Itoh  Naoki

Osada  Makoto

Takahashi Keisuke G.

Tanimoto  Saeko

English

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Tanimoto, Saeko

Itoh, Naoki

Osada, Makoto

Takahashi, Keisuke G.

Tohoku University Repository (TOUR) / 東北大学機関リポジトリ

Agglutination of Bacteria by a Lectin in theHemolymph of the Pacific Oyster Crassostreagigas著者 Tanimoto  Saeko, Itoh  Naoki, Osada  Makoto,Takahashi  Keisuke G.journal orpublication titleTohoku journal of agricultural researchvolume 60number 3-4page range 83-90year 2010-03URL http://hdl.handle.net/10097/48070Tohoku Joumal of Agriclユltural ResearchVol. 60 No. 3-4, March 2010Phtnted in JapanA亀虫lutination of Bacteria by a Lectin in the Hemolymphof the Pacific Oyster Crassost7･ea glgaSSaeko TANIMOTO, Naoki ITOH, Makoto OsADA and Keisuke a. TAKAHASIIILaboratory of AqucELnLltural Biobgy, Graduate School of Agricultural Science,Tohoku Universuy, Sendai 981-8555, Japan(Received, February 15, 2010)SummaryCel1-free hemolymph (plasma) from the Pacific oyster, Crassostrea gigas,contains two lectins with the ability to agglutinate ERBC (Gigalin E) and hun!anRBC (Gigalin H)･ We investigated whether a. gigas plasma can agglutinate nlnebacterial species, includingfive marine bactcria･ All vertebrate erythrocytes(RBCs) tested (ERBC, GRBC and SRBC) were agglutinated by C･ gigas plasma.Only Gigalin E required calcium ions forglobqonnis, B･ subtilis, D･ aquamarina, E･agglutinated by a. gigas plasma, whereastubiashii were not. Pre-incubation of a.strongly reduced titers when the adsorbedactivlty toward live cells of the threeincubation with ERBC had no effect onThe agglutinating activity offixed cellshemagglutinating activity (HA)･ A･cold, SI Putrefcm'ens and S･ aureus wereM. halophilus, V. lginolyttcus and V･gtgas plasma with GRBC resulted inplasma was titrat冶d for agglutinatlngbacterial sp cies. In contrast, pre-subsequent agglutination of bacteria･of S･ aureus did not change due topre-adsorpt10n With either ERBC or GRBC. These results indicate that thehemolymph component(S) responsible for erythrocyte agglutination also appeartobe involved in bacterialagglutination･ One component is probably Gigalin H, asindicated by the adsorptlOn Of hemagglutinating activity by GRBC･Key words : lectin, hemolymph, aggllltination, oyster, Crassostrea g叩aSLectins are protein complexes with carbohydrate-specific binding propertiesthat are widely expressed in plants, lnVertebrates,and vertx)brates and may servea wide variety of physiological functions (Sharon and Lis, 1972). Invertebratesare believed to possess e銃cient host defense mechanisms by virtue of theirhumoral　defense molecules because they lack antibody-mediated acquiredimmune systems (Arason, 1996)･Lectins′may act as recognition molecules forhost defense activities such as aggregation and opsonization in invertebrates,including bivalve molluscs (Vasta and Marchalonis, 1984 ; Vasta et al･, 1994 ;Olafsen, 1995 ; Wang et al" 2007).Lectins are good candidates for the recogni-8384 S. Tan,-moto et al.Lion role becausethey can bind and opsonize foreign materialwith recognitionspecificity for `pathogen-associated molecular patterns'(PAMPs) (Medzhitov andJaneway, 2000).Invertebrate lectins have been found in hemolymph plasma and bound tohemocyte membranes (Vasta et alリ1982 ; Renwrantz and Stahmer, 1983 ; Tasumiand Vasta, 2007). Lectins have bejm isolated and characterized from thehemolymph of many species of bivalve molluscs･ In oysters, two lectins (GigalinsEand H) have