Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils

INFECTION AND IMMUNITY, July 2005, p. 4161–4170 Vol. 73, No. 7 0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.7.4161–4170.2005 Copy igh © 2005, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Supe oxide P oduc ion in Galle ia mellonella Hemocy es: Iden i ica ion o P o eins Homologous o he NADPH Oxidase Complex o Human Neu ophils Da id Be gin, 1 † Eme P. Ree es, 1 † Julie Renwick, 1 F ans B. Wien jes, 2 and Ke in Ka anagh 1 * Medical Mycology Uni , Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, NUI Maynoo h, Co. Kilda e, I eland, 1 and Cen e o Molecula Medicine, Uni e si y College London, 5 Uni e si y S ee , London WC1E 6JJ, Uni ed Kingdom 2 Recei ed 4 Janua y 2005/Re u ned o modi ica ion 11 Feb ua y 2005/Accep ed 7 Ma ch 2005 The insec immune esponse has a numbe o s uc u al and unc ional simila i ies o he inna e immune esponse o mammals. The objec i e o he wo k p esen ed he e was o es ablish he mechanism by which insec hemocy es p oduce supe oxide and o asce ain whe he he p o eins in ol ed in supe oxide p oduc ion a e simila o hose in ol ed in he NADPH oxidase-induced supe oxide p oduc ion in human neu ophils. Hemocy es o he g ea e wax mo h (Galle ia mellonella) we e shown o be capable o phagocy osing bac e ial and ungal cells. The kine ics o phagocy osis and mic obial killing we e simila in he insec hemocy es and human neu ophils. Supe oxide p oduc ion and mic obial killing by bo h cell ypes we e inhibi ed in he p esence o he NADPH oxidase inhibi o diphenyleneiodonium chlo ide. Immunoblo ing o G. mellonella hemocy es wi h an ibodies aised agains human neu ophil phox p o eins e ealed he p esence o p o eins homologous o gp91 phox , p67 phox , p47 phox , and he GTP-binding p o ein ac 2. A p o ein equi alen o p40 phox was no de ec ed in insec hemocy es. Immuno luo escence analysis localized insec 47-kDa and 67-kDa p o eins h oughou he cy osol and in he pe inuclea egion. Hemocy e 67-kDa and 47-kDa p o eins we e immunop ecipi a ed and analyzed by ma ix-assis ed lase deso p ion ioniza ion— ime o ligh analysis. The esul s e ealed ha he hemocy e 67-kDa and 47-kDa p o eins con ained pep ides ma ching hose o p67 phox and p47 phox o human neu ophils. The esul s p esen ed he e indica e ha insec hemocy es phagocy ose and kill bac e ial and ungal cells by a mechanism simila o he mechanism used by human neu ophils ia he p oduc ion o supe oxide. We iden i ied p o eins homologous o a numbe o p o eins essen ial o supe oxide p oduc ion in human neu ophils and demons a ed ha signi ican egions o he 67-kDa and 47-kDa insec p o eins a e iden ical o egions o he p67 phox and p47 phox p o eins o neu ophils. Neu ophils play a pi o al ole in he inna e immune esponse o mammals by phagocy osing and des oying in ading mic o- o ganisms (39). Se um-opsonized mic obes can be killed wi hin he con ines o he phagocy ic acuole, a p ocess which is dependen upon he gene a ion o eac i e oxygen species (ROS) (55), p edominan ly supe oxide (O 2 ⫺ ) and hyd ogen pe oxide (H 2 O 2 ) (2). In addi ion o he dismu a ion p oduc s o O 2 ⫺ , his anion has also been shown o be indi ec ly espon- sible o he ac i a ion o ca ionic g anule enzymes, such as elas ase and ca hepsin G, which hen pa icipa e in he des uc- ion o he mic obes (55). The NADPH oxidase complex o neu ophils is a highly egula ed mul icomponen sys em, and he absence o his sys- em o an abno mali y in i esul s in disease, such as ch onic g anuloma ous disease (CGD) (33), which is cha ac e ized by a p o ound p edisposi ion o in ec ion. The plasma memb ane and he memb ane o he speci ic g anules o neu ophils con- ain a la ocy och ome b( la ocy och ome b 558 ), which is in- co po a ed in o he memb ane o he phagocy ic acuole (59). Fla ocy och ome bis a he e odime composed o gp91 phox and p22 phox (phox indica es phagocy e oxidase) a a mola a io o 1:1 (68). gp91 phox con ains NADPH and la in adenine dinu- cleo ide binding si es in i s soluble cy osolic domain (61). The ac i a ed la ocy och ome b 558 wo ks as he ca aly ic edox cen e , whe e elec ons a e ans e ed om NADPH o mo- lecula oxygen o gene a e O 2 ⫺ . A numbe o homologues o his p o ein ha e been disco e ed, including Nox1 o Nox5 (10, 45), p138Tox in hy oid (23), enox in he kidney (30), and bohA in plan s (35). p22 phox is equi ed o gp91 phox s abili y and appea s o bind soluble cy osolic ac i a ing ac o s (64). These ac o s include p67 phox , p47 phox , and he small GTP- binding p o ein ac in he GTP-bound o m, which oge he wi h amphiphiles such as sodium dodecyl sul a e (SDS) and memb anes o pu e la ocy och ome induce elec on anspo in i o, in wha is known as he “cell- ee sys em” (13). An- o he p o ein, p40 phox (69), also binds s ongly o p67 phox . p40 phox becomes phospho yla ed when he NADPH oxidase is ac i a ed; howe e , he ole o his phox p o ein is con o e - sial since bo h s imula o y (66) and inhibi o y e ec s (17) on he oxidase ha e been obse ed. These cy osolic p o eins in- e ac wi h each o he (28, 29, 70), wi h ac (19, 20), and wi h he la ocy och ome (15, 16, 21) h ough a numbe o S c homology 3 (SH3), p oline- ich, e a icopep ide epea , and PC mo i s (53). The immune sys em o insec s exhibi s a high deg ee o s uc u al and unc ional simila i y wi h he inna e immune sys em o mammals (41, 57, 67). The insec hemolymph con- * Co esponding au ho . Mailing add ess: Medical Mycology Uni , NICB, Depa men o Biology, NUI Maynoo h, Co. Kilda e, I eland. Phone: 353-1-7083859. Fax: 353-1-7083845. E-mail: ke in.ka anagh @may.ie. † D.B. and E.P.R. con ibu ed equally o his wo k. 4161 ains hemocy es, which unc ion in a manne simila o ha o phagocy es o humans (54). A leas six ypes o hemocy es ha e been iden i ied in insec s such as Galle ia mellonella ( he g ea e wax mo h) (7), and plasma ocy es and g anulocy es a e he mos abundan phagocy ic cell ypes. NADPH oxidase and NO syn hase pa hways ha e been obse ed in he sel -de ense sys em o My ilus gallop o incialis (1), and p oduc ion o ROS has also been de ec ed in hemocy es; e idence o bo h O 2 ⫺ (32) and i s dismu a ion p oduc , H 2 O 2 , has been ound in plasma ocy es o D osophila melanogas e la ae (51) and G. mellonella (62). Simila i ies be ween e eb a e and in e eb a e immune cells ex end o he signal ansduc ion pa hways ha igge hei ac i a ion. In phagocy ic cells o e eb a es he e a e wo pa hways leading o NADPH oxidase ac i a ion, and hese pa hways a e Ca 2⫹ and p o ein kinase C (PKC) dependen o independen and a e ac i a ed by pho bol-12-my is a e-13-ac- e a e (PMA) and concana alin-coa ed pa icles, espec i ely (18, 72). In bi al e mollusks, pho bol es e s ha e been shown o s imula e he hemocy e NADPH oxidase pa hway di ec ly a he le el o PKC (3), whe eas lamina in has been epo ed o in e ac wi h be a-1,3-glucan-binding p o eins on he hemocy e su ace in a h opods and mollusks and o ac i a e he whole oxidase pa hway (63). The simila i ies be ween he oxida i e bu s pa hways o insec hemocy es and mammalian neu ophils aise he possi- bili y ha he complexes ha gene a e he ROS migh also con ain homologous componen s. Recen epo s ha e p o- ided e idence o he in ol emen o p o eins homologous o human neu ophil p47 phox and p67 phox (5, 31), which suppo supe oxide gene a ion by he NADPH oxidase NOX and a e e med NOXO1 and NOXA1. In addi ion, bo h phox p o eins ha e been de ec ed immunologically in cul u ed plan cells and possibly pa icipa e in he soybean cell oxida i e bu s (24). Thus, an examina ion o whe he memb ane and cy osolic phox p o eins a e p esen in insec hemocy es would p o ide a s in- gen es o he homology be ween he insec and mammal oxidase complexes. Mos immunological da a o insec s a e da a o D. melanogas e ; howe e , in G. mellonella, a ious immuno ele an p o ein molecules ha e been desc ibed, and his sys em is being inc easingly used as a model o assessing he i ulence o a ange o mic oo ganisms (41). La ae o G. mellonella ha e been used o e alua e he pa hogenici y o lipopolysaccha ide-de icien mu an s o Pseudomonas ae ugi- nosa (22), and he e is a good co ela ion be ween he i u- lence o P. ae uginosa in Galle ia la ae and he i ulence o P. ae uginosa in mice (40). La ae o G. mellonella ha e been employed o di e en ia e be ween pa hogenic and nonpa ho- genic ungi (56) and yeas s (11), and a s ong co ela ion be- ween he i ulence o Candida albicans mu an s in insec s and he i ulence o C. albicans mu an s in mice has been es ab- lished (8). Gi en he inc eased use o insec s o e alua ing mic obial i ulence (8, 11, 22, 40, 41, 56) and he need o educe he numbe o e eb a es used in such es ing (4), i is essen ial o demons a e ha insec s a e a alid al e na i e o he use o mammals (41). In he wo k p esen ed he e we compa ed he abili ies o hemocy es o G. mellonella and human neu ophils o engul and kill bac e ial and ungal cells. Using immunolog- ical and ma ix-assis ed lase deso p ion ioniza ion— ime o ligh (MALDI-TOF) analyses, we obse ed he p esence o bo h p47 phox and p67 phox p o eins in insec hemocy es, which may pa icipa e in he oxida i e bu s o hese hemocy es. This inding u he s eng hens he simila i ies be ween he oxida- i e bu s pa hways in he wo cell ypes and suppo s he use o G. mellonella as an al e na i e o mammals in in i o pa ho- genici y assays. MATERIALS AND METHODS Chemicals. All chemicals and eagen s we e he highes pu i y and we e pu chased om Sigma Ald ich Chemical Co. L d., Do se , Uni ed Kingdom, unless indica ed o he wise. Mic obial s ains and cul u e condi ions. S aphylococcus au eus (o iginally isola ed om a wound in ec ion in S . James’s Hospi al, Dublin, I eland) was cul u ed in Lu ia-Be ani (LB) b o h (Di co Labo a o ies, Spa ks, Md.) a 37°C and 200 pm in an o bi al incuba o . S ocks we e main ained on LB aga (2% [w / ol] aga [Di co Labo a o ies]). C. albicans MEN (a kind gi om D. Ke - idge, Camb idge, Uni ed Kingdom) was cul u ed in YEPD b o h (2% [w / ol] glucose, 2% [w / ol] Bac o pep one [Di co Labo a o ies], 1% [w / ol] yeas ex ac [Oxoid L d., Basings oke, England]) a 30°C and 200 pm in an o bi al shake . S ocks we e main ained on YEPD aga pla es (YEPD b o h supple- men ed wi h 2% [w / ol] aga ). P epa a ion o human neu ophils and insec hemocy es. No mal human neu ophils we e sepa a ed om blood collec ed in 10-ml hepa inized acuum ubes (BD Vacu aine Sys ems, Plymou h, Uni ed Kingdom) by dex an sedi- men a ion and Ficoll-Hypaque (Axis-Shield PoC AS, Oslo, No way) cen i uga- ion (58). Residual e y h ocy es we e emo ed by hypo onic lysis in wa e o ob ain a suspension con aining 95% neu ophils. The cells we e esuspended in phospha e-bu e ed saline (PBS) (pH 7.4) con aining 5 mM glucose and used immedia ely. Insec hemocy es we e ha es ed om six h-ins a la ae o G. mellonella (Mealwo m Company, She ield, Uni ed Kingdom). La ae we e s o ed in wood sha ings in he da k a 15°C, and insec s weighing be ween 0.2 and 0.4 g we e selec ed. La al hemolymph ( h ee o ou la ae ga e 1 ml) was bled in o 9 ml o insec physiological saline (IPS) (65) con aining 10 mM EDTA and 30 mM sodium ci a e as an icoagulan s. A popula ion