Genetic evidence for a protective role of the
peritrophic matrix against intestinal bacterial
infection in Drosophila melanogaster
Takayuki Kuraishi1, Olivier Binggeli, Onya Opota, Nicolas Buchon, and Bruno Lemaitre1
Global Health Institute, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland
Edited by Alexander S. Raikhel, University of California, Riverside, CA, and approved August 17, 2011 (received for review April 14, 2011)
The peritrophic matrix (PM) forms a layer composed of chitin and
glycoproteins that lines the insect intestinal lumen. This physical
barrier plays a role analogous to that of mucous secretions of the
vertebrate digestive tract and is thought to protect the midgut
epithelium from abrasive food particles and microbes. Almost
as an immune barrier has never been addressed by a genetic ap-
proach. Here we show that the Drosocrystallin (Dcy) protein, a pu-
PM formation. A loss-of-function mutation in the dcy gene results
bacterial ingestion a higher level of expression of antibacterial pep-
tides was observed in dcy mutants, pointing to an influence of this
matrix on bacteria sensing by the Imd immune pathway. Moreover,
with the entomopathogenic bacteria Pseudomonas entomophila
and Serratia marcescens. Dcy mutant flies also succumb faster than
wild type upon ingestion of a P. entomophila toxic extract. We
show that this lethality is due in part to an increased deleterious
action of Monalysin, a pore-forming toxin produced by P. entomo-
phila. Collectively, our analysis of the dcy immune phenotype indi-
cates that the PM plays an important role in Drosophila host
of pore-forming toxins on intestinal cells.
gut|innate immunity|insect immunity|entomopathogens
nition and control (1). This is also true for insects such as Dro-
sophila, which live on decaying matter such as rotting fruits and
ingest large quantities of microbes. Some studies have started to
investigate the mechanisms underlying the gut defense to bac-
terial infection in Drosophila. These studies have indicated that
(i) reactive oxygen species (ROS) production through the en-
zyme Duox, (ii) production of antibacterial peptides through the
Imd pathway, and (iii) maintenance of gut homeostasis through
regulation of stem cell activity are all essential elements of the
gut defense to infection (2).
Oral ingestion of bacteria induces the rapid synthesis of ROS
in the Drosophila gut by an NADPH oxidase called Duox (3).
Ingested bacteria were shown to persist throughout the intestinal
tract of Duox RNAi flies, which indicates a predominant role
of ROS in the elimination of ingested microbes. Complemen-
tary to this ROS response, several antimicrobial peptides (e.g.,
Diptericin) are produced in the gut under the control of the Imd
pathway (4). This local immune response is triggered by the
recognition of Gram-negative peptidoglycan by the pattern rec-
ognition receptor PGRP-LC (peptidoglycan recognition protein
LC) (5) and was shown to contribute to host survival upon in-
testinal infection with several pathogenic bacteria (6–8). Finally,
efficient and rapid recovery from bacterial infection is possible
only when clearance of bacteria from the gut is coordinated with
epithelium renewal to repair damage caused by infection. Epi-
thelium renewal of the Drosophila gut is stimulated by the release
ecause the gut epithelium is in contact with microorganisms,
it must be armed with efficient systems for microbial recog-
of the cytokine Upd3 from damaged enterocytes, which then
activates the JAK/STAT pathway in intestinal stem cells to pro-
mote both their division and differentiation, establishing a ho-
meostatic regulatory loop (9, 10). Interestingly, both Imd pathway
activity and epithelium renewal are also stimulated at a basal level
by the indigenous gut microbiota (10).
The peritrophic matrix (PM) forms a layer composed of chitin
and glycoproteins that lines the insect midgut lumen (11, 12).
Although structurally different, it plays a role analogous to that of
mucous secretions of the vertebrate digestive tract and is thought
to protect the midgut epithelium from abrasive food particles and
microbes. Studies in insects have suggested that the PM plays a
role in the defense against ingested pathogens. However, most of
14). Diptera such as Drosophila have a type II PM that is contin-
uously produced by specific cells of the cardia, a specialized organ
at the anterior of the midgut (15). As the PM grows posteriorly, it
encloses the food passing through it all along the digestive tract.
To date almost nothing is known about PM functions in Dro-
sophila, and specifically its function as an immune barrier has
that a protein with a chitin-binding domain, Drosocrystallin (Dcy)
contributes to PM formation in Drosophila adults. A loss-of-
function mutation in dcy results in a reduction of PM width and
renders flies more susceptible to infections with the entomopa-
thogenic bacteria Pseudomonas entomophila. We show that this
lethality is due in part to an increased deleterious action of
Monalysin, a pore-forming toxin produced by P. entomophila.
plays an important role in Drosophila host defense against in-
testinal pathogens by preventing the action of toxins on gut cells.
