Download Drosophila Infections in for Resistance to Gram

Cutting Edge: The Toll Pathway Is Required
for Resistance to Gram-Positive Bacterial
Infections in Drosophila
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of June 16, 2017.
Sophie Rutschmann, Ahmet Kilinc and Dominique
Ferrandon
J Immunol 2002; 168:1542-1546; ;
doi: 10.4049/jimmunol.168.4.1542
http://www.jimmunol.org/content/168/4/1542
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References
●
Cutting Edge: The Toll Pathway Is Required for
Resistance to Gram-Positive Bacterial Infections in
Drosophila1
Sophie Rutschmann, Ahmet Kilinc, and Dominique Ferrandon2
T
he Drosophila host defense consists of both humoral and
cellular reactions (1). The cellular arm of the response is
mediated by hemolymphatic cells, plasmatocytes, that
phagocytose microorganisms. A septic injury triggers several protease cascades, some of which induce melanization reactions at the
site of injury. These reactions are thought to contribute to host
defense by the production of reactive oxygen species that may
participate in the killing of microorganisms (2, 3). However, an
essential feature of the humoral response is the secretion by the fat
body, a functional equivalent of the liver, of potent antimicrobial
peptides. These peptides are active against a limited range of microorganisms (4). For instance, Drosomycin is active on filamen-
tous fungi, whereas Diptericin was purified as an antiGram-negative peptide in the fly Phormia terranovae (5, 6).
Drosophila Diptericin and Attacin are thought to be active on
Gram-negative bacteria. In addition to its main anti-Gram-negative
activity, Cecropin has been reported to be also active on fungi (7).
Defensin is the peptide that displays the major anti-Gram-positive
activity in Drosophila (P. Bulet, personal communication) (8).
Mutants which affect the regulatory pathways that control the inducibility of the antimicrobial peptide genes have been characterized. Interestingly, such mutants were found to succumb rapidly to
microbial infections. These findings suggested, but did not demonstrate, that antimicrobial peptides are major effectors of Drosophila innate immunity.
Genetic analysis has shown that the expression of antimicrobial
peptide genes is controlled by at least two distinct pathways (1, 9).
The Toll pathway regulates the transcription of Drosomycin, and
partially that of Defensin (10 –12). This regulatory pathway appears to be preferentially activated by Gram-positive bacteria and
fungi (11, 13). Toll pathway mutant flies succumb rapidly to fungal
infections but not to a Gram-negative bacterial challenge (10, 11).
Conversely, flies mutated for genes of the immune deficiency
(imd)3 pathway are susceptible to Gram-negative infections, but
are as resistant as wild-type flies to natural infections with spores
of the entomopathogenic fungus Beauveria bassiana. The imd
pathway controls the expression of all known Drosophila antibacterial peptides, including Diptericin and Defensin (14 –22). However, neither pathway appeared to be required for resistance
against Micrococcus luteus, a microorganism that was classically
used as a Gram-positive bacterial inducer (this work and Ref. 16).
In this report, we show that mutants of the Toll pathway, but not
of the imd pathway, succumb to other Gram-positive bacterial
infections.
Materials and Methods
Fly strains
Institut de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9022 du
Centre National de la Recherche Scientifique, Strasbourg, France
Received for publication October 29, 2001. Accepted for publication December
14, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Centre National de la Recherche Scientifique,
National Institutes of Health Grant 1PO1 AI44220-02, the French Ministère de
l’Education Nationale, de la Recherche et de la Technologie (Programme de Recherche
Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires).
2
Address correspondence and reprint requests to Dr. Dominique Ferrandon, Institut
de Biologie Moléculaire et Cellulaire, Unité Propre de Recherche 9022 du Centre
National de la Recherche Scientifique, 15, rue R. Descartes, F67084 Strasbourg, Cedex, France. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists
●
Fly cultures were grown on standard medium at 25°C. yellow (y) white (w)
DD1; cinnabar brown (cn bw) flies were used as wild-type control. They
carry both pdipt-LacZ and pdrom-GFP transgenes on the X chromosome
(DD1) (11). Dif1, key1, spzrm7, and Df(2L)J4b lines are described in Flybase
(http://flybase.harvard.edu/).