been purified from the Pacific oyster, Crassostrea gigas (Hardy etal., 1977 ; 01afsen et al., 1992),and a calcium-dependent lectin (C-type lectin) hasbeen purified from the Chilean oyster, Ostrea chilensis (Minamikawa el al･, 2004)･In addition, lectin genes inthe Pacific oyster have been analyzed･ The fullcoding sequences of C-type lectin (CgCLeC-1) and galectin (CgGal) from C･ gigashave been obtained, and tissue expressionanalyses have been conducted (Yama-ura et al., 2008). Some bivalve lectins act as opsonins (Renwrantz and Stahmer,19g5 ; olafsen et al., 1992) and agglutinate environmental bacteria (Arimoto andTripp, 1977 ; Tamplin and Fisher, 1989 ; Fisher and I)iNuzzo, 1991 ; Tunkijjanu-kij et al., 1997). Therefore, 1ectins appear to play a criticalrole in the host-defense IneChanisms of bivalve molluscs, bothby recognlZing, binding to andagglutinating foreignmicroorganisms and by opsonizlng foreign particles foruptake by phagocytic hemocyt8S.A number of lect,ins described to date haぶbeen limit冶d by the use ofvertebrate erythrocytes (red blood cells ; RBCs) in agglutination assays (Fisherand DiNuzzo, 1991). RBCs are obviously not potentialinvaders. Furthermore,RBC surfaces exhibit only 7 0f over 100 monosaccharides found in nature (Yeaton,1981). In marine bivalve molluscs, investigation intothe functional role oflectins must employ potent invasive organisms, such as marine bacteria･ GigalinsE and H fromthe hemolymph of C･ gigas agglutinate bacteria, including Vibn'oanguillarum (01afsen el al., 1992) ; however, little isknown about the interactionsof C･ gigas lectins with marine bacteria (Mori et al･, 1984)･ We investigatedwhether a. gigas plasma can agglutinate marine bacteria, including pathogenicVibrio species.Materials and MethodsAnimalsSpecimens of the Pacific oyster, Crassostrea gもgaS, Withan average shell heightof 12.4±2.7 cm were obtained from hanglng-Culture beds in Matsushima andOnagawa Bays, MiyagiPrefecture, Japan･ They were then transferred to ourlaboratory and held in 180-I aquariumsfilled withcirculating,filtered artificialseawater (MARINE ART BR, Senju Seiyaku C0., Osaka, Japan) for five to sevendays. Water temperature was maintained at 15±loC･Bacten'al Agglutination by an Oyster Lectin　　　　　　85Preparation of Cell-free HemolymphHemolymph was withdrawn from the adductor muscle using a tuberculinsyrlnge With a　26-gauge, 015-inch needle. The collected hemolymph wascentrifuged at 290×g for lOmin at 40C to remove the hemocytes. Aftercentrifugation, the supernatant (plasma) was used for the hemagglutination assays.ErythrocytesHemagglutination assays of the plasma samples were performed using animalRBCs. Equine blood, goose blood and sheep blood stored in AIsever'S solutionwere obtained from Nippon Biotest Laboratories (Tokyo, Japan).Hemagglutinating Activity (HAJ AssayEquine erythrocytes (ERBC), goose erythrocytes (GRBC)and sheep eryth-rocytcs (SRBC) used as agglutination test particles were placed in u-bottomed 96-wellmicrotitcr plates. Twelve oysters were used for this assay. A plasmasample (25 ill) was serially diluted two-fold with TBS buffer (50 mM TriS-HCl,PII 8.0, containing 150 mM NaCl) or Ca-TBS buffer (TBS plus 10 mM CaC12, PH8.0). The sample was then mixed with a 2% erythrocyte suspension (25FLl) inTBS. Activity was expressed as hemagglutinating (HA) titer, defined as thereciprocal of the highest dilution of plasma glVlng a positive reaction after 2 h atroom temperature (21-24oC). Median HA titer was