consis ing o 100% hemocy es was pelle ed by cen i uga ion a 500 ⫻g o 5 min in a Beckman GS-6 cen i uge a oom empe a u e. The cells we e washed once in IPS and inally esuspended in PBS con aining 5 mM glucose. In i o phagocy osis o C. albicans by human neu ophils and insec hemo- cy es. C. albicans was opsonized using human blood plasma (55) o cell- ee hemolymph dilu ed 1/10 in IPS (1). Phagocy osis was measu ed by incuba ing 1 ⫻ 10 7 neu ophils o 1 ⫻10 7 hemocy es wi h 2 ⫻10 6 C. albicans cells in a apidly s i ed chambe o a Cla k ype oxygen elec ode a 37°C (60). An aliquo was emo ed immedia ely a e addi ion o he yeas cells ( ime ze o) and a e 15, 30, 45, o 60 min o incuba ion. The pe cen ages o iable neu ophils and hemocy es be o e and a e phagocy osis we e asce ained by he ypan blue exclusion me hod, and an app oxima ely 8% educ ion in iabili y was obse ed o e 60 min o incuba ion o bo h cell ypes. Th ee hund ed neu ophils o hemocy es we e examined mic oscopically, and he numbe o phagocy osing cells was asce ained. The mean ⫾s anda d e o numbe o in e nalized yeas cells pe neu ophil o hemocy e was de e mined. In i o killing o S. au eus and C. albicans in neu ophils and hemocy es. Neu ophils (1 ⫻10 7 cells) o hemocy es (1 ⫻10 7 cells) we e incuba ed a 37°C in PBS (600 ␮l) in a apidly s i ed chambe . Se um-opsonized S. au eus (1 ⫻10 7 cells) o C.albicans (2 ⫻10 6 cells) was added, and killing was measu ed as desc ibed p e iously (55) in he p esence o absence o diphenyleneiodonium chlo ide (DPI) (5 ␮M) (12). The pe cen ages o iable cells be o e and a e PMA s imula ion we e ou inely assessed, and he alue was ound o dec ease by app oxima ely 10 and 2% o neu ophils and hemocy es, espec i ely. E e y 15 min an aliquo (100 ␮l) was emo ed om he chambe and placed in o chilled LB b o h o bac e ia o in o YEPD b o h o yeas cells. The cell suspensions we e dilu ed and pla ed on o LB aga o YEPD aga pla es as app op ia e. The esul s we e calcula ed by de e mining he means ⫾s anda d e o s om a leas h ee expe imen s in which colony coun s we e de e mined in iplica e o each sample, and he da a we e exp essed as pe cen ages o he o iginal numbe a ime ze o. Oxygen consump ion and cy och ome c educ ion. Oxygen consump ion was measu ed in a empe a u e-con olled (37°C) chambe ha was a ached o an oxygen elec ode o which sodium di hiona e was added o calib a ion, assuming 4162 BERGIN ET AL. INFECT.IMMUN. ha he no mal oxygen con en o wa e is 230 nmol/ml (60). An aliquo o cells (10 6 o 10 7 cells in 1 ml) was placed in he chambe and allowed o each a s eady s a e o espi a ion. Cells we e s imula ed wi h PMA (1 ␮g/ml) in he p esence o absence o DPI (5 ␮M). P oduc ion o supe oxide by 2 ⫻10 6 hemocy es a e s imula ion wi h PMA (1 ␮g/ml) was measu ed by de e mining he supe oxide dismu ase (SOD) (50 ␮g/ ml)-inhibi able educ ion o cy och ome c(2). Abso bance a 550 nm was e- co ded wi h a Beckman DU640 spec opho ome e o e 30 min. Elec opho esis and immunoblo ing. Samples we e un on SDS-polyac yl- amide gel elec opho esis (PAGE) minigels (8 o 12.5% polyac ylamide), and p o ein p o iles we e isualized by Coomassie blue s aining. Fo Wes e n blo - ing, he p o ein was ans e ed o a ni ocellulose memb ane using a semid y blo e o 1ha 1.4mA/cm 2 . Rabbi and goa polyclonal an ise a aised agains human p40 phox , p47 phox , and p67 phox (69) we e used a a dilu ion o 1/1,000 o 1 h. An ibodies o gp91 phox , PKC ␦(San a C uz Bio echnology, Inc., San a C uz, Cali .), and ac 2 we e used a a dilu ion o 1/1,000 and incuba ed o 16 h a 4°C. The an ibodies o ac 2 we e speci ic and did no c oss- eac wi h ac 1 (San a C uz Bio echnology). Ho se adish pe oxidase-conjuga ed donkey an i- abbi im- munoglobulin G (IgG) (1/5,000 dilu ion; Ame sham Biosciences UK L d., Buck- inghamshi e, England) o abbi an i-goa IgG (Calbiochem, Me ck Biosciences L d., No ingham, Uni ed Kingdom) was used o de ec eac i e bands wi h he enhanced chemiluminescence sys em (Pie ce Bio echnology, Inc., Rock o d, Ill.). F ac iona ion and immunop ecipi a ion o hemocy es and neu ophils. Di- isop opyl luo ophospha e (1 mM) was added o pelle ed hemocy es and neu- ophils (1 ⫻10 8 cells) and incuba ed on ice o 10 min. The cells we e esus- pended in 200 ␮l o b eak bu e [10 mM KCl, 3 mM NaCl, 4 mM MgCl 2 ,10mM pipe azine-N,N⬘-bis(2-e hanesul onic acid) (PIPES); pH 7.2] (55) con aining p o ease inhibi o s (10 ␮g/ml leupep in, peps a in A, ap o inin, and N-␣-p- osyl- L-lysine chlo ome hylke one hyd ochlo ide [TLCK]), sonica ed (Bandelin Sono- puls; Bandelin Elec onics, Ge many) h ee imes o 5 s, and cen i uged a 200 ⫻g o 10 min a 4°C. The pos nuclea supe na an (PNS) was laye ed on op o a discon inuous suc ose g adien (17% and 34% [w /w ] suc ose) and cen i uged a 50,000 ⫻g o 10 min a 4°C. The cy osolic ac ion was emo ed om abo e he 17% (w /w ) suc ose laye , and memb anes we e eco e ed om he op o he 34% (w /w ) suc ose laye . P o eins in hese di e en ac ions we e sepa- a ed by SDS-PAGE and Wes e n blo ed. Fo immunop ecipi a ion, p echilled 2⫻solubiliza ion bu e (2% [ ol/ ol] T i on X-100, 300 mM NaCl, 2% [w / ol] sodium deoxychola e, and 0.2% [w / ol] SDS in b eak bu e wi h p o ease inhibi o s [10 ␮g/ml leupep in, peps a in A, ap o inin, and TLCK]) was added o he PNS o 2 ⫻10 8 cells a a a io o 1:1. P ima y an ibody aised agains he ele an p o eins was added o 1honice, and his was ollowed by incuba ion wi h econs i u ed p o ein A-Sepha ose beads o e nigh a 4°C. The p o ein A-Sepha ose was washed h ee imes in p echilled 1⫻solubiliza ion bu e con aining p o ease inhibi