Dcy Is a PM Protein Induced upon Oral Bacterial Infection. A micro-
array analysis indicated that a gene named drosocrystallin (dcy)
encoding a chitin-binding protein is induced in the Drosophila
adult gut upon oral infection with the Gram-negative bacterium
Erwinia carotovora 15 (Ecc15) (16). Real-time quantitative PCR
(qPCR) analysis confirmed that dcy is up-regulated soon after
infection and reaches its maximum 4 h after infection, at a level
approximately sevenfold higher than in unchallenged condition
(Fig. 1A). A previous microarray study indicated that dcy expres-
sion is not controlled by the Imd pathway (16). In agreement with
Author contributions: T.K., O.B., O.O., N.B., and B.L. designed research; T.K., O.B., O.O.,
and N.B. performed research; O.O. contributed new reagents/analytic tools; T.K., O.B.,
O.O., N.B., and B.L. analyzed data; and T.K. and B.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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| 1 of 6
FlyAtlas (17),we found that dcy mRNA is strongly enriched in the
midgut and heads of adult flies (Fig. 1B).
The dcy gene encodes one transcript for a 477-aa protein
with a predicted molecular mass of 55.9 kDa. This protein was
initially named Drosocrystallin because of its strong expression
in the Drosophila compound eye, where it is thought to be a
structural component of the corneal lens (18, 19). Dcy contains a
signal peptide and a chitin-binding domain. Its strong expression
in the midgut suggested that Dcy could be a component of the
PM. To address this hypothesis, we examined the localization of
Dcy in the midgut with an anti-Dcy serum. The immunostaining
analysis showed that a signal is observed on the PM with the anti-
Dcy serum in wild-type intestines, suggesting that Dcy protein
is a component of the PM in adult Drosophila (Fig. 1C).
Loss of dcy Compromises PM Permeability. To gain insight into the
physiological role of dcy, we used a Drosophila strain, dcyMB08319
(referred to as dcy1), with a Minos transposon inserted in the first
intron of dcy (Fig. S1A). The amount of dcy mRNA in dcy1ho-
mozygous flies or in flies carrying the dcy1allele over a deficiency
[Df(2L)Exel6030] was less than 10% of that of wild-type flies (Fig.
S1B). Dcy1flies fail to induce any dcy expression after oral in-
fection with Ecc15 (Fig. 1A). In addition, immunostaining of
midgut sections from dcy1adults with the anti-Dcy serum gave
only a faint signal in the PM (Fig. 1C). These results indicate that
dcy1is a strong loss-of-function mutation of dcy. We also gener-
ated a precise excision line of dcy1by remobilization of the Minos
transposable element. This line, referred to as dcyRev, expressed a
normal level of dcy and was used in addition to the Oregon R
strain as a wild-type control strain in the following experiments.
Dcy1homozygous mutants are viable, fertile, and do not show
any external morphological defect. dcy1mutants are apparently
not blind because they can orient themselves according to the
light (Fig. S1C). Nevertheless, the dcy1have a shorter lifespan,
starting to die 3 to 4 wk after emergence (Fig. S1D). Interestingly,
transmission electron microscopy of sections of anterior midguts
revealed that the thickness of the PM in dcy1adults is only ap-
proximately half that of control flies (Fig. 2A). We next in-
vestigated the effect of the dcy1mutation on the PM permeability
by feeding adults with FITC-labeled dextran molecules of differ-
ent sizes as described in ref. 13. The occurrence of FITC signals in
close contact with the intestinal epithelium was interpreted as the
resultofmolecules crossing thePM into theectoperitrophicspace
(between the PM and epithelium). Fig. 2B and Fig. S2 show that
ingested 70- and 150-kDa FITC-labeled dextran molecules were
observed in contact with the epithelial cells of wild-type flies,
whereas 500-kDa and almost all 250-kDa dextran molecules
Interestingly, we observed that in dcy1mutants, 250-kDa and
a large proportion of 500-kDa molecules were found in close vi-
cinity to the gut epithelium (Fig. 2B, Right, and Fig. S2). These
results indicate that the PM of dcy1mutants tends to be more
permeable than that of wild-type flies.
Dcy Is Required for the Defense Against Oral Infection with
Entomopathogenic Bacteria. We next used dcy1flies to analyze
the contribution of the PM to the protection against oral bac-
terial infection. P. entomophila is a natural bacterial pathogen of
Drosophila (20). Oral infection with P. entomophila at a high
dose induces a strong local and systemic immune response in
Drosophila but is still highly pathogenic because it quickly
induces a blockage of food uptake and irreversible gut damage
(10). Fig. 3A shows that dcy1mutants exhibit a higher suscepti-
bility than wild-type to oral infection with a lethal dose of
P. entomophila. The dcy mutants even succumb to the infection
with a fourfold-diluted P. entomophila solution, which is not le-
thal for wild-type flies (Fig. S3A). Several lines of evidence in-
dicate that this increased susceptibility is indeed due to the dcy
(A) Dcyexpression uponEcc15 oralinfection inwild-type and dcy mutantflies.
Dcy mRNA was measured by real-time qPCR in whole flies at indicated time
points, and results are shown as a relative Dpt/rpL32 ratio. (B) Real-time qPCR
analysis of dcy mRNA expression from the indicated tissues of wild-type
Drosophila. Note that adult carcasses do not include gut. Data are represen-
tativeofthree(A) ortwo (B) independent experiments (shownareerror bars).