calculated and recorded.Each assay was performed in duplicate. Controls received TBS or Ca-TBS inplace of oyster plasma.Cross-adsorp払on AssaysLectins in a. g甘gaS Plasma were examined for cross-reaction with ERBC,GRBC and SRBC. For each test, a 200-Ill plasma sample was added to a Jtubecontaining 400 /Jl of packed RBCs･ Tubes were gently rotated for 2 h at 4DCandthen centrifuged at 500× g for 5min at 20oC. The resulting supernatant wasplaced inmicrotiter plates, serially diluted, and tQSted by HA assays with freshRBCs. Twenty oysters were used forthis assay. Each assay was done induplicate･Bacteria and Culture Condl,'tionsNine selected bacterial　strains we/re examined for agglutination byhemolymph plasma : Arthrobacter globqormis (IAM 12137), Bam'llus subtilis (IAM1026), Deleya aquamarina (IAM 12645), Escherichia cold (IAM 1264), Man'nococcushalophilus (IAM 12844), Shewanella putrefaciens (IFO　3908), Staphylococcusaureus (IFO 3761), Vibrio alginolyticus (ATCC 19108)and V･ tubiashii (ATCC19106). Five of these species were marine bacteria : D･ aquamarina, M･ halo-86 S. Tanimoto et al.philus, S. putrefaciens, V. alginolyticus and V tubiashii･ IAM strains were kindlysupplied by the Institute of Molecular and Celllllar Biosciences of The Universityof Tokyo, Japan･ IFO strains were purchased from the Institute for Fermenta-tion, Osaka, Japan･ ATCC strains were obtained from the American TypeCulture Collection, Rockville, MI), USA. Before bacterial agglutinating activity(BA) assays, marine bacterialstrains Were cultured on marine agar 2216 (I)ifcoLaboratories, Detroit, MI, USA) at 26oC. The four non-marine strains werecultured on tryptic soy agar (Difco Laboratories) at 260CI After two days ofgrowth, colonies were suspended in sterile balanced salt solution (BSS) formulatedto be compatible with oyster physiology (44616 mM NaCl, 14･5 mM KCl, 1412 mMMgSO4･7H20, 10･6 mM MgC12･6H20, 8･6 mM CaC12, 310mM NaIICO3, 0･08mMNaH2PO. and 5.6 mMglucose, pH 7･8 ; Takahashi and Mori, 2000) at approxi-mately 109 colony-forming units (CFUs)/ml･For some experiments, S. putrefdm'ensand S. aureus were treated with 1%glutaraldehyde for 12 hrs at room temperature･ Aftcrfixation,the bacterial cellswere washedthree times and resuspended in BSS･ This experiment demonstratedthatglutaraldehydefixation did not affect BA･BA AssayWe detcrminedthe ability of cell-free oyster plasma to agglutinate bacteria･First, 50 ill of plasma was serially diluted two-fold with Ca-TBS in flat-bottomed96-Well plates, mixed with the bacterial suspension (25FLl, 109 CFU/ml) andincubated for 2 h at 250C with gentle shaking every 30 min･ After incllbation, theresulting plates were observed under an inverted microscope at　400×magnification (Eclipse TEIOOO ; Nikon Corp., Tokyo, Japan) to determinewhether the bacterialcells were agglutinatcd･ Five replicates of each agglutina-t,ion assay wereperformed･To determine the agglutinatlng Specificity of lectins for bacteria, wealsoperformed BA assays using supernatant pre-adsorbed withRBCs (ERBCandGRBC). Live cells of A. globLformis, D･ aquaman-naand SI Putrefwiensandglutaraldehyde-fixed cells of S･ aureus were used in these assays･ Each assay wasdone in duplicate･ Five oysters were used for these assays･Results and DiscussionHemagglutinating Specがcity and VariabilityHemolymph from a. gigas contained lectins with agglutinating activity for avariety of cells, including a panel of RBCs. All RBCs tested (ERBC, GRBC andSRBC) were agglutinated by C･ gigas