o s and hen boiled o 3 min in a minimum olume o 2⫻Laemmli sample bu e (44). Immuno- p ecipi a es we e subjec ed o SDS-PAGE and s ained wi h Coomassie blue o isualize p o eins in ended o MALDI-TOF mass spec ome ic (MS) analysis. Immuno luo escence mic oscopy. Immuno luo escence mic oscopy was pe - o med as desc ibed p e iously (34). In b ie , hemocy es we e collec ed om Galle ia la ae in IPS and allowed o adhe e o me hanol-cleaned glass co e - slips. The cells we e ixed wi h 4% pa a o maldehyde o 10 min and washed in PBS. Then he cells we e pe meabilized wi h 0.2% ( ol/ ol) T i on X-100 in PBS o 10 min, washed in PBS, and blocked wi h 10 mM NaBH 4 o 1 h. A e washing wi h PBS, he cells we e incuba ed wi h p ima y an ibody agains human p47 phox o p67 phox o e nigh a 4°C. The slides we e washed in PBS and incu- ba ed wi h luo escein goa an i- abbi IgG seconda y an ibody (S a ech Scien- i ic L d., Camb idgshi e, England) o 1 h. The con ols o his expe imen included cells alone and cells exposed only o seconda y an ibody. MALDI-TOF mass spec ome y. Mass spec ome y o ypsin-diges ed p o- eins was pe o med using an E an MALDI-TOF spec ome e (Ame sham Biosciences, F eibu g, Ge many). All 1.5-ml ubes we e siliconized wi h Sigma- co e, and all eagen s we e high-pe o mance liquid ch oma og aphy g ade. Fo pep ide analysis, Coomassie blue-s ained p o ein bands we e excised om an SDS-PAGE gel and diced inely, be o e hey we e incuba ed wi h a minimum olume o ace oni ile (Romil L d., Camb idge, Uni ed Kingdom) o 5 o 10 min. The ace oni ile was eplaced wi h 100 mM NH 4 HCO 3 , and he p epa a ion was incuba ed o 5 min, a e which an equal olume o ace oni ile–100 mM NH 4 HCO 3 (1:1) was added o 15 min. The gel agmen s we e d ied in a acuum cen i uge (He o-Hol en, Jouan No dic A/S). Di hio h ei ol (10 mM) in 100 mM NH 4 HCO 3 (100 ␮l) was added, and each sample was incuba ed o 1ha 37°C, a e which he supe na an was emo ed and he gel pieces we e washed wice (10 min each) wi h 75 ␮l o 100 mM NH 4 HCO 3 ; he inal supe na an was disca ded. Iodoace amide (50 mM) in 100 mM NH 4 HCO 3 (100 ␮l) was added and incuba ed a 37°C o 20 min in he da k, be o e he p epa a ion was washed once wi h 100 mM NH 4 HCO 3 and once wi h ace oni ile–100 mM NH 4 HCO 3 (1:1). Gel pieces we e d ied o 30 min in a acuum cen i uge. Su icien diges- ion bu e (1 ␮g ypsin, 20 ␮l50mMNH 4 HCO 3 , 5 mM CaCl 2 ) was added o co e he gel pieces, which we e incuba ed o e nigh a 37°C. Samples we e cen i uged a 12,000 ⫻g o 10 min, and he supe na an was emo ed. Residual pep ides we e ex ac ed om he gel pieces by adding 50 ␮l ex ac ion bu e (1% [ ol/ ol] i luo oac eic acid, 60% [ ol/ ol] ace oni ile) and incuba ing he p ep- a a ion o 30 min a 37°C be o e cen i uga ion and emo al o he supe na an . All supe na an s we e combined, and he p epa a ion was concen a ed o 50 ␮l in a acuum cen i uge. Samples we e mixed a a 1:1 a io wi h a sa u a ed solu ion o ␣-cyano-4-␣-cyano-4-hyd oxycinnaminic acid in 0.25% ( ol/ ol) i - luo oac eic acid–50% ( ol/ ol) ace oni ile, and 0.3 o 0.5 ␮l was applied o a MALDI a ge slide o MALDI-TOF MS analysis. The sequence o neu ophil p47 phox and p67 phox was ob ained om he Na ional Cen e o Bio echnology In o ma ion websi e (h p://www.ncbi.nlm.nih.go ), and he heo e ical pep ide masses we e de e mined by using MS Diges (h p://p ospec o .ucs .edu/ucs h ml4 .0/msdiges .h m). S a is ical analysis. S a is ical compa isons we e made wi h S uden ’s es using he Sigma S a s a is ical analysis package, e sion 1.00 (SPSS Inc., Chi- cago, Ill.). A P alue o ⬍0.05 was conside ed signi ican . RESULTS In i o phagocy osis by human neu ophils o insec hemo- cy es. Phagocy osis and in e naliza ion o mic obes by neu o- phils in ol e he o ma ion o ilopodia ha con ac and su - ound and engul he mic oo ganism. The a es o phagocy osis o C. albicans by human neu ophils and insec hemocy es we e compa ed (Fig. 1). Neu ophils in e nalized se um-opsonized C. albicans in i o, and 18% ⫾0.88% o he neu ophils had phagocy osed cells a e 30 min o incuba ion. A e 1 h, 72% ⫾2.3% o he neu ophils we e obse ed o ha e phagocy osed cells. The mean numbe o Candida cells in e nalized pe neu- ophil emained cons an o e ime (1.25 Candida cells pe neu ophil) a a Candida cell/neu ophil a io o 1:5. Hemo- cy es phagocy osed hemolymph-opsonized C. albicans, and 21.6% ⫾2.0% o he hemocy es had phagocy osed cells a e 30 min and 66.85% ⫾2.8% o he hemocy es had phagocy- osed cells a e 60 min. No signi ican di e ences in he a es o in e naliza ion we e obse ed be ween neu ophils and he- mocy es (P⫽0.26), and he mean numbe o Candida cells FIG. 1. Kine ics o phagocy osis by neu ophils and hemocy es: in i o phagocy osis o se um- o hemolymph-opsonized C. albicans (2 ⫻ 10 6 cells) by neu ophils (1 ⫻10 7 cells) (A) and hemocy es (1 ⫻10 7 cells) (B). The scale on he igh yaxis indica es he numbe o in e - nalized yeas cells pe 100 phagocy ic cells. O e 60 min, he numbe o phagocy osing neu ophils (ba s) o in e nalized Candida cells (E) was no signi ican ly di e en om he numbe o hemocy es (ba s) (P⫽0.374 and P⫽0.26). Simila esul s we e ob ained in wo inde- penden expe imen s. The da a a e means ⫾s anda d e o s o h ee de e mina ions. VOL. 73, 2005 SUPEROXIDE PRODUCTION IN G. MELLONELLA HEMOCYTES 4163 in e nalized by hemocy es emained cons an (1.18 Candida cells pe hemocy e) a a hemocy e/Candida cell a io o 1:1. In i o killing assays. In o de o de e mine whe he hu- man neu ophils and insec phagocy es kill in e nalized mi- c obes in simila ways, he mic obicidal ac i i ies