for at in a containing and acid in were in for and in in Theromacin and theromyzin were using a secondary and as described were of immune by peptides. of and at different and were subjected to on a model was as described The of to theromacin or theromyzin was and the of peptide was by the with the The divided by the of hemolymph to the of theromacin in the coelomic fluid of the and of Theromacin and and theromyzin were isolated from leech body fluid in A sequence of the molecules then was by In and mass revealed a deduced mass of for theromacin and for theromyzin, that the by were fully the amino acid of the peptides and to on the cDNA was using a and acid deduced from the cDNAs to that theromacin is a cationic 75-amino acid peptide of and that theromyzin is an anionic 86-amino acid peptide of in revealed no sequence similarities of peptides with other known peptides or that the mass to the theromacin sequence deduced from the cDNA was 10 in of the mass of the peptide The in mass was to the of the 10 cysteine residues in the sequence in disulfide In contrast with the mass of theromyzin was in with the that theromyzin is a peptide of also were deduced from the cDNA The revealed that 1) theromacin and theromyzin are at the by a putative hydrophobic signal peptide, and and that 2) the for signal is most after the the for theromacin and after the the for This to the that mature peptides be and can be to the of the of the of the cysteine residues in disulfide the mass of theromacin was subjected first to with and then by mass The mass revealed that the to the to a mass of consistent with the of 10 not to a mass not This that the 10 cysteine residues of theromacin are in disulfide the of the primary structure of the antibacterial peptide showing that theromacin not a cysteine described in other known antimicrobial peptides, the of the disulfide array was by of the peptide The from the were by in the was in with the of the at to at one to the in the theromacin The revealed the existence of an in the at In the an in the at was deduced from the observed at A at that two disulfide in the at on the presence of two disulfide in this was not to the and disulfide The then was The detected at to the to the revealed the presence of an in the the the to the are with the array of and array of not present similarities with determined in other peptides or Theromacin is a novel cysteine-rich antimicrobial peptide. of Theromacin and liquid growth the theromacin was active against M. minimal activity was E. or F. at the theromacin was with M. at a 10 the of the bacteria were in theromacin a The of antibacterial was theromacin was that disulfide an in the activity of the Theromyzin was to be active against M. The growth of bacteria on the was observed theromyzin was for the minimal showing that the activity against M. and of Theromacin and Theromyzin gene expression of theromacin and theromyzin during immune response was by of the in leeches was first at and after or bacteria or M. in the theromacin and theromyzin is by Gram-positive Gram-negative or The expression levels were high after infection and are with a bacterial with a In no were observed between and that with bacteria induces a and of theromacin and theromyzin mRNA levels and that this enhancement not on the of septic This variation of theromacin and theromyzin upon bacterial challenge also has been by a C. C. Vandenbulcke F. D. Y. M. Biochem. J. Scholar). and to the of theromacin and theromyzin in we the of the gene expression using and with the theromacin on leech Both genes are expressed and in large cells in animals. they we named them large fat cells Interestingly, are in contact with containing coelomic fluid from which theromacin and theromyzin peptides were of were with of we that the of animals observed by be to the in the then to the by was in or in other The induction was by leeches with bacteria or The were with The signal under as an of and The of the was to the transcript in the enhancement of the theromacin transcript levels was observed in the of the of induction in was with bacteria with for both genes These gene expression studies were to animals or not with or with a of Gram-positive and Gram-negative bacteria. on of leeches was using theromacin and theromyzin The are in the of theromacin and theromyzin in the of animals. The expression was after bacteria after for theromacin and for signal was in other cells or contrast to animals no or were observed in leeches The of the with theromyzin were detected in the control by this only. we that several that during the be of theromacin and theromyzin gene In of this leeches were and after the blood which to the of the of leeches were and with the theromacin and theromyzin cDNA The in a of both theromacin and theromyzin after the blood These the cells in theromacin and theromyzin mRNA that in of the of i.e. or the enhancement of theromacin and theromyzin was observed in This that the leech an in the of antimicrobial molecules comparable to the insect fat of Theromyzin and Theromacin and of in the the of theromacin and theromyzin in anti-infectious and were in leeches subjected to bacterial challenge. and were by on the of leeches Theromacin was in in the intestinal and at the epidermis level. Theromyzin was observed also in the of and a in coelomic in contact with the using theromacin and theromyzin and was detected in the and the of the Interestingly, coelomic the body fluid from which theromacin and theromyzin were These in of an of the antibacterial peptides from the to the body fluid of the the of on a variation of theromacin and theromyzin was liquid coelomic fluid was at and after or bacteria and of body fluid theromacin and theromyzin were by The in demonstrated an of the levels of theromacin and theromyzin after bacteria or challenge. These rapid and were in leech by bacteria and for theromacin and theromyzin, by and for theromacin and theromyzin, theromacin and theromyzin to and These are in with the and that theromacin and theromyzin exert their antibacterial activity into the body fluid of the can to antimicrobial peptides of theromacin and theromyzin peptides are in tissue this and are released into the coelomic fluid after septic B. Reichhart J.M. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. Scholar). In to their in the systemic response, theromacin and theromyzin have a in defense. the structures in the epithelia in contact with the the epithelium, and The at the epidermis to for the peptides in the the animals. This also was on the during the we a enhancement of by leech after bacterial challenge. and of the two at the as theromacin and by and that the two molecules isolated from the to theromacin and theromyzin not described both the and of two novel antibacterial peptides from the body fluid of the leech tessulatum. These are a cationic peptide and theromyzin, an anionic peptide with The peptide deduced from the theromacin and theromyzin genes putative signal peptides, that mature peptides to molecules. in the of this study, the genes encoding peptides are by immune challenge in a tissue by the the several of antimicrobial peptides containing cysteine residues have been described in In invertebrates, most of them the disulfide array of the insect/arthropod P. Hetru C. J.L. Hoffmann D. Immunol. Scholar). In to cysteine residues of theromacin not this the in that the peptide has no significant with other known peptides. theromacin can be as a new cysteine-rich antimicrobial peptide. cysteine-rich antimicrobial peptides different from the insect/arthropod have been reported in other such as the nematode A. suum (9Kato Y. Komatsu S. J. Biol. Chem. 1996; 271: 30493-30498Google Scholar). This that cysteine-rich antibacterial peptides be in which to the by Kato et al. Y. H. Zhang H. Biochem. J. 2002; Scholar). antibiotic cysteine-rich molecules in plants, or they a The majority of antimicrobial peptides including of the cysteine-rich family a their with the bacterial Y. Biopolymers. 2002; Scholar). that theromyzin, in contrast to is an anionic This is the first report of an anionic antibacterial peptide in invertebrates. In antimicrobial peptides with anionic properties were in the and in (4Brogden K.A. De Lucca A.J. Bland J. Elliott S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 412-416Google Scholar). properties are anionic of the of and as a for a family of antimicrobial peptides in also the presence of for activities D. G. 2000; Scholar). studies that the antimicrobial activities of a that from the of the peptide with both and D. G. 2002; Scholar). The of residues at the of theromyzin in of structures between the leech antibacterial peptide and Based on we can that the active of theromyzin be to the in and residues and that theromyzin a for Theromacin and theromyzin genes are expressed in in the is after bacterial challenge. These a of the theromacin gene to that of the insect antimicrobial peptides in the genes encoding antibiotic peptides are a septic and their expression is a septic B. Reichhart J.M. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. Scholar). no in gene expression was observed after Gram-positive or Gram-negative that the antibacterial response of Theromyzon is This also has been in Drosophila the of Lemaitre et al. B. Reichhart J.M. Hoffmann J.A. Proc. Natl. Acad. Sci. U. S. A. Scholar) demonstrated that the antimicrobial response of the fruitfly among classes of microorganisms and a response that is to the This in a natural of the leech also its antimicrobial In addition, that during the also be of the theromacin and theromyzin gene the leeches their we that such factors in this in the induction of theromacin and theromyzin This of induction been reported for the Drosophila and M. G. Biochem. Biol. 1996; Scholar) present evidence that an in the response of the gene during the to a in and after a septic The of theromacin and theromyzin is followed by a rapid release of the peptides into the coelomic fluid after septic theromacin and theromyzin their antimicrobial activities a systemic the presence of peptides in the intestinal epithelial cells and at the epidermis that leech antimicrobial peptides also a in epithelial defense. The of antibiotic molecules in the also has been reported in insects and in vertebrates where they provide a rapid local immune response against in during C.L. E. Scholar, C.L. M. Annu. Rev. Biochem. Scholar, J. S. C. P. Biochem. Biol. 2001; Scholar). The of theromacin and theromyzin in leech the local response reported in in which antibacterial peptides in the bacteria and/or infection C.L. M. Annu. Rev. Biochem. Scholar). In addition, a the after we a of leech theromacin and theromyzin against bacteria during The of allows to present a for the of antimicrobial peptides in the immune system of the provokes an of that bacteria present in the of the The bacteria then be by theromacin and theromyzin present in the the theromacin and theromyzin gene expression and the of the gene products from the into the body fluid of the theromacin and theromyzin exert their antimicrobial a systemic This in with the action of to the in the body The are in contrast with reported for other lophotrochozoans such as the M. galloprovincialis and the (13Cho J.H. Park C.B. Yoon Y.G. Kim S.C. Biochim. Biophys. Acta. 1998; 1408: 67-76Google Scholar, G. Vandenbulcke F. B. M. Roch P. J. Sci. 2000; Scholar). In antibiotic peptides are expressed constitutively and stored in no specific like the fat body is The and of active theromacin and theromyzin by a specific tissue and the rapid up-regulation on infection for the first time in a lophotrochozoan an antimicrobial response developed by holometabola insects Y. Immunol. Scholar, M. S. S. G. D. Hoffmann J.A. P. J. Biol. Chem. Scholar). the a between insects and In i.e. in or in antibiotic peptide genes are by bacterial challenge M. D. R. C. A. Hoffmann J.A. P. J. Biol. Chem. 2001; Scholar). In no inducible system was in like the These in arthropods, such inducible of antimicrobial peptide gene have at the time of the of from insects. The between the antibacterial response of the leech and that of also is by the functional between the leech and the insect fat body, which the to for are to J. for to for the of F. to J. C. and de for to the and to J. for the in