www.sciencemag.org/cgi/content/full/310/5749/848/DC1

Supporting Online Material for

A Direct Role for Dual Oxidase in Drosophila Gut Immunity

Eun-Mi Ha, Chun-Taek Oh, Yun Soo Bae, Won-Jae Lee*

*To whom correspondence should be addressed. E-mail: lwj@ewha.ac.kr

Published 4 November 2005, Science 310, 848 (2005) DOI: 10.1126/science.1117311

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S7 References

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Supporting Online Material.

A Direct Role for Dual Oxidase in Drosophila Gut Immunity.

Eun-Mi Ha, Chun-Taek Oh, Yun Soo Bae, Won-Jae Lee

Materials and Methods

Expression and purification of recombinant Peroxidase-homology domain (PHD)

of dDuox.

The PHD of dDuox (encoding amino acids 1 to 566 of dDuox) was subcloned into

pMT/V5-His vector (pMT/V5-His-PHD), under the control of the metallothionein

promoter (Invitrogen), to generate COOH terminal V5-His-tagged PHD. Drosophila

immunocompetent Schneider 2 (S2) cells (ATCC CRL-1963) were maintained exactly

as described previously (S1). Transfection was performed according to standard CaPO4

protocols (S2) and transfected cells were selected with 300 µg/ml of hygromycin-B

(Invitrogen, USA) for 6 weeks as described previously (S3). Expression was induced in

cells by addition of CuSO4 to the culture medium at a final concentration of 500 µM.

Cells were induced for 48 hr before use. To purify the recombinant PHD from the

culture medium of S2 cells stably expressing the recombinant PHD, nickel-

nitrilotriacetic acid (Ni+-NTA)-agarose resin was used according to manufacturer’s

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protocols (Qiagen, Chtsworth, CA, USA). The PHD protein was eluted from the resin

with 250 mM of imidazole, and dialyzed with 50 mM sodium phosphate buffer (pH 7).

Constructs and fly strains.

A 510bp PCR fragment (encoding amino acids 976 to 1145 of dDuox) and a 504

bp PCR fragment (encoding amino acids 547 to 714 of dNox) were used to generate the

dDuox-RNAi and dNox-RNAi construct, respectively. In order to eliminate potential

problems with cross-silencing, we verified that these dsRNAs had no significant perfect

matches of 19 to 21 nucleotides to other sequences in the fly genome by using BLAST

analysis. These head-to tail inverted repeats were subcloned into the pUAST vector (S4)

to yield the pUAST-dDuox-RNAi and pUAST-dNox-RNAi constructs. In these RNAi

constructs, the hairpin loop sequence between the head-to-tail inverted repeats was

replaced with an intronic spacer to maximize target gene silencing (S5). The human

Duox1 and 2 were subcloned into the pUAST vector to yield the pUAST-hDuox1 and

pUAST-hDuox2, respectively. Full length of dDuox and dDuox-∆PHD mutant lacking

PHD (accomplished by deleting the region corresponding amino acids 1 to 566 and by

adding a signal peptide in the NH2 terminus) were also subcloned into the pUAST

vector to yield pUAST-dDuox and pUAST-dDuox-∆PHD, respectively. These constructs

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were then used to generate transgenic animals by P element-mediated transformation.

These constructs were microinjected into w1118 -expressing embryos (S6). IRC-RNAi

flies were described (S7). The cad-GAL4 flies expressing GAL4 mainly in the intestine

were described (S8) and the c564-GAL4 flies, which express GAL4 in the fat body and

hemocytes, were described (S9, S10). Da-GAL4 flies expressing GAL4 in the whole

body were described (S11). The DD1 (drosomycin-GFP, diptericin-lacZ) flies were

described (S12)

Peroxidase activity assay.

Various amounts of recombinant PHD (4, 8 and 16 µg) were incubated with the

reaction mixture (100 µl), consisting of H2O2 (0.01%) and 3, 5, 3’, 5’-

Tetramethylbenzidine (0.2 mg/ml) in citric acid buffer (pH 5.5) as described (S13).

After incubation at room temperature for 20 min, the reaction was finished by the

addition of 100 µl of 2 M H2SO4. The absorbance was measured at 450 nm (reference:

620 nm). Human leukocyte myeloperoxidase (sigma) was used as a positive control.