o pu i ied neu ophils and hemocy es we e de e mined in i o. The ki- ne ics o bac e ial and ungal killing a e shown in Fig. 2 o a a io o neu ophils o hemocy es o bac e ial cells o 1:1 o o a a io o neu ophils o hemocy es o yeas cells o 5:1. Killing o S. au eus by neu ophils occu ed quickly, and o e 60% ⫾ 3.9% o he bac e ia we e killed a e 15 min (Fig. 2A), as p e iously desc ibed (55). Bac e ial killing by hemocy es oc- cu ed mo e slowly; 19% ⫾5.5% o he bac e ia we e killed a e 15 min, and 38.3% ⫾3.5% o he bac e ia we e s ill ali e a e 30 min (Fig. 2B). In e es ingly, he e was a di e ence in he pa e ns o killing o S. au eus and C. albicans.A 15min hemocy es killed C. albicans well and S. au eus weakly, while he opposi e was obse ed o neu ophils (Fig. 2C and D). A e 15 min o incuba ion hemocy es had killed 68.7% ⫾ 7.0% o he yeas cells, and only 6.6% o he cells emained iable a e 60 min. Killing o C. albicans by neu ophils was slowe , and less han 50% o he cells we e killed a e 30 min. The addi ion o DPI (an inhibi o o NADPH oxidase [12]) g ea ly impai ed he abili ies o bo h neu ophils and hemo- cy es o kill S. au eus and C. albicans. Killing o S. au eus by neu ophils and hemocy es was educed by 54% (P⫽0.001) and 34% (P⬍0.001), espec i ely, and killing o C. albicans by neu ophils and hemocy es was educed by 46% (P⫽0.002) and 35% (P⫽0.013), espec i ely, a 60 min compa ed o he con ol. These obse a ions con i m he inding ha oxidase ac i i y is equi ed by bo h neu ophils and hemocy es o de- s oy bac e ia (Fig. 2A and B) and yeas cells (Fig. 2C and D). Howe e , he obse a ion ha killing by cells ea ed wi h DPI was impai ed a he han o ally inhibi ed con i ms he conclu- sion ha bo h oxidase ac i i y and nonoxida i e g anule p o- ease ac ion a e necessa y o gene a ion o su icien mic obi- cidal ac i i y o con ol yeas and s aphylococcal in ec ions in i o (55). DPI inhibi s oxygen consump ion and cy och ome c educ- ion by insec hemocy es. Since DPI has been used o docu- men simila i y be ween he oxida i e bu s complexes o neu o- phils and o he cell ypes (1), he e ec on oxygen consump ion and supe oxide p oduc ion by insec hemocy es was examined. The cha ac e is ics o he bu s o oxygen consump ion by neu ophils and hemocy es a e addi ion o PMA (1 ␮g/ml) a e summa ized in Fig. 3. A e addi ion o PMA o neu o- phils (1 ⫻10 7 cells/ml), he e was a lag o abou 60 s be o e oxygen consump ion commenced, a e which i apidly in- c eased un il i eached a linea a e a e abou 120 s. The lag in oxygen consump ion inc eased o abou 150 s o hemocy es (1 ⫻10 7 cells/ml) o neu ophils (1 ⫻10 6 cells/ml), and 70 ␮M oxygen was consumed a e 600 s. Oxygen consump ion by bo h cell ypes was inhibi ed in he p esence o DPI (5 ␮M) when i was added 3 min p io o he s imulus. The basis o he p oposal ha s imula ed neu ophils gen- e a e O 2 ⫺ is he obse a ion ha hese cells educe cy o- ch ome cin solu ion and ha his educ ion is inhibi ed by SOD (2). Incuba ion o hemocy es wi h cy och ome cand PMA was ound o lead o educ ion o he cy och ome, and he O 2 ⫺ p oduc ion was 0.25 ⫾0.03 ␮M/min/10 6 hemocy es (Fig. 4). The O 2 ⫺ p oduc ion was educed o nea ly con ol alues when 5 ␮M DPI o 50 ␮g/ml SOD was added o he hemocy e suspension. These dec eases in O 2 ⫺ gene a ion we e s a is ically signi ican ( o DPI, P⫽0.003; o SOD, P⫽ 0.005) compa ed o he O 2 ⫺ gene a ion o PMA-ac i a ed cells. Wes e n blo analysis o insec hemocy e NADPH oxidase p o eins. Immunoblo analysis was pe o med o cha ac e ize FIG. 2. Kine ics o bac e ial and ungal killing by human neu o- phils and insec hemocy es. Se um- o hemolymph-opsonized S. au eus cells (1 ⫻10 7 cells) (A and B) o C. albicans cells (2 ⫻10 6 cells) (C and D) we e exposed o 1 ⫻10 7 neu ophils (A and C) o hemocy es o G.mellonella (B and D). Killing was also measu ed in he p esence (䊐 and ■) and in he absence (‚and Œ) o DPI (5 ␮M) added 3 min p io o ini ia ion o phagocy osis. Mic obial su i al is exp essed as a pe - cen age o he con ol a ime ze o, and he da a a e means ⫾s anda d e o s. The da a a e om a ep esen a i e expe imen pe o med in iplica e. FIG. 3. Inhibi ion o he hemocy e oxida i e bu s by DPI. The a e o oxygen consump ion by neu ophils (1 ⫻10 7 cells [F]o 1⫻10 6 cells [‚]) was compa ed o he a e o oxygen consump ion by insec hemocy es (1 ⫻10 7 cells) (䊐) a e s imula ion wi h PMA (1 ␮g/ml). Oxygen consump ion by neu ophils (1 ⫻10 6 cells) (E) and hemocy es (1 ⫻10 7 cells) (■) was also measu ed in he p esence o DPI (5 ␮M) added 3 min p io o elici a ion. The da a a e means o h ee ials ca ied ou on sepa a e days. 4164 BERGIN ET AL. INFECT.IMMUN. he componen s o he insec hemocy e NADPH oxidase sys- em. Human neu ophil memb anes and cy osol ac ions (Fig. 5A) we e employed. Hemocy e memb anes we e no obse ed on he con en ional 17% and 34% (w /w ) suc ose g adien , and he e o e, o al hemocy e PNS, con aining a mix u e o cellula memb anes and cy osol, was employed. By Wes e n blo ing, abbi polyclonal an ibodies agains human p40 phox , p47 phox , p67 phox (69), and gp91 phox p o eins we e used o iden- i y he ele an p o eins in hemocy e lysa e. In he neu ophil ac ion, gp91 phox mig a ed on SDS-PAGE gels as a b oad band, an elec opho e ic p ope y cha ac e is ic o glycop o- ein (38), a an appa en molecula mass o 90 kDa (Fig. 5B). Using an ibodies o human gp91 phox , an immuno eac i e band was de ec ed in he insec hemocy e PNS a a molecula mass o abou 77 kDa, possibly sugges ing ha his an ibody ecog- nizes a egion o gp91 phox ha has emained cons an , al hough he mass o he p o ein in humans