(C) Transversal sections of wild-type or dcy adult anterior midgut were ana-
lyzed by immunostaining with an anti-Dcy serum. Arrows indicate the PM
(Scale bars, 50 μm.)
Dcy expression is induced in the midgut upon oral bacterial infection.
adult anterior midguts derived from wild-type or dcy1mutant flies were ob-
servedbytransmission electron microscopy. A2 andA5 are magnified viewsof
A1 and A4. A4 and A6 show another section at high magnification. Arrows
indicate the PM. M, mucus; EC, enterocytes; L, lumen with ingested food.
(Scale bars, 10 μm in A1 and A4, 1 μm in A2, A3, A5, and A6.) Right: Quanti-
tativemeasurements ofthethicknessof thePM inwild-type ordcy1flies.Data
show means and SEs from six and nine different midgut sections for the
dcy1flies. Adult flies were fed with 70-kDa or 500-kDa FITC-labeled dextran
beads. Guts were dissected and examined under a fluorescence microscope.
The FITC signal is retained in the lumen if the dextran beads cannot pass
through the PM. The FITC signal is observed in contact with epithelial cells
(indicated as positive) if beads can cross the PM. Bar graph shows the number
of “positive” guts for each genotype when 500-kDa molecules were fed.
Means and SEs from four independent experiments are shown. *P < 0.05.
The dcy mutation induces PM defects. (A) Left: Ultrathin sections of
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| www.pnas.org/cgi/doi/10.1073/pnas.1105994108Kuraishi et al.
mutation and not to the genetic background. First, revertant
dcyRevflies do not show the increased susceptibility, and Df(dcy)/
dcy1flies exhibit the same phenotype as the homozygous dcy1
mutants (Fig. 3A). Second, another genetic background, a y,w,
Diptericin-lacZ, Drosomycin-GFP; dcy1line, has the same sus-
ceptibility as dcy1flies (Fig. S3B). Third, an in vivo RNAi si-
lencing of dcy in the intestine using a midgut-specific GAL4
driver (genotype: NP1-GAL4; UAS-dcy-IR) also results in higher
susceptibility to P. entomophila (Fig. S3C). Fourth, over-
expression of dcy in the midgut using the NP1-GAL4 driver
(genotype: dcy1/dcy1; NP1-GAL4/UAS-dcy) rescued the dcy1
susceptibility phenotype (Fig. 3B). Fig. 3C shows that the dcy1
mutants also succumb more rapidly than wild-type when orally
infected with Serratia marcescens Db11, another entomopatho-
genic bacterium (7). Finally, dcy1mutant flies have a wild-type
resistance to Ecc15, a nonlethal Gram-negative bacterium, upon
septic injury (Fig. S3D), indicating that the survival phenotype of
the dcy1mutant upon oral bacterial infection is not caused by a
general weakness of dcy1mutant flies.
Both resistance and tolerance mechanisms contribute to main-
tain gut integrity upon bacterial infection. Resistance mecha-
nisms involve the activation of various immune responses that
directly target pathogens, whereas tolerance mechanisms im-
prove the capacity to survive infection without acting on bacterial
elimination. (21). We observed that the number of P. entomo-
phila detected in the gut 18 h after infection (when flies start to
die) was roughly similar in dcy1and wild-type (Fig. 3D). This
result indicates that the dcy1mutation does not affect bacterial
clearance but rather the tolerance of flies to P. entomophila in-
fection. This is consistent with a role of the PM in protecting the
gut epithelium from lethal infection. This also indicates that the
dcy1phenotype was not caused by a feeding defect because dcy1
and wild-type flies ingested the same quantity of P. entomophila,
as determined by cfu assay at 30 min after ingestion (Fig. 3D).
Moreover, the triglyceride and glycogen stores of the dcy1un-
challenged mutants were not significantly different from wild-type
(Fig. S4), indicative of normal digestive capacity.
Together, our results show that expression of dcy in the midgut
is required for defense against oral infection with entomopatho-
Enhanced Activation of the Imd Pathway in the dcy Mutant. The Imd
plays a role in the resistance against both P. entomophila and S.
marcescens (6–8). This prompted us to analyze the effect of the
dcy1mutation on the Imd pathway. For this, we compared the
controlled by the Imd pathway, in dcy1and wild-type flies. Real-
time qPCR revealed that dcy1flies show a stronger induction of
Dpt in the gut upon oral infection with P. entomophila (Fig. 4A),
notably at early time points of infection. This higher induction
was not specific to P. entomophila because similar results were
obtained when flies were fed with the nonlethal strain Ecc15 (Fig.
S5). P. entomophila leads not only to a local but also to a strong
systemic fat body immune response (20). Both real-time qPCR
and X-gal staining with flies carrying a Dpt-lacZ reporter gene
revealed higher levels of systemic Dpt expression in dcy1mutant
flies compared with wild-type upon P. entomophila infection (Fig.
4 A and B). The observation that the dcy1mutation leads to a
stronger overall activation of the Imd pathway in infected flies
indicates that the PM influences bacterial sensing in the gut.