One unit will produce an increase in absorbance of 1.0 per minute at pH 7.0 and 25 °C,

calculated from the initial rate of reaction using guaiacol as substrate. Results were

expressed as the mean and standard deviations of three different experiments.

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Measurement of total in vivo ROS.

The intestine of individual adult fly was rapidly hand-dissected in PBS

containing aminotriazol (2 mg/ml). The dissected intestines were cut into small pieces

(~ 2 mm) and pooled in 50 µl of H2O containing aminotriazol (2 mg/ml). For each

measurement, five or ten intestines were used. The sample was centrifuged for 5 min at

3000 x g. The resultant supernatant (40 µl) was further used for the colorimetric

quantitative determination of diffused ROS. The ferric–xylenol orange assay in the

presence of 100 mM sorbitol was used as describe previously (S14). The change in

absorbance of xylenol orange at 560 nm was determined. Results were expressed as the

mean and standard deviations of three different experiments.

Measurement of in vitro superoxide-generating activity.

Intestines from control flies or Duox-RNAi flies were dissected and lysed by

sonication. The sonicate was centrifuged for 10 min at 5,000 x g. The resultant

supernatant was further centrifuged for 1 hr at 100,000 x g. The pellet was used as the

membrane fraction for the measurement of superoxide production as described

previously (S15). Membrane fraction (50 µg of protein) were incubated in triplicate with

enhanced luminol-based substrate, lucigenin (500 µM) and 2.5 mM NADH in PBS

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buffer, pH 7.4, at 37°C, and luminescence was measured for 30 min using a

luminometer (Microlumat Plus LB96V, Berthold Tech.). In some experiment, CaCl2

were added in the reaction mixture at final concentration of 10-5~10-9 M. To inhibit

superoxide-generating activity, DPI and ethylene glycol-bis(β-aminoethylether)-

N,N,N’,N’-tetraacetic acid (EGTA) were added in the reaction mixture at the final

concentration of 10 µM and 10 mM, respectively. Results were expressed as the mean

and standard deviations of three different experiments.

In vitro chloride-dependent microbicidal activity assay.

The halide dependence of myeloperoxidase activity was tested essentially

according to the method of Klebanoff and Shepard (S16). Microbial cells (at a density

of 1×106 ampicillin-resistant E. coli) were mixed with different amounts of recombinant

PHD (8 or 16 µg) in 0.02 M PBS (pH 7.0) containing 300 µM H2O2 and 100 mM NaCl.

To see the effect of chloride, microbicidal assay was also performed in the presence or

absence of 100 mM NaCl. In a negative control experiment, PHD and/or H2O2 were

omitted in the assay mixture. After 1hr of incubation at 37 ºC, the suspensions were

serially diluted and spread onto Luria-Bertani plates containing ampicillin (100 µg/ml).

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The numbers of colony forming units (CFUs) were calculated. Results were expressed

as the mean and standard deviations of three different experiments.

In vivo bacterial persistence assay.

Natural infection was induced using spectinomycin-resistant Ecc15-GFP. Ecc15-

GFP persistence was measured by plating appropriate dilutions of the homogenates of

five surface-sterilized intestines, collected at different times after infection. The

microbes were grown on LB agar plates containing spectinomycin (100 µg/ml). The

numbers of colony-forming units (CFUs) were then obtained at each time point after

infection. Results were expressed as the mean and standard deviations of three different

experiments.

Protein carbonylation and lipid peroxidation assay

Protein carbonylation and lipid peroxidation constitute the principal biochemical

consequences of cellular oxidative attack (S17, S18). Therefore, we have assessed the

levels of oxidative damage in ingested bacteria recovered from the intestines of either

the wild type flies or the dDuox-RNAi flies. To compare the levels of oxidative stress of

ingested microbes, adult male flies (control flies and dDuox-RNAi flies) were naturally

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infected with Ecc15-GFP. At 24 hr post-infection, ingested Ecc15-GFP bacteria were

recovered from dissected intestines.