and he mass o he p o ein in insec s a e di e en . On Wes e n blo s o he essen ial cy osolic componen s o he NADPH oxidase, p67 phox and p47 phox s ained a single band in samples o human neu ophil cy osol, which mig a ed like immuno eac i e bands in insec hemocy e PNS (Fig. 5C and D). These esul s indica e ha immunologically ela ed p o- eins ha ing he same molecula masses as p47 phox and p67 phox a e p esen in insec phagocy ic cells. Only one o he phox p o- eins, p40 phox , was no de ec ed in he insec hemocy es (Fig. 5E). Ano he cy osolic componen which is equi ed o comple- men p47 phox and p67 phox and memb anes in he cell- ee assay is he small GTP-binding p o ein ac. ac 2 is exp essed p e- dominan ly in myeloid cells and is he physiologically ac i e molecule in neu ophils (42). Using polyclonal an ibodies spe- ci ic o ac 2, a single band (Fig. 5F) was de ec ed wi hin neu- ophil cy osol and insec hemocy e PNS, again indica ing he p esence o an immunologically ela ed p o ein. Ac i a ion o he NADPH oxidase is associa ed wi h phos- pho yla ion o p47 phox , p67 phox , and p40 phox . A numbe o kinases ha e been implica ed in he ac i a ion o he oxidase, including PKC (14, 25). Using abbi polyclonal an ibodies o PKC ␦, we success ully iden i ied his kinase in cy osol o hu- man neu ophils and PNS o insec hemocy es as an immuno- eac i e band a 80 kDa (Fig. 5G). To u he s eng hen he e idence ha O 2 ⫺ p oduc ion in insec phagocy es is associa ed wi h p o eins which a e homol- ogous o componen s o he NADPH oxidase complex o hu- man neu ophils, luo escence mic oscopy was used o de e - mine he dis ibu ion o p47 phox and p67 phox in hemocy es. Cells ha had a ached o a glass co e slip and sp ead we e ex- FIG. 4. E ec o an NADPH oxidase inhibi o on in i o PMA- s imula ed gene a ion o O 2 ⫺ in hemocy es o G. mellonella. The p o- duc ion o O 2 ⫺ by uns imula ed hemocy es (1 ⫻10 6 cells/ml) (Con ol) and PMA (1 ␮g/ml)-ac i a ed cells was measu ed using he educed cy och ome cassay. P oduc ion o O 2 ⫺ by hemocy es was inhibi ed in he p esence o DPI (5 ␮M) and sca enged in he p esence o SOD (50 ␮g/ml). FIG. 5. Immunoblo ing o hemocy es o G.mellonella wi h an i- bodies o human neu ophil phox p o eins. (A) Coomassie blue-s ained SDS-PAGE gel (12.5% polyac ylamide) o neu ophil cy osol (lane 1) and memb anes (lane 2). The p o ein p o ile o neu ophil PNS (lane 3) was compa ed o he p o ein p o ile o PNS o insec hemocy es (lane 4). The posi ions o molecula weigh ma ke s a e indica ed on he le . (B) Elec opho e ically sepa a ed neu ophil memb ane p o- eins and insec hemocy e PNS ans e ed o ni ocellulose and p obed wi h abbi an ise um o gp91 phox . The esul s show ha he e was an immuno eac i e band a 90 kDa o neu ophil memb anes and a band a 77 kDa o insec hemocy e PNS. (C o G) Neu ophil cy osol and insec hemocy e PNS p obed wi h abbi an ise a o p67 phox (C), p47 phox (D), p40 phox (E), ac 2 (F), and PKC ␦(G). The esul s e- ealed immunologically ela ed p o eins ha ing he same molecula mass o all p obed p o eins excep p40 phox , which was no de ec ed in insec hemocy es. VOL. 73, 2005 SUPEROXIDE PRODUCTION IN G. MELLONELLA HEMOCYTES 4165 amined. The phox p o eins we e loca ed p edominan ly h ough- ou he cy osol o he cell and in he pe inuclea egion (Fig. 6). A simila dis ibu ion o p47 phox and p67 phox in uns imula ed neu ophils has been desc ibed p e iously (34). Immunop ecipi a ion o p47 phox and p67 phox om hemo- cy es and neu ophils. To de e mine he iden i ies o he im- muno eac i e bands iden i ied by Wes e n blo ing, immuno- p ecipi a ion was pe o med using abbi polyclonal an ibodies o p67 phox and p47 phox . Coomassie blue-s ained gels a e shown in Fig. 7A, in which insec p o eins ha ing molecula masses equi alen o hose o p47 phox and p67 phox a e isible. P o eins we e ans e ed o a ni ocellulose memb ane and p obed wi h goa polyclonal an ibodies o he ele an p o eins. Figu e 7B shows ha bo h neu ophils and hemocy es ga e posi i e signals wi h goa an ibodies aised agains p47 phox and p67 phox . P o ein bands om he immunop ecipi a ion we e cu om he gel and analyzed by MALDI-TOF MS. MALDI-TOF analysis o insec p o eins. The immunop e- cipi a ed bands ha we e c oss- eac i e wi h he ele an an- ibodies o human neu ophil p47 phox and p67 phox we e ex- cised om he Coomassie blue-s ained gels and diges ed wi h ypsin o MALDI-TOF analysis. In he MALDI-TOF spec- um, pep ides we e obse ed ha had monoiso opic (m/z ol- e ance, ⬍1 Da) alues e y simila o hose o a heo e ical diges o p47 phox and p67 phox o human neu ophils. In he yp ic diges o he insec p o ein band ha was immuno eac i e wi h an ibodies o p67 phox , 10 o 206 pep ides (5%) we e iden i ied ha we e simila o pep ides o p67 phox o human neu ophils (Fig. 8A). This esul ed in o al p o ein co e age o 26%. Fou o hese pep ides we e loca ed in he conse ed domains o p67 phox , wo pep ides we e loca ed in he i s SH3 domain, and wo o he pep ides we e loca ed in PB1 FIG. 6. Dis ibu ion o p47 phox and p67 phox homologues in un- s imula ed hemocy es: dis ibu ion o 47-kDa (A) and 67-kDa (B) in- sec p o eins in hemocy es adhe ing o glass slides. The dis ibu ion o he p o eins was p edominan ly pe inuclea (indica ed by an a ow) and h oughou he cy osol. (magni ica ion, ⫻400). FIG. 7. Immunop ecipi a ion o p67 phox and p47 phox om human neu ophils and hemocy es o G.mellonella. Pos nuclea supe na an s we e p epa ed om 2 ⫻10 8 neu ophils o hemocy es and solubilized in bu e as desc ibed in he ex . (A) Immunop ecipi a ion was ca ied ou using abbi polyclonal se a agains p67 phox and p47 phox , he p ep- a a ion was esuspended in 60 ␮l o Laemmli sample bu e , and 25 ␮l was analyzed by SDS-PAGE and Coomassie blue s aining. The posi- ions o immunop ecipi a ed p67 phox and p47 phox om neu ophils (N) and hemocy es (H) a e indica ed by he a ows. (B) Immunop e- cipi a ion p epa a ions we e Wes e n blo ed using goa polyclonal se a agains p67 phox and p47 phox . In wo o he expe imen s simila immunop ecipi a ion esul s we e ob ained. 