Septic injuries and survival experiments
Septic injuries were performed at 20°C by pricking adult flies with a thin
tungsten needle previously dipped into a concentrated culture of the following bacteria: Escherichia coli 1106, M. luteus (CIP A270), or a mixture
of both, or Enterococcus faecalis. All bacterial strains are generous gifts
3
Abbreviations used in this paper: imd, immune deficiency; Dif, dorsal-related immunity factor; IKK, I␬B kinase; TLR, Toll-like receptor, key, kenny; spz, spätzle.
0022-1767/02/$02.00
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In Drosophila, the response against various microorganisms
involves different recognition and signaling pathways, as well
as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the
defense against filamentous fungi and controls the expression
of antifungal peptide genes. The gene coding for the only
known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival
experiments to Gram-positive bacteria have been performed
with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such
infections. In this study, we report that the Toll pathway, but
not that of immune deficiency, is required for resistance to
other Gram-positive bacteria and that this response does not
involve Defensin. The Journal of Immunology, 2002, 168:
1542–1546.
The Journal of Immunology
1543
from H. Monteil (University Louis Pasteur, Strasbourg, France), except for
Bacillus megaterium and Bacillus thuringensis (CIP 53137) that were
kindly donated by J. Millet and A. Klier (Institut Pasteur, Paris, France).
Survival experiments were performed in the same conditions for each
genotype tested. Groups of 25 adult females, ages from 5 to 7 days, were
challenged with E. coli or E. faecalis, grown at 29°C for E. coli infections
and at 25°C for E. faecalis, and transferred to fresh vials every 2 days. The
flies that died within 3 h following the injury were not considered in the
analysis.
RNA preparation for Northern blot analysis
Northern blot analysis was conducted as previously described (14) except
that total RNA was prepared from 15 flies using a TRIzol (Life Technologies, Rockville, MD) extraction protocol.
Phagocytosis tests
Sensitivity of Toll pathway mutant flies to other Gram-positive
bacterial strains
The M. luteus strain we use was initially chosen for its low pathogenicity to humans. We therefore tested other Gram-positive
Results
The Drosophila systemic antimicrobial response is not necessary
to resist M. luteus infections
In the course of a large genetic screen of the second chromosome,
we have identified mutants in both pathways of the humoral response: the dorsal-related immunity factor (Dif) in the Toll pathway and kenny (key) in the imd pathway (11, 15). We and others
have previously shown that DIF is the transcription factor that
mediates the activation of the Toll pathway in adults (11, 12). key
encodes the Drosophila homologue of the vertebrate I-␬B kinase ␥
(IKK␥) gene. Its corresponding protein has been shown to form a
complex with the Drosophila IKK␤ kinase that phosphorylates the
Rel transcription factor Relish (23). Relish is the transactivator of
the imd pathway (18). Dif and key mutants present the usual phenotypes of strong loss of function mutations in their respective
pathways. In addition, both of them resist a challenge with M.
luteus (data not shown). To determine whether the response to
Gram-positive bacteria is dependent on both pathways as reported
by Leulier et al. (16) in a previous study, we constructed a Dif-key
double mutant strain. Thus, we had generated mutant lines that
inactivate one, the other, or both pathways in the same genetic
background: this allowed us to compare rigorously their phenotypes. We have analyzed the expression of the antimicrobial peptide genes in response to a challenge with a mixture of E. coli
(Gram negative) and M. luteus in these mutant flies. As expected,
the immune inducibility of all tested antimicrobial genes is drastically reduced in the Dif-key double mutants (Fig. 1). Remarkably,
we found that Dif-key double mutants are as resistant as our wildtype reference strain to M. luteus infection (data not shown). Since
the systemic antimicrobial response is virtually abolished in this
background, this finding suggests that the cellular response may be
sufficient to dispose of this microorganism (17).
FIGURE 2. Toll pathway mutants are sensitive to Gram-positive (E.
faecalis) but not to Gram-negative (E. coli) infections. A, Five- to 7-dayold flies were infected using a thin tungsten needle previously dipped in a
concentrated E. faecalis solution. Batches of 25 flies were kept in vials at
25°C, counted every day (D), and vials were changed every 2 days. Survival rates are expressed in percentage of flies still alive 3 h after injury.
These experiments have been repeated at least three times. Similar results
have been obtained with S. saprophyticus and P. acidolactici Gram-positive bacteria. The genotypes are indicated on the right. J4, deficiency which
uncovers Dif and dorsal (12). The Dif 1/J4 flies show that the susceptibility
to E. faecalis is not due to a second site mutation on the Dif chromosome.