Total bacterial lysates (100 µg protein) were subjected to protein carbonylation

assay and lipid peroxidation assay. To measure the level of protein carbonylation,

lysates were denatured and derivatized in 3% SDS, 10 mM 2,4-dinitrophenylhydrazine

(DNPH) dissolved in 10% trifluoroacetic acid (tissue:DNP; 1:3 ratio). After incubation

for 30 min at room temperature with occasional stirring, an equal volume of

neutralization solution (2 M Tris, 30% glycerol) was added. DNP-derivatized protein

samples were analyzed by Western blot analysis and DNP-reactive carbonylated

proteins were detected with rabbit anti-DNP antibody (Chemicon, USA). The same blot

was also probed with anti-GFP antibody for a loading control of bacterial protein

extracts

To measure the level of lipid peroxidation, spectrophotometric assay for

malondialdeyde (MDA) was performed essentially as described in Esterbauer et al.(19)

by using BIOXYTECH® MDA-586™ (OxisResearch™, Portland, USA). The lipid

peroxidation level in the total bacteria extract (100 µg) was then analyzed by

determination of MDA concentration by reacting a chromogenic reagent, N-methyl-2-

phenylindole (NMPI) at 45 ºC. One molecule of MDA reacts with 2 molecules of NMPI

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to yield a stable carbocyanine dye with a maximum absorption at 586 nm. The amount

of MDA in tissue lysates was determined by a standard curve prepared with serial

dilutions of MDA. Results were expressed as the mean and standard deviations of three

different experiments.

Microbial Infection

Natural microbial infection was performed essentially as described previously

(S7). Briefly, adult flies (age: 3-4 day) were dehydrated for 2 hr without food and then

transferred into a vial containing filter paper hydrated with 5 % sucrose solution

containing concentrated microbe solution (~1010 CFUs/ml). Exponential microbial

culture (OD600=1.0) was used for all experiments. Filter papers were changed everyday.

The flies fed sucrose only were used as a control. All animals were incubated at 25 °C.

In all cases, survival in three or more independent cohorts of about 25 flies each was

monitored over time. Results are expressed as the means and ± S.D. (p < 0.05).

Microorganisms used in this study were Ecc15, Escherichia coli, Salmonella

typhimurium, Micrococcus luteus, Saccharomyces cerevisiae. Ecc15 strain can colonize

the apical side of the Drosophila gut epithelium and activate immune system (S20).

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Green fluorescence protein-tagged Ecc15 (Ecc15-GFP) strain was used to examine the

in vivo bacterial persistence in the intestine.

Real-Time PCR analysis

To quantify the amount of gene expression, fluorescence real-time PCR was

performed with the double-stranded DNA dye, SYBR Green (Perkin Elmer, Boston

MA). Primer pairs for dDuox (sense, 5’-TAG CAA GCC GGT GTC GCA ATC AAT-

3’; antisense, 5’-ACG GCC AGA GCA CTT GCA CAT AG-3’), dNox (sense, 5’-TAG

CCG AGC CGA ACA GGG TCA ACT-3’; antisense, 5’-GAG CGC AGG AAT GTG

GGT CGT C-3’), diptericin (sense, 5’-GGC TTA TCC GAT GCC CGA CG-3’;

antisense, 5’-TCT GTA GGT GTA GGT GCT TCC C-3’) and control Rp49 (sense, 5’-

AGA TCG TGA AGA AGC GCA CCA AG-3’; antisense, 5’-CAC CAG GAA CTT

CTT GAA TCC GG-3’) were used to detect target gene transcripts. SYBR Green

analysis was performed on an ABI PRISM 7700 system (PE Applied Biosystems)

according to manufacturer’s instructions. All samples were analyzed in triplicate, and

the levels of detected mRNA were normalized to control Rp49 mRNA values. The

normalized data were used to quantify the relative levels of a given mRNA according to

cycling threshold analysis (S21). The target gene expression in the uninfected wild type

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flies (0 hr) was taken arbitrarily as 100, and the results were presented as relative

expression levels.

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Supporting Text.

The genotypes of the flies used in this study.

The genotypes of the flies used in the Fig. 1B were as follows: Cont. (cad-

GAL4/+); dNox-RNAi (whole body) (Da-GAL4/UAS-dNox-RNAi); dDuox-RNAi

(whole body) (Da-GAL4/UAS-dDuox-RNAi); dDuox-RNAi (intestine) (cad-GAL4/+;

UAS-dDuox-RNAi/+); dDuox-RNAi (fat body/hemocytes) (c564-GAL4/+; UAS-dDuox-

RNAi/+).