4166 BERGIN ET AL. INFECT.IMMUN. and he second SH3 domain (Fig. 8B). Se en o 152 pep ides we e ob ained om he yp ic diges o he insec p o ein immuno eac i e wi h an ibodies o p47 phox , yielding 16% p o- ein co e age (Fig. 8C). One o he pep ides iden i ied was ound o lie a he s a o he highly conse ed PX domain o p47 phox (Fig. 8D). DISCUSSION Insec s a e a e y success ul g oup o animals and ha e colonized almos all habi a s on he plane excep he seas. Thei abili y o colonize such a di e se ange o en i onmen s can be explained pa ially by hei e icien immune esponse. The e is a s ong s uc u al and unc ional simila i y be ween he insec immune sys em and he inna e immune sys em o mammals (41, 57, 67). This has been exploi ed in ecen yea s by employing insec s o assessing he i ulence o a ange o pa hogenic bac e ia and ungi. La ae o G. mellonella ha e been used o e alua ing he i ulence o mu an s o P. ae ugi- nosa (22) and o di e en ia ing be ween pa hogenic and non- pa hogenic yeas species (11). A s ong co ela ion has been es ablished be ween he i ulence o bac e ia (40) and mu an s o C.albicans in G. mellonella and he i ulence o hese o ganisms in mice (8). Since he immune esponse o insec s is simila o he inna e immune esponse o mammals, insec s ha e been ecognized as alid al e na i es o in i o es ing o mic obial mu an s and may be use ul o assessing he e icacy o new an i ungal and an ibac e ial d ugs (36). Phagocy osis equi es sequen ial signal ansduc ion e en s, and he ecep o s on he su ace o hemoly ic plasma ocy es and g anulocy es a e simila o ecep o s on mammalian phagocy es (67). Immuni y- ela ed p o eins and mechanisms ha a e simila in insec s and mammals ha e been iden i ied, FIG. 8. Ma ching pep ides o hemocy e 67-kDa and 47-kDa p o eins o human neu ophil phox p o eins. (A) Ma ching pep ides o hemocy e 67-kDa p o ein o human p67 phox ha s ongly eac ed wi h bo h abbi and goa an i-human p67 phox in immunop ecipi a ion. (B) Regions o p67 phox in ol ed in p o ein-p o ein in e ac ions wi h ma ching pep ides o hemocy e 67-kDa p o eins (indica ed by a ows). (C) Ma ching pep ides o hemocy e 47-kDa p o ein o human p47 phox ha s ongly eac ed wi h bo h abbi and goa an i-human p47 phox . (D) Regions o p47 phox in ol ed in p o ein-p o ein in e ac ions. Ma ching pep ides o hemocy e 47-kDa p o ein o neu ophil p47 phox in he PX domain is indica ed by an a ow. VOL. 73, 2005 SUPEROXIDE PRODUCTION IN G. MELLONELLA HEMOCYTES 4167 and he simila i ies include he ema kable s uc u al and unc- ional simila i ies be ween he sys ems media ing D osophila Toll and mammalian in e leukin-1 ecep o -media ed signal- ing (47). A u he a ea o de ense whe e di ec compa isons can be d awn is in he syn hesis o a b oad ange o an imic o- bial pep ides which play a c ucial ole in comba ing in ec ion, and simila classes o p o eins ha e been ound in e eb a es and in e eb a es (67). The p oposed oxic p oduc s o NADPH oxidase in neu o- phils include supe oxide, which quickly dismu a es o o m hyd ogen pe oxide. The pe oxide and supe oxide can eac o p oduce hyd oxyl adicals in he p esence o me al ions, and hyd ogen pe oxide can also se e as a subs a e o myelope - oxidase-media ed oxida ion o halides (37). The e has been an accumula ion o e idence ha ROS a e p oduced in he he- molymph and hemocy es o many insec s. The in i o gene - a ion o ROS by hemocy es was s udied by elec on spin es- onance spec oscopy (62) and ni oblue e azolium educ ion (32). The ROS de ec ed o da e include ni ic oxide, supe ox- ide, and hyd ogen pe oxide, and he la e wo ROS a e e- qui ed o he p oduc ion o he highly eac i e hyd oxyl ad- ical ei he h ough he Habe -Weiss eac ion o Fen on’s eac ion (37). In his s udy he p oduc ion o ROS by hemo- cy es was u he cla i ied and ex ended o include he quan- i ica ion o oxygen consump ion and supe oxide p oduc ion by ac i a ed hemocy es. Incuba ion o he hemocy es wi h PMA (1 ␮g/ml) o 10 min esul ed in a signi ican inc ease in he consump ion o oxygen (70 ␮M) compa ed o ha in unac i- a ed con ol cells and cells exposed o DPI p io o s imula- ion. The p ocess o phagocy osis in insec hemocy es is no ully unde s ood, bu plasma ocy es and hemocy es ha e been shown o ha e ecep o s simila o hose o mac ophages (27). Se e al s udies ca ied ou wi h bi al e hemocy es ha e shown he abili y o hese cells o phagocy ose immedia ely a e con- ac wi h a a ie y o nonsel ma e ials, including bac e ia (48, 52) and yeas cells (49). Ano he s udy ca ied ou wi h he same cell ype demons a ed ha phagocy osis is associa ed wi h lysosomal NADPH oxidase ac i i y o memb anes (71). In ou s udy he e was a ema kable simila i y be ween he a es o phagocy osis o opsonized C. albicans by neu ophils and hemocy es (Fig. 1). Phagocy osis had a clea ime-dependen index, and he pe cen ages o cells ha phagocy osed opso- nized yeas cells in 60 min we e compa able o he wo im- mune cell ypes. Fu he mo e, signi ican educ ions in supe - oxide p oduc ion (Fig. 4) and mic obial killing (Fig. 2) we e obse ed when he hemocy es we e incuba ed wi h DPI. These obse a ions con i m he sugges