Dcy Mutation Affects Neither Resistance to ROS Nor Gut Repair Mech-
anisms. Recent studies have demonstrated that the activation of
epithelium renewal is required in the gut to compensate for
damage caused by infectious agents. (9, 10, 22). Along this line,
infection with high doses of P. entomophila leads to a loss of gut
disrupt epithelium renewal through excessive damage to the gut.
This raised the possibilities that the dcy mutant might be more
susceptibility of the dcy mutant to P. entomophila. To examine
these possibilities, we orally administrated paraquat, a potent in-
ducer of ROS, or bleomycin, a DNA-damaging agent that dam-
ages the gut, thus increasing stem cell proliferation (10, 23). Both
compounds have molecular sizes (paraquat, 0.257 kDa; bleomy-
cin, 1.4 kDa) small enough to easily cross the PM. We observed
that dcy1flies show survival rates similar to wild-type flies upon
feeding with either paraquat or bleomycin (Fig. S6 A and B).
To determine whether the dcy1mutation impacts gut repair,
we quantified the level of epithelial renewal in flies upon in-
fection with the strain Ecc15. We choose this strain because,
unlike P. entomophila, oral infection with Ecc15 triggers a high
level of epithelial renewal without affecting flies’ viability. We
first examined the level of epithelium renewal itself upon Ecc15
oral infection by counting the number of dividing cells along the
midgut using an anti-phosphohistone H3 (anti-PH3) antibody as
an indicator of mitotic activity (10). No difference was observed
between wild-type and dcy1flies (Fig. S7A). Signals from both
JAK-STAT and EGFR pathways control stem cell proliferation
and thereby gut repair and homeostasis after oral infection (16).
We monitored the activation of these pathways by quantifying
transcripts for upd3, Socs36E (JAK-STAT pathway), and Keren
and argos (EGFR pathway) upon oral infection with P. ento-
mophila. Fig. S7B shows that the dcy mutants express wild-type
levels of these genes in response to Ecc15 infection.
These results indicate that the dcy1mutation does not induce a
higher sensitivity to oxidative burst and does not affect the ability
to repair the gut upon damage.
pathogenic bacteria. (A) Survival analysis of wild-type, dcyRev, homozygous
dcy1, and dcy1/Df(dcy) hemizygous flies upon oral infection of P. entomo-
phila. Means and SEs of four independent experiments are shown (P <
0.0001, log–rank test). (B) Survival analysis of wild-type, homozygous dcy1,
and the dcy1mutants expressing the dcy gene in the midgut upon oral in-
fection of P. entomophila. Means and SEs of three independent experiments
are shown (P < 0.0002, log–rank test). (C) Survival analysis of wild-type,
dcyRev, and homozygous dcy1flies upon oral infection with S. marcescens
Db11. Graphs show the means of 60 flies, bars show the SE. This experiment
was repeated three times and yielded similar results (P < 0.0001, log–rank
test). (D) Bacterial persistence in wild-type and dcy1flies. Bacterial persis-
tence in dcy1and dcyRevat 30 min and 18 h upon oral infection with
P. entomophila (OD600= 100), as the number of cfu per fly. No difference
was observed between the two strains. Each histogram corresponds to the
average of three independent experiments.
Dcy is required for protection against oral infection with entomo-
Kuraishi et al. PNAS Early Edition
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Increased Susceptibility of dcy Mutants to Ingested Bacterial Toxin.
It has been proposed that the PM is a barrier protecting the
midgut epithelium from secreted bacterial virulence factors such
as toxins or proteases (12). We therefore speculated that toxic
compounds secreted by P. entomophila could have a more
damaging effect in the dcy1mutant owing to the higher perme-
ability of the PM. Membrane-filtered extracts from sonicated
P. entomophila cells were fed to wild-type and dcy1adults. We
found that up to 80% of the dcy1mutants succumbed to this
treatment, whereas no lethality was observed in wild-type flies
fed with the same extract (Fig. 5A). Virulence factors of P.
entomophila required for Drosophila infection include a secreted
metalloprotease (AprA) that protects against antimicrobial
peptides, and a pore-forming toxin named Monalysin that par-
ticipates in the damage to intestinal cells (6, 24). Both AprA and
Monalysin are regulated by the GacS-GacA two-component
system that regulates the production of secreted proteins and
metabolites (6). Fig. 5B shows that a P. entomophila extract from
a gacA mutant no longer kills the dcy1mutant flies, whereas
extracts from an aprA mutant retain their wild-type pathogenicity
on dcy1flies (Fig. 5C). Interestingly, extracts from the monalysin
(mnl) mutant P. entomophila less efficiently kill dcy1mutant flies
(Fig. 5D), implying that this pore-forming toxin is partially re-
sponsible for the killing activity of the extract. The observations
that (i) Monalysin contributes to the killing activities of the ex-
tract and (ii) the dcy1mutation increases the susceptibility to the
P. entomophila extract indicate that the PM provides protection
against ingested toxins, especially pore-forming toxins.