B, An experiment similar to that described in A was conducted at 29°C with
the Gram-negative bacteria E. coli.
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Indian ink (Pébéo, Gemenos, France) diluted 1/50 in PBS was injected
using a Nanoject microinjector (Drummond Scientic, Broomall, PA) in
wild-type or Dif1 third instar larvae hemocoel. The phagocytosis of indian
ink by the sessile blood cells was monitored 2 h later. FITC-labeled Staphylococcus aureus were purchased from Molecular Probes (Eugene, OR)
and prepared according to the manufacturer’s instructions. Briefly, 32.2 nl
of FITC-labeled bacteria were injected in the abdomen on the ventral lateral side using a Nanoject (Drummond Scientific). If needed, 400 nl of a
0.4% trypan blue solution (Sigma-Aldrich, St. Louis, MO) was injected in
the thorax 30 – 45 min after the injection of bacteria. To saturate the phagocytic system, 200 nl of Surfactant-free Red CML latex beads (0.28 ␮M
diameter; Interfacial Dynamics, Eugene, OR) was injected in the abdomen
4 – 6 h before the experiment. Injected flies were observed using a Leica
MZ FLIII dissecting microscope and photographs were taken using a digital charge-coupled device Spot RT color camera (Diagnostic Instruments,
Sterling Heights, MI).
FIGURE 1. The immune inducibility of antimicrobial genes is drastically reduced in Dif-key double mutants. Batches of 15 females of each
indicated genotype have been injured with a mixture of Gram-negative (E.
coli 1106) and Gram-positive (M. luteus) bacteria. Northern blot analysis
was performed as described in Materials and Methods. The same Northern
blot has been hybridized sequentially to Diptericin, Cecropin, Attacin, Defensin, Metchnikowin, Drosomycin, and Ribosomal Protein 49 (RP 49)
probes. RP 49 was used as loading control. The genotypes and the time
points (in hours) are indicated at the bottom (0, unchallenged). WT ⫽
wild-type reference strain with a second chromosome marked by cinnabar
(cn) and brown (bw) in which the Dif and key mutants were generated.
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CUTTING EDGE: Toll-DEPENDENT RESISTANCE TO GRAM-POSITIVE INFECTIONS IN Drosophila
FIGURE 3. Induction of antimicrobial peptides genes by E. faecalis suggests that Defensin is not needed for Drosophila host defense against this
pathogen. Batches of 15 females of each genotype have been injured with E. coli or E. faecalis. Northern blot analysis has been performed as described
in Materials and Methods. The same Northern blot has been hybridized sequentially to Drosomycin, Defensin, Diptericin, and RP 49 probes. The genotypes
are indicated on the top, whereas the inducers and the time of incubation (in hours) are indicated at the bottom. U. C., unchallenged. The wild-type control
in this experiment is the cnbw/J4 transheterozygous strain.
Effectors of the Gram-positive antibacterial response?
It has been reported that Gram-positive bacteria are stronger inducers of the Toll pathway than Gram-negative bacteria (11, 13).
Thus, our finding that this pathway is also involved in the host
defense against Gram-positive infections sheds a new light on this
observation.
To correlate the E. faecalis sensitivity phenotype observed in
Toll pathway mutants to the pattern of antimicrobial peptide genes
expression, we performed Northern blot analysis on flies challenged with this microorganism (Fig. 3). As expected, we found
that Drosomycin is more strongly induced by E. faecalis than by E.