ion o p e ious wo ke s (55) ha phagocy osis o o eign ma e ials by hemocy es s imula es he espi a o y bu s pa hway in ol ing NADPH oxidase ac i - i y and illus a e ha supe oxide p oduc ion is necessa y o e icien mic obial killing by bo h neu ophils and hemocy es. The unc ional simila i ies be ween hemocy es and phago- cy ic neu ophils p omp ed us o sea ch o componen s o he NADPH oxidase o neu ophils in insec hemocy es. In a p e- ious in es iga ion, he p esence o p47 phox and p67 phox in plan cells was examined (24), and he esul s indica ed ha he e was a di ec co ela ion be ween he p esence o he wo phox p o eins and a quan i a i e oxida i e bu s . In ou s udy an immunological app oach was aken, in which we p obed hemocy e cell lysa es wi h polyclonal an ibodies aised agains he human o ms o gp91 phox , p40 phox , p47 phox , and p67 phox . In he immunoblo s shown in Fig. 5 a s ong b oad band o gp91 phox in insec hemocy es is isible, bu i mig a ed on SDS gels a a lowe molecula mass, app oxima ely 70 kDa. The e a e h ee main subg oups o he NADPH oxidase am- ilies (NOXs), and he gp91 phox sub amily consis s o Nox1, gp91 phox , Nox3, and Nox4, all o which ha e molecula masses o app oxima ely 65 kDa, a molecula mass simila o ha o he immuno eac i e p o ein o insec hemocy es. I his p o ein has a mechanism simila o ha o gp91 phox , we would expec ha i would anspo elec ons ac oss memb anes, wi h oxy- gen as he p obable ecipien , esul ing in he o ma ion o supe oxide. p67 phox is a 59.7-kDa p o ein and is ich in mo i s in ol ed in p o ein-p o ein in e ac ions (Fig. 8A). This p o ein is abso- lu ely equi ed o induce elec on anspo h ough la ocy o- ch ome, and a de ec in he gene is known o gi e ise o he au osomal o m o CGD (33). As de e mined by Wes e n blo - ing (Fig. 5C) and immunop ecipi a ion (Fig. 7), he an ibodies aised agains neu ophil p67 phox eac ed wi h a p o ein ha was a simila size in he insec hemocy e lysa es. MALDI-TOF analysis o his p o ein ga e ise o a numbe o pep ides, some o which ell wi hin he wo SH3 and PB1 domains o neu o- phil p67 phox . The impo ance o hese domains is appa en , as he C- e minal SH3 domain o p67 phox is equi ed o binding o he p oline- ich mo i o p47 phox (28, 46) and p67 phox binds o p40 phox h ough he PB1 domain (50). Pep ides o hese binding mo i s we e ound in he insec 67-kDa p o ein, and by analogy wi h p67 phox , i would be expec ed ha his p o ein has he abili y o in e ac wi h o he p o eins, al hough he main binding pa ne , p40 phox , was no de ec ed in he insec cell lysa e. The equi emen o p40 phox in he neu ophil NADPH oxidase emains obscu e. S udies done in se e al di e en lab- o a o ies ha e sugges ed bo h nega i e (17) and posi i e eg- ula o y oles (66) and, mo e ecen ly, a ole in he ansloca- ion o p47 phox and p67 phox o he acuola memb ane upon ac i a ion (43). Like p67 phox , p47 phox is essen ial o unc ional NADPH oxidase ac i i y and has p e iously been shown o be missing in mos cases o au osomal ecessi e CGD (33). p47 phox has a molecula mass o 44.6 kDa, and phospho yla ion o his p o- ein has been co ela ed wi h ac i a ion o he oxidase and occu s a he C- e minal end, in which 9 o 10 se ine phos- pho yla ion si es ha e been iden i ied by phosphopep ide se- quencing and si e-di ec ed mu agenesis (26). Se e al kinases a e known o phospho yla e p47 phox in i o; among hese kinases is p o ein kinase C, which was success ully iden i ied in insec hemocy es lysa e (Fig. 5G). The esul s o Wes e n blo - ing (Fig. 5D) and immunop ecipi a ion analyses (Fig. 7) e- ealed he p esence o a p o ein immunologically simila o p47 phox o neu ophils in he hemocy es o G. mellonella. As de e mined by immunohis ochemis y, he insec 47-kDa p o- ein was ound h oughou he cy oplasm and he pe iphe y o he cell (Fig. 6). This dis ibu ion o p47 phox has p e iously been obse ed in unac i a ed neu ophils, and a possible eg- ula o y ole o p47 phox in he eo ganiza ion o he cy oskel- e on accompanying supe oxide gene a ion has been sugges ed (34). MALDI-TOF analysis o he insec 47-kDa p o ein ga e ise o a numbe o pep ides which co e ed 16% o neu ophil 4168 BERGIN ET AL. INFECT.IMMUN. p47 phox (Fig. 8C and D), including a po ion o he p47 phox PX domain (in ol ed in phosphoinosi ide binding). The minimal equi emen o in i o cell- ee oxidase ac- i i y has been ecognized as lipid- econs i u ed cy och ome (neu ophil memb ane ex ac o ecombinan ), p47 phox , p67 phox , and ac (in he GTP-bound s a e) oge he wi h NADPH and anionic amphiphiles such as a achidonic acid o SDS (9). ac 2 is he p edominan o m in neu ophils and was also ound in insec hemocy es (Fig. 5F). The e o e, in his s udy we iden i ied immunologically ela ed p o eins (gp91 phox , p67 phox , p47 phox , and ac) o all he key p o eins ha a e equi ed in he cell- ee oxidase sys em. The indings p esen ed in his pape illus a e he simila i ies a he cellula le el be ween he human inna e immune e- sponse and he insec immune esponse. Gi en he c i ical ole o he inna e immune esponse in p o ec ing mammals om mic obial in ec ion and he high deg ee o s uc u al and unc- ional simila i y be ween he mammalian and insec inna e immune esponses, s udying he insec esponse o in ec ion can p o ide da a compa able o da a which may be ob ained using mammals (41). La ae o G. mellonella ha e been used o e alua e he i ulence o a ange o bac e ia and ungi, and mo e ecen ly o he insec species such as D.melanogas e ha e been used o e alua e he pa hogenici y o Aspe gillus species (6). 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