Proteomic analyses have revealed the complexity of the insect
PM that is composed of chitin microfibrils embedded in a matrix
of proteins and glycoproteins (12, 25, 26). PM-associated pro-
teins include mucins and peritrophin proteins. The Drosophila
in dcy1flies compared with wild-type flies. (A) Dpt expression
in the midgut (Left) or the fat body (Right) of wild-type and
dcy1flies upon oral infection with P. entomophila was mea-
sured by real-time qPCR at the indicated time points. Data are
the mean of four independent experiments, and error bars
show the SE. **P < 0.01 vs. dcyRev. Results are shown as a
percentage of the Dpt/rpL32 ratio normalized to the levels
observed in wild-type flies collected 8 h after septic injury (SI)
with P. entomophila. (B) β-Galactosidase staining reveals lacZ
gene expression in the fat body of wild-type or dcy1flies
carrying a Dpt-lacZ reporter. Flies were collected 4 h after oral
infection with P. entomophila.
Dpt expression upon oral bacterial infection is higher
extract. (A) Survival analysis of dcyRevand dcy1flies upon feed-
ing of the extract from P. entomophila (Pe extract) or buffer
(PBS 1% Triton X-100). (B) Survival analysis of wild-type, dcyRev,
and gacA P. entomophila derivatives. (C) Survival analysis of
wild-type, dcyRev, and dcy1flies upon feeding with extracts de-
rived from wild-type and AprA P. entomophila derivatives. (D)
with extracts derived from wild-type and monalysin (mnl) P.
entomophila derivatives. In A–D, graphs show the means of 60
flies, and bars show the SE. These experiments were repeated
three times andyielded similar results (*P < 0.0001,**P < 0.005,
log–rank test. ns, not significant).
Dcy1flies succumbed to ingestion of a P. entomophila
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| www.pnas.org/cgi/doi/10.1073/pnas.1105994108 Kuraishi et al.
genome contains approximately 25 genes encoding chitin-bind-
ing domain proteins and 36 genes encoding mucin-like proteins
(27). To date, none of these genes have been studied in the
context of the PM, and little is known about the role of mucus
and the PM in Drosophila gut homeostasis and immunity. Here
we show that the Dcy protein, a putative component of the eye
lens of Drosophila, contributes to adult PM formation. Although,
we cannot exclude that Dcy also exist as free molecules in the
gut, four lines of evidences support that Dcy is an integral
component of the PM: (i) the presence of a chitin-binding do-
main, suggesting that Dcy associates with chitin fibrils of the PM;
(ii) the staining of the PM using an anti-Dcy antibody; (iii) the
thinner PM observed in dcy1mutants; and (iv) the higher per-
meability of the PM in dcy1mutants. Because dcy is expressed in
the midgut and not in the cardia, it is likely that this protein is
directly incorporated to the PM after its synthesis. The obser-
vation that a strong loss-of-function mutation in dcy reduces the
PM width by half and increases its permeability to larger mole-
cules indicates that despite the high number of structural pro-
teins associated with the PM, Dcy is an essential component of
the PM. Dcy cannot be considered sensu stricto as a peritrophin,
owing to the absence of characteristic cystein arrangement in its
chitin-binding domain. The dual function of this protein in both
eye and gut is intriguing. Of note, no clear Dcy paralog is
encoded by the Drosophila genome. In addition, no homolog can
be found outside the Drosophilidae family, suggesting that Dcy
has evolved to fulfill a function specific to this clade.
Using the dcy1mutation, we were able to indirectly assess a role
for the PM in Drosophila host defense. Our observation that dcy
mutants are highly susceptible to infection with P. entomophila
and S. marcescens points to a protective role of the PM in host
defense against entomopathogenic bacteria. We observed that
ingestion of a P. entomophila extract is sufficient to induce le-
thality in dcy1mutant flies but not in wild-type flies. This supports
a role of the PM in limiting the diffusion of a bacterial toxin.
Interestingly, a P. entomophila extract from the pore-forming
toxin monalysin-deficient strain seems less toxic to dcy flies. This
indicates that the PM provides an effective protection against the
action of this pore-forming toxin. β-Pore-forming toxins such as
Monalysin have the ability to multimerize into circular polymers,
a step required for pore formation in targeted cells (28). This
suggests that the PM could function as a sieve blocking the action
of this class of toxins. A role of the PM in the protection against
pore-forming toxins is also supported by studies in other insects.
Hayakawa et al. (29) showed that Bombyx mori is sensitive to the
Cry1Aa toxin and resistant to Cry1Ac, both insecticidal toxins of
Bacillus thuringiensis. This difference correlates with the capacity
of Cry1Aa to pass through the PM faster than Cry1Ac in an
in vitro assay. It was also reported that the activity of Cry1Ac toxin
on Helicoverpa armigera larvae is enhanced by B. thuringiensis
Enhancin, a metalloprotease that degrades PM-associated mucins
(30). The involvement of a pore-forming toxin in P. entomophila
virulence, together with the well-characterized action of B. thur-
ingiensis cytotoxin Cry, have recently led to the notion that pore-
forming toxins constitute an efficient arm to promote bacterial
colonization of the insect gut (24, 31). Our present studies suggest
that the PM provides an important barrier to counteract the ac-
tion of this class of toxins.