plasma (Table 1)I A survey of 12 individ-ualoystcrs showed much higher IIA titers for ERBC, ranging from 1,024 to 65,536(median, 16,384; n-12), than for the other two RBCsI Interestingly, onlyBacterial Agglutination by an Oyster Lectin　　　　　　87Table l･ Agglutination of three vertebrate erythrocytes (RBCs) by C･ gigas plasmaAgglutination with erythrocyteE qu ine Goose SheepPlasma wit,h Ca2十　　16,384 (512-65,536)　128 (16-512)　　　2,048 (0-8,192)Plasma　　　　　　　　　　2 (0-8)　　　　　128 (8-512)　　1,024 (04096)Values in the Table demonstrate medianhemagglutinating (IIA) titersand range ofHA titers of different samples (n- 12)･ERBC agglutinating activity in C･ gigas plasma required Ca2'･ Plasma fromdifferent individualoystcrs varied widely in HA titer for SRBC. Five of twelveindividual oysters had very hightiters, whereas the remainlng OyStX)rS Showed lowor zero titers for SRBC. Previous studies have shownthat a. gigがhemolymphcontains two erythrocyte lectins with the ability to agglutinate ERBC (Gigalin E)and human RBC (Gigalin H) (Hardy et al･, 1977). Most C-type lectins identifiedin invertebrates are Ca2㌧dependent (Wang et al･, 2007). Thus, Gigalin E mightbe a C-type lectin.A dsorption StudiesPlasma lectins demonstrated agglutinating activitJy for a variety of RBCs(Table2). ERBC incompletely adsorbed HA activity for SRBC and did notadsorb that for GRBC. SRBC variably left a proportion of HA activity unadsor-bed. GRBC adsorbed most HA activity for SRBC, but HA activity for ERBCwas unaffected. It has been observed that GRBC completely adsorbs IIA activltyfor HRBC (Nakamura, unpublished data). Therefore, HA activity for GRBCwas produced by Gigalin H. This study reconfirms that two lectins, Gigalins Eand H, Occur in C･ gigがhemolymph･Agglutination of BacteriaTo establish whether a. glgaS Plasma lectins play a role in preventlngTable21 Cross-adsorption tests of C･ gigas plasma with diqerent RBCsAdsorbing RBCERBC GRBC SRBC128　　　　　　　　　6416,3848　　　　　　　　64256None (Ca-TBS only)　　　16,384　　　　　128　　　　　　512Values in the Table demonstrate median HA titer (n-20). - ; HA titcrswere not detected.88 S, Tanimoto et al.Tables. Buterial agglutinating (BA) activity of C･ gigas plasmatou)ard nine bacterial strainsTreatment Strain Median BA tiber RangeLive Arthrobacter globqormisBGm'llus subtilisDeleya aquamarinaEscherichia coldMarinococcus halophilusShewanella putrefbciensStaphylococcLLS aureuSVibrio alginolyt-sVibrio tubiashii4　8　2　4　　　　　2　402　　3　　1　5 1　一日H 512 2,0484-1616-1284-8512-1,0242-4F ixed Shewanella pu trefhciensStaphylococcus aureus　　　　　　　64　　　　　64- 128ー; not detected.bactcrialinfection, we investlgatedthe ability of C･ gigas plasma to agglutinatebacteria, including pathogenic species･ A･ glob%formis, B･ subtilis, A aquamarina,E. colt, S. putrefuiens and S･ aureus were agglutinated by C･ gigas plasma,whereas M. halophilus, V. alginolyticus and V･ tubiがhii were not (Table 3)･Agglutinating activities for A･ globqomw'予and S･ putrefaoiens were remarkablyhigh･ S･ putrefaciens cells were not agglutlnatCd by fixation with glutaraldehyde,while丘Ⅹed cells of A aureus were strongly agglutinatcd･ Because the agglutinat-1ng abilities of lectins are heterogeneous in structure and ligand binding, differentbacterial surface moieties may be involved inthis process (Canesi et all, 2002)･Gigalins Eand H from C･ gigas hemolymph agglutinate bacteria, including Vibrioanguillarum (01afsen et alリ1992)･ In the American oyster (C･ virginica), Tam-plinand Fisher (1989) have reported that