The genotypes of the flies used in the Fig. 1C were as follows: Cont. (Da-

GAL4/+); dDuox-RNAi (UAS-dDuox-RNAi/Da-GAL4).

The genotypes of the flies used in the Fig. 1D were as follows: Cont. (Da-

GAL4/+); dDuox-RNAi (UAS-dDuox-RNAi/Da-GAL4); IRC-RNAi (UAS-IRC-RNAi/+;

Da-GAL4/+); dDuox-RNAi + IRC-RNAi (UAS-IRC-RNAi/+; UAS-dDuox-RNAi/Da-

GAL4).

The genotypes of the flies used in the Fig. 1E were as follows: Cont. (cad-

GAL4/+); dDuox-RNAi (cad-GAL4/+; UAS-dDuox-RNAi/+); IRC-RNAi (UAS-IRC-

RNAi/cad-GAL4); dDuox-RNAi + IRC-RNAi (UAS-IRC-RNAi/cad-GAL4; UAS-dDuox-

RNAi/+).

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The genotypes of the flies used in the Fig. 2A-2B were as follows: Cont. (Da-

GAL4/+); dDuox-RNAi (UAS-dDuox-RNAi/Da-GAL4).

The genotypes of flies used in the Fig 3B were: Cont (cad-GAL4/+); dDuox-

RNAi (cad-GAL4/+; UAS-dDuox-RNAi/+); dDuox-RNAi + dDuox (cad-GAL4/UAS-

dDuox; UAS-dDuox-RNAi/+); dDuox-RNAi + hDuox1 (cad-GAL4/UAS-hDuox1; UAS-

dDuox-RNAi /+); dDuox-RNAi + hDuox2 (cad-GAL4/UAS-hDuox2; UAS-dDuox-RNAi

/+); dDuox-RNAi + dDuox-∆PHD (cad-GAL4/UAS-dDuox-∆PHD; UAS-dDuox-RNAi

/+).

The genotypes of flies used in the 3C-3D were: Cont (Da-GAL4/+); dDuox-

RNAi (UAS-dDuox-RNAi/Da-GAL4); dDuox-RNAi + dDuox (UAS-dDuox/+; UAS-

dDuox-RNAi/Da-GAL4).

The genotypes of flies used in the Fig. 3E were: Cont (Da-GAL4/+); dDuox-

RNAi (UAS-dDuox-RNAi/Da-GAL4); dDuox-RNAi + dDuox (UAS-dDuox/+; UAS-

dDuox-RNAi/Da-GAL4); dDuox-RNAi + dDuox-∆PHD (UAS-dDuox-∆PHD/+; UAS-

dDuox-RNAi/Da-GAL4).

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Supplementary Figures.

Fig. S1. Schematic presentations of dNox and dDuox. The Drosophila genome was

determined to contain one Nox homologue and one Duox homologue. Schematic

presentations for their molecular organizations, predicated on the data concerning

human Nox and Duox.

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Fig. S2. dNox and dDuox expressions are determined to be significantly decreased

in the intestines of flies carrying dNox-RNAi and dDuox-RNAi, respectively.

Control adult male flies (cad-GAL4/+), dDuox-RNAi flies (cad-GAL4/+; UAS-dDuox-

RNAi/+) and dNox-RNAi flies (cad-GAL4/+; UAS-dNox-RNAi/+) were used in the

present experiment. In order to quantify the amount of gene expression, fluorescence

real-time PCR was performed as described in the SOM Methods. The target gene

expression in the intestines of control flies was taken arbitrarily as 100, and the results

are shown as relative expression levels. Results are expressed as the mean of three

different experiments.

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Fig. S3. Natural infection experiments with various microbes. Natural infection

experiment was performed using a variety of microorganisms (Escherichia coli,

Saccharomyces cerevisiae, Micrococcus luteus, Salmonella typhimurium). Flies

carrying cad-GAL4/+; UAS-dDuox-RNA/+ (dDuox-RNAi) were used in this experiment.