The observation that dcy as well as several peritrophin genes
are induced upon ingestion of bacteria (16) also points to the
existence of active mechanisms that reinforce the role of the PM
barrier during infection. This indicates that the PM is not just a
passive physical barrier but can be remodeled during gut in-
fection. However, our study does not address whether PM pro-
tection against P. entomophila pore-forming toxin is mediated by
its impermeability to Monalysin or by its capacity to bind and
sequester this toxin. In support of the second hypothesis, Abedi
and Brown (32) discovered that the PM excreted by Aedes aegypti
larvae that are resistant to dichlorodiphenyltrichloroethane was
laden with the insecticide. This finding led to the notion that this
matrix may play a role in sequestering and possibly detoxifying
Production of ROS in the gut by Duox in response to bacteria
inflicts damage to the intestinal epithelium that is repaired
through stem cell proliferation(10). Although someof theseROS
compounds could be inactivated by antioxidant enzymes (33), it
has been proposed that the PM could serve as a “sacrificial anti-
oxidant” through the scavenging of ROS (34, 35). In opposition to
this idea, we did not observe a higher susceptibility of dcy mutants
to ROS produced by paraquat. This does not rule out a role of the
PM as antioxidant but suggests that in our infection model the
main defensive role of the PM is to limit the action of bacterial
toxins. Moreover, the gut repair capacity through epithelium re-
newal is not affected by the dcy mutation in flies orally infected
with Ecc15 or flies that ingested Bleomycin, thus reinforcing the
idea that the PM protection is specific for a certain type of threat,
such as that linked to pore-forming toxins.
Recent studies in Drosophila have revealed that multiple
regulatory mechanisms are required to precisely control the level
of Imd pathway activity in the gut. These include Pirk, a protein
interacting with PGRP-LC and with amidase PGRPs, which both
restrict the activation of the immune pathway by indigenous flora
(5, 36). Here, we observed that disruption of the PM affects the
level of Imd pathway activity in response to infection. Dcy1
mutants exhibit enhanced gut and systemic immune responses to
Gram-negative bacteria. This indicates a role of the PM in the
fine-tuning of Imd pathway activity during bacterial infection. An
attractive hypothesis is that the PM could limit the diffusion from
the gut lumen to epithelial cells of peptidoglycan, the bacterial
elicitor recognized by the Imd pathway.
In conclusion, our studies ascribe important functions to the
PM in Drosophila host defense against bacteria by limiting
the effect of bacterial toxins and reducing Imd pathway activity.
The importance of the PM function is even underestimated
in our study because the dcy1mutation reduces but does not
eliminate the PM. Our study is also in line with those in verte-
brates that revealed the key role of mucus in gut homeostasis and
mucosal immunity (37). Indeed, deletion of the large gel-forming
mucin Muc2 in mice allows the direct contact of bacteria with the
epithelia cells, thus provoking colon inflammation. It now seems
that both mucus in mammals and PM in insects provide an im-
portant protection against the action of pathogens and influence
the immune reactivity of the digestive tract. A better compre-
hension of the physiological role of the PM is essential to un-
derstand insect gut homeostasis. Moreover, we must keep in
mind that the PM is an attractive target for insect pest man-
agement strategies (11, 12). We expect that this study will open
the route for a genetic dissection of PM function in Drosophila
that could be useful in other insects of economic or global
Materials and Methods
Fly Stocks. Oregon R flies were used as wild-type flies. Dcy1(MB08319, Fig. S1)
and Df(2L)Exel6030 were obtained from the Bloomington Drosophila Stock
Center. Canton S, w1118, RelishE20(RelE20), NP1-Gal4, and Diptericin-LacZ fly
lines are described in ref. 16. The UAS-CG16963-IR RNAi line from the Vienna
Drosophila RNAi Stock Center and UAS-yellow (Bloomington Center) or w1118
were used as control. A full-length cDNA of dcy (CG16963_cDNA gold
RH66281 from the Drosophila Genomics Resource Center) was inserted in the
pENTR Gateway entry clone (Invitrogen) and then subcloned in the pTW
transgenesis vectorused for generating transgenic flies according to standard
procedures. Fly line carrying the transgene on the third chromosome was
established and used as UAS-dcy. F1 progeny carrying both the UAS construct
and the Gal4 driver were transferred to 29 °C at late pupal stage for optimal
efficiency of the UAS/Gal4 system. To obtain a revertant of dcy1, the Minos-
element MB08319 was mobilized by a Minos transposase source (38), and
precise excision line (referred to as dcyrev) was isolated. Drosophila stocks and
Kuraishi et al.PNAS Early Edition
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crosses were maintained at 25 °C in tubes containing standard fly medium Download full-text
(maize flour, dead yeast, agar, and fruit juice) devoid of living yeast.