V･ cholerae strains belonging to 01serovars and biovars are predominantly agglutinated by ce111free plasma, but 79Other bacterial strains, representlng 26 Species, are not agglutinated. Further-more, these authorsalso indicate that some other Vibrio species, such as TT.trWlnがcus and V･ parahaemolyticus, are not agglutinated by C･ virginica plasma･Similar results have been obtained by Fisher and DiNuzzo (1991). Inthis study,bacteriathat are pathogenic for bivalve larvae, TT･ alginolytwus and γ･ tubiashii,were not agglutinated by C･ gigas plasma･ The effects of oyster lectins ontheecology of vibrios in marine and estuarine waters remain unelear･Pre-incubation of C･ gigas plasma with GRBC resulted in strongly reducedtitcrs when the adsorbed plaぷma Was titrated for agglutinating activity towardlive cells of the three bacterialspecies (Table 4). In contrast, pre-incubation withERBC had no effect on subsequent agglutination of bacterial The agglutinatlngBacterial Agglutination by an Oyster Lectin　　　　　　89Table4. BA liter of C･ gigas plasma folhwing adsorption with ERBC and GRBCTreatment Strain RBC Median BA titer RangeL ive ERBC 1,024　　　　512-2,048Arthrobacter globqormis GRBC　　　　　4　　　　　0- 16None 1 ,024　　　　5 12-2,048ERBCDeleya aquamarina GRBCNoneERBC　　　　　　256　　　　　256-512Shewanella putrefbciens GRBC　　　　　4　　　　　2- 16None　　　　　　　256　　　　　256-5 12ERBCStaphybcoccus aureus GRBCNoneactivlty Of丘Ⅹed cells of S･ aureus did not change due to pre-adsorptlOn With eitherERBC or GRBC. These results indicate thatJ the hemolymph component(S)responsible for erythrocyte agglutinationalSo appear to be involved in bacterialagglutination (BA). One component is probably Gigalin H, as indicated by theadsorption of hemagglutinatlng activity by GRBCI Gigalin H wasalmost com-pletely removed with GRBC by centrifugation, and BA activity of the resultingsupernatant was markedly reduced for live cells of all bacterialspecies t冶StCd･Gigalin H exhibits specificity for sialic acid higha毘nity for sialic acid residues inglycoproteins (IIardy et al･, 1977)･ In a study of C･ m'rginica lectins, the IT･cholerae ligand has been suggested to be a heat-stable N-acetyl-neuramic acid-likeresidue (Tamplinand Fisher, 1989). Cross-adsorption tests showed that C･ vigasplasma in'cubated with ERBC eliminated only IIA,and BA titer was not reduced.Gigalin E apparently did not agglutinate the bacteria examined in this study･BA ability forfiⅩed cells of S･ aurews was not reduced by pre-treatment withRBCs･ This result implies that IIAand a part of BA activlty may Involvedifferent components of C･ g甘gaS hemolymph･ As described above, previousinvestigations have found only two lectins (Gigalins E and H) in C･ gigashemolymph (IIardy et all, 1977; 01afsen et al･, 1992). It remains to be detcr一mined whether other agglutinatlng Substapces are present in C･ g甘gaS hemolymph･ReferencesArason, G.J., Fish Shelljish Zmmunol･, 6, 277-289 (1996)Arimoto, R. and Tripp, M.R., J･, Znvertebr･ Pathol･, 30, 406-413 (1977)90 S. Tanimoto et al.Canesi, L., Gallo, G., Gavioli, M.,and Pruzzo, C., Micros･ ResI Tech･, 57, 469-476(2002)Fisher, W.S.and DiNuzzo, A.R., J. Znvertebr. 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Agglutination of Bacteria by a Lectin in the Hemolymph of the Pacific Oyster Crassostrea gigas

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Agglutination of Bacteria by a Lectin in the Hemolymph of the Pacific Oyster Crassostrea gigas

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