The flies carrying cad-GAL4/+ alone (Cont.) were used as controls. At least, two

different transgenic lines carrying UAS-dDuox-RNAi were used in this study, and all

gave similar results. The results were expressed as means and standard deviations of

three different experiments with one representative transgenic line.

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Fig.S4. Unlike Duox-RNAi flies, NF-κB pathway mutant flies are completely

unaffected following natural infection. The mutant flies for two NF-κB pathways,

Toll and IMD pathways, were used. For Toll pathway mutants (S22-S25), persephone

mutant (psh), spaetzle mutant (spzrm7), PGRP-SA mutant (PGRP-SAseml) and p65-like

NF-κB/Dif mutant (Dif1) were used. For IMD pathway mutants (S26-S28), p105-like

NF-κB/relish mutant (RelE20), Dredd mutant (DreddB118) and PGRP-LC (PGRP-LCE12)

mutant were used. Control adult male flies (Da-GAL4/+) or dDuox-RNAi flies (UAS-

dDuox-RNAi/Da-GAL4) were also used. Natural infection was performed with Ecc15.

Results are expressed as the mean of three different experiments.

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Fig. S5. The dDuox-dependent ROS are not involved in the control of the NF-κB-

dependent antimicrobial peptide gene expression in the gut. (A) Real time PCR

analysis. The dDuox-RNAi flies (dDuox-RNAi/Da-GAL4) were subjected to natural

infection (24 hr) with Ecc15. Control flies (Da-GAL4/+) and IMD pathway mutant flies

(DreddB118) were also used as controls. Quantitative real-time PCR analysis of diptericin

gene transcription was performed using the intestines. The diptericin expression in the

tissue of non-infected control flies was taken arbitrarily as 100, and the results are

shown as relative expression levels. Results are expressed as the mean of three different

experiments. (B) In vivo diptericin-LacZ reporter analysis in the intestines. The dDuox-

RNAi flies carrying diptericin-LacZ (DD1; Da-GAL4/dDuox-RNAi) were subjected to

natural infection (24 hr) with Ecc15. Control larvae (DD1;Da-GAL4) and IMD pathway

mutant larvae (DD1; RelE20) were also used as controls. Histochemical staining of

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diptericin-LacZ activity was performed as describe previously (S29). Arrows indicate

the infection-induced diptericin-LacZ staining.

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Fig. S6. NF-κB pathways are not involved in the basal and infection-induced

expression of intestinal dDuox. Wild type and IMD pathway mutant (DreddB118) flies

were subjected to natural infection (0, 3 and 24 hr) with Ecc15. Quantitative real-time

PCR analysis of dDuox gene transcription was performed using the dissected intestines.

The level of dDuox expression in the tissue of non-infected control flies was taken

arbitrarily as 100, and the results are shown as relative expression levels. Results are

expressed as the mean of three different experiments.

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Fig. S7 Protein carbonylation and lipid peroxidation of the ingested bacteria

recovered from the intestines. (A) The reduced protein carbonylation of the ingested

Ecc15-GFP recovered from the intestines of the dDuox-RNAi (UAS-dDuox-RNAi/Da-

GAL4) flies. Flies (both the dDuox-RNAi flies and the control flies) were naturally

infected with Ecc15-GFP bacteria for 24 hr, and the ingested Ecc15-GFP bacteria were

then recovered from the dissected intestines. The total bacterial proteins (100 µg) were

then derivatized into 2,4-dinitrophenylhydrazone (DNP-hydrazone), then subjected to

Western blot analysis using anti DNP-antibody (upper panel). The same blot was also

probed with anti-GFP antibody for a loading control of bacterial protein extracts (lower

panel). (B) The reduced lipid peroxidation level of the ingested Ecc15-GFP recovered

from the intestines of the dDuox-RNAi flies. Bacterial extracts were prepared exactly as

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described above. The lipid peroxidation level in the total bacterial extract (100 µg) was

then analyzed by determination of malondialdehyde (MDA) concentration. Ecc15-GFP

with no treatment was used as a negative control, and Ecc15-GFP treated for 2 min with

H2O2 was used as a positive control. The values were expressed as relative MDA

concentrations, taking the MDA concentration of the untreated Ecc15-GFP arbitrarily

set to 100. Results are expressed as the mean and standard deviations of three different

experiments.

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