Bacterial Stocks and P. entomophila Protein Extracts. P. entomophila L48 (20)
was grown in LB for all experiments. P. entomophila mutated for the gacA,
aprA, and mnl are described elsewhere (6, 24). The Ecc15 strain was de-
scribed previously (20). They were grown at 29 °C and allowed to reach the
stationary phase. Cells were then concentrated at OD600= 200 except when
indicated. The solution was added. S. marcescens strain Db11 (7) was grown
at 37 °C and used as pellets of OD600= 200. Pellets were not washed before
use. For P. entomophila extracts, stationary phase cultures of wild-type and
mutant P. entomophila were concentrated by centrifugation. The cell pellet
was washed with PBS and adjusted to OD600= 200 in PBS with 1% Triton X-
100. The pellets were sonicated, recentrifuged, and filtered with a 0.22-μm
Infection and Survival Assays. Septic injuries were performed by pricking
adults in the thorax with a thin needle dipped into a concentrated bacterial
pellet. For oral infection, female flies were starved for 2 h at 29 °C and then
placed in a fly vial with food solution. The food solution was obtained by
mixing a pellet of bacteria (OD600= 200, corresponding to 1.3 × 1011bac-
teria/mL), solution of paraquat (10–50 mM, Sigma), or 500 μg/mL bleomycin
(Sigma) with a solution of 5% sucrose (1:1), added to a filter disk that
completely covered the surface of standard fly medium. Flies were main-
tained at 29 °C, and mortality was monitored as at different time points.
Survival assays were performed with 60–80 flies for each genotype.
Antibodies, Immunohistochemistry, and X-Gal Staining. Anti-PH3 staining was
performed as previously described (16). An Anti-Dcy antibody was raised by
immunizing rats with the carboxyl end of Dcy protein (amino acid positions
402–477), which was expressed in Escherichia coli as a fusion protein with
GST and affinity-purified. Immunohistochemistry, electron microscopy, and
X-gal staining are described in SI Materials and Methods.
Dextran Feeding Assay. FITC-dextran beads (Sigma) were dissolved in 2.5%
sucrose and filtrated with Sephadex G-10 (GE Healthcare) and used for
feeding experiments. Female flies were starved for 2 h at 29 °C in an empty
vial and then placed in a normal fly vial covered with a filter paper soaked
with the dextran solution. After 15 min at 29 °C, guts were dissected in PBS,
and FITC signal was observed under a fluorescent microscope (Zeiss). Images
were captured with a Leica DFC300FX camera and the Leica Applica-
Real-Time qPCR. Total RNA was extracted from whole flies, cuticles of flies as
fat bodies, dissected midguts, or other tissues by using TRIzol (Invitrogen).
Real-time qPCR was performed using SYBR Green I (Roche) on a LightCycler
2.0 as described previously (16). The amount of mRNA detected was nor-
malized to rpL32 control values. Primers used to monitor mRNA quantifi-
cation can be obtained on request.
Statistics. Statistical analyses were done by Student t test or log–rank test,
and P values of <0.05 were considered significant.
ACKNOWLEDGMENTS. We thank our colleagues J. P. Boquete and Danielle
Brandalise for technical assistance; Nichole Broderick for helpful comments
on the manuscript; D. Ferrandon (Strasbourg) for the S. marcescens Db11;
the Bloomington Stock Center, the Vienna Drosophila Research Center, and
the National Institute of Genetics for fly stocks; and S. Rosset and G. Knott
(Ecole Polytechnique Fédérale de Lausanne BioEM Facility) for assistance
with histological analysis. The B.L. laboratory is supported by a European
Research Council Advanced Investigators grant, the Bettencourt-Schueller
foundation, and Swiss National Science Foundation Grant 3100A0-12079/1.
1. Sansonetti PJ (2004) War and peace at mucosal surfaces. Nat Rev Immunol 4:953e964.
2. Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu
Rev Immunol 25:697e743.
3. Ha EM, Oh CT, Bae YS, Lee WJ (2005) A direct role for dual oxidase in Drosophila gut
immunity. Science 310:847e850.
4. Tzou P, et al. (2000) Tissue-specific inducible expression of antimicrobial peptide
genes in Drosophila surface epithelia. Immunity 13:737e748.
5. Zaidman-Rémy A, et al. (2006) The Drosophila amidase PGRP-LB modulates the im-
mune response to bacterial infection. Immunity 24:463e473.
6. Liehl P, Blight M, Vodovar N, Boccard F, Lemaitre B (2006) Prevalence of local immune
response against oral infection in a Drosophila/Pseudomonas infection model. PLoS
7. Nehme NT, et al. (2007) A model of bacterial intestinal infections in Drosophila
melanogaster. PLoS Pathog 3:e173.
8. Ryu JH, et al. (2006) An essential complementary role of NF-kappaB pathway to mi-
crobicidal oxidants in Drosophila gut immunity. EMBO J 25:3693e3701.
9. Jiang H, et al. (2009) Cytokine/Jak/Stat signaling mediates regeneration and ho-
meostasis in the Drosophila midgut. Cell 137:1343e1355.
10. Buchon N, Broderick NA, Chakrabarti S, Lemaitre B (2009) Invasive and indigenous
microbiota impact intestinal stem cell activity through multiple pathways in Dro-
sophila. Genes Dev 23:2333e2344.
