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). This wo k suppo s he alidi y o using insec s o ob ain
da a compa able o he da a ob ained by using mammals o
assessing he i ulence o mic obial pa hogens.
ACKNOWLEDGMENTS
This wo k was suppo ed by unding om he Highe Educa ion
Au ho i y h ough PTRLI 3.
We g a e ully acknowledge Alan Mu phy o assis ance wi h he
MALDI-TOF MS analysis.
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