coli and that its expression is dramatically reduced in spz and Dif
flies, but not in key mutants. In agreement with previous experiments performed using M. luteus as an elicitor, the mild Diptericin
inducibility obtained with an E. faecalis challenge is abolished in
key, but not in spz and Dif flies. Strikingly, the expression of the
main anti-Gram-positive peptide gene, Defensin, reaches similar
low levels in all tested mutant backgrounds after immunization
with E. faecalis. We conclude that Defensin is not necessary for
resistance against this Gram-positive bacterium since key mutants
do not express Defensin at high levels, yet are resistant to this
microorganism. This observation suggests that other presently unknown mechanisms could play a role in the defense against this
infection. Indeed, several peptides with unidentified function were
detected in the hemolymph of infected flies, and most of these are
controlled by the Toll pathway (25). None of these Toll-controlled
peptides appear to have an obvious antimicrobial activity (P. Bulet,
FIGURE 4. Phagocytosis of FITC-labeled S. aureus is not affected in Dif mutant flies. A–D, Wild-type flies at 64-fold magnification. E–H, Dif1 mutant
flies at 64-fold magnification. A and E, Flies injected with FITC-labeled S. aureus. The fluorescence is seen all over the fly, but is concentrated in multiple
foci that may correspond to islands of sessile hemocytes. These are easy to visualize on the dorsal vessel. B and F, Same as A and E except that trypan
blue was injected 30 min after the injection of labeled bacteria. Most of the background fluorescence has disappeared except in the sessile hemocytes.
Exposure time has been doubled as compared, respectively, to A and E. C and G, Same as A and E except that flies have been previously injected with
latex beads to saturate the phagocytic apparatus of hemocytes. The fluorescence is no longer concentrated in foci and single bacteria can be seen flowing
in the hemolymph, especially in the transparent legs and wings. D and H, Same as C and G except that trypan blue has been injected 30 min after the
injection of labeled bacteria. The remaining fluorescence is much weaker.
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strains: Bacillus megaterium, Staphylococcus hemolyticus, Pediococcus acidolactici, E. faecalis, Staphylococcus saprophyticus,
and B. thuringiensis. The first two strains behaved as M. luteus. In
contrast, flies infected with B. thuringiensis succumbed too rapidly
to note a significant difference between the different mutant fly
strains. However, for P. acidolactici, E. faecalis, and S. saprophyticus infections, we observed that Dif mutant flies died more rapidly than wild-type control and key flies (Fig. 2 and data not
shown). Interestingly, Dif-key mutant flies succumb at the same
rate as Dif flies, indicating further that the key gene is not required
for resistance to such Gram-positive infections.
To ascertain whether the whole Toll pathway is required for the
resistance to these Gram-positive bacteria, we tested mutants of the
most upstream known component of this pathway: spätzle (spz)
(24). This gene codes for the putative ligand of the Toll receptor.
Indeed, spz mutant flies are also sensitive to E. faecalis infections
(Fig. 2). We conclude from these experiments that the Toll pathway is necessary for resistance against Gram-positive bacterial infections, whereas the imd pathway appears to be dedicated to the
response against Gram-negative bacteria.
The Journal of Immunology
Conclusion
In summary, we have shown that the Toll pathway is required for
resistance to some Gram-positive bacterial infections and that the
imd pathway is not. The former had been previously shown to play
a fundamental role in the defense against filamentous fungi. In
addition to its immunological functions, this pathway also controls
the establishment of dorso-ventral polarity in the Drosophila embryo (24, 27). In vertebrates, the Toll pathway has been implicated
in the activation of NF-␬B following the detection of microbial
components by several distinct members of the Toll-like family of
receptors (TLR) (28 –30). For instance, TLR4 mediates the activation of NF-␬B by LPS, the main component of the outer membrane of Gram-negative bacteria, whereas TLR2 plays a similar
role in response to peptidoglycan, the major constituent of the
Gram-positive cell wall (31, 32). In contrast, the same Toll receptor is required for the response to both fungal and Gram-positive
bacterial elicitors in Drosophila. This suggests that in this system,
the recognition step that discriminates between these distinct
pathogens takes place further upstream of the Toll receptor. Indeed, the peptidoglycan recognition protein SA has recently been
shown to play a major role in the activation of the Toll pathway
(33). Although the mechanisms that allow the identification of
Gram-positive bacteria have long been unknown but are beginning
to be deciphered, we paradoxically do not yet understand well the
effector arm of the Drosophila immune response. Indeed, the systemic antimicrobial response (through Defensin) and the cellular
response do not appear to play a major role in the defense against
some virulent Gram-positive bacterial strains. A major challenge
of the coming years will be to understand how the Toll pathway
manages its multiple roles in Drosophila host defense.
Acknowledgments
We thank Jules Hoffmann for his continuous interest in our work;
J. Hoffmann, Marie Gottar, and Vanessa Gobert for critical reading of this
manuscript; and René Lanot for help with the phagocytosis test. Martine
Schneider and Raymonde Syllas are gratefully acknowledged for their
technical help.
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