11. Lehane MJ (1997) Peritrophic matrix structure and function. Annu Rev Entomol 42:
12. Hegedus D, Erlandson M, Gillott C, Toprak U (2009) New insights into peritrophic
matrix synthesis, architecture, and function. Annu Rev Entomol 54:285e302.
13. Edwards MJ, Jacobs-Lorena M (2000) Permeability and disruption of the peritrophic
matrix and caecal membrane from Aedes aegypti and Anopheles gambiae mosquito
larvae. J Insect Physiol 46:1313e1320.
14. Wang P, Granados RR (2000) Calcofluor disrupts the midgut defense system in insects.
Insect Biochem Mol Biol 30:135e143.
15. King DG (1988) Cellular organization and peritrophic membrane formation in the
cardia (proventriculus) of Drosophila melanogaster. J Morphol 196:253e282.
16. Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B (2009) Drosophila
intestinal response to bacterial infection: Activation of host defense and stem cell
proliferation. Cell Host Microbe 5:200e211.
17. Chintapalli VR, Wang J, Dow JA (2007) Using FlyAtlas to identify better Drosophila
melanogaster models of human disease. Nat Genet 39:715e720.
18. Janssens H, Gehring WJ (1999) Isolation and characterization of drosocrystallin, a lens
crystallin gene of Drosophila melanogaster. Dev Biol 207:204e214.
19. Komori N, Usukura J, Matsumoto H (1992) Drosocrystallin, a major 52 kDa glyco-
protein of the Drosophila melanogaster corneal lens. Purification, biochemical char-
acterization, and subcellular localization. J Cell Sci 102:191e201.
20. Vodovar N, et al. (2005) Drosophila host defense after oral infection by an en-
tomopathogenic Pseudomonas species. Proc Natl Acad Sci USA 102:11414e11419.
21. Schneider DS, Ayres JS (2008) Two ways to survive infection: What resistance and
tolerance can teach us about treating infectious diseases. Nat Rev Immunol 8:
22. Chatterjee M, Ip YT (2009) Pathogenic stimulation of intestinal stem cell response in
Drosophila. J Cell Physiol 220:664e671.
23. Amcheslavsky A, Jiang J, Ip YT (2009) Tissue damage-induced intestinal stem cell di-
vision in Drosophila. Cell Stem Cell 4:49e61.
24. Opota O, et al. (2011) Monalysin, a novel β-pore-forming toxin from the Drosophila
pathogen Pseudomonas entomophila, contributes to host intestinal damage and le-
thality. PLoS Pathog, in press.
25. Tellam RL, Wijffels G, Willadsen P (1999) Peritrophic matrix proteins. Insect Biochem
Mol Biol 29:87e101.
26. Dinglasan RR, et al. (2009) The Anopheles gambiae adult midgut peritrophic matrix
proteome. Insect Biochem Mol Biol 39:125e134.
27. Syed ZA, Härd T, Uv A, van Dijk-Härd IF (2008) A potential role for Drosophila mucins
in development and physiology. PLoS ONE 3:e3041.
28. Gonzalez MR, Bischofberger M, Pernot L, van der Goot FG, Frêche B (2008) Bacterial
pore-forming toxins: The (w)hole story? Cell Mol Life Sci 65:493e507.
29. Hayakawa T, Shitomi Y, Miyamoto K, Hori H (2004) GalNAc pretreatment inhibits
trapping of Bacillus thuringiensis Cry1Ac on the peritrophic membrane of Bombyx
mori. FEBS Lett 576:331e335.
30. Fang S, et al. (2009) Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac
protein to Helicoverpa armigera larvae by degrading insect intestinal mucin. Appl
Environ Microbiol 75:5237e5243.
31. Vallet-Gely I, Lemaitre B, Boccard F (2008) Bacterial strategies to overcome insect
defences. Nat Rev Microbiol 6:302e313.
32. Abedi ZH, Brown AWA (1961) Peritrophic membrane as vehicle for DDT and DDE
excretion in Aedes aegypti larvae. Ann Entomol Soc Am 54:539e542.
33. Ha EM, et al. (2005) An antioxidant system required for host protection against gut
infection in Drosophila. Dev Cell 8:125e132.
34. Barbehenn RV, Stannard J (2004) Antioxidant defense of the midgut epithelium by
the peritrophic envelope in caterpillars. J Insect Physiol 50:783e790.
35. Summers CB, Felton GW (1996) Peritrophic envelope as a functional antioxidant. Arch
Insect Biochem Physiol 32:131e142.
36. Lhocine N, et al. (2008) PIMS modulates immune tolerance by negatively regulating
Drosophila innate immune signaling. Cell Host Microbe 4:147e158.
37. Johansson ME, et al. (2008) The inner of the two Muc2 mucin-dependent mucus layers
in colon is devoid of bacteria. Proc Natl Acad Sci USA 105:15064e15069.
38. Metaxakis A, Oehler S, Klinakis A, Savakis C (2005) Minos as a genetic and genomic
tool in Drosophila melanogaster. Genetics 171:571e581.
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