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Originally published In Press as doi:10.1074/jbc.M313856200 on February 25, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21121-21127, May 14, 2004
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Differential Activation of the NF-{kappa}B-like Factors Relish and Dif in Drosophila melanogaster by Fungi and Gram-positive Bacteria*

Marika Hedengren-Olcott{ddagger}§, Michael C. Olcott||, Duane T. Mooney**, Sophia Ekengren{ddagger}{ddagger}§§, Bruce L. Geller**, and Barbara J. Taylor{ddagger}

From the Departments of {ddagger}Zoology, ||Biochemistry and Biophysics, and **Microbiology, Oregon State University, Corvallis, Oregon 97331, the §Wenner-Green Institute, Stockholm University, S-106 91 Stockholm, Sweden, and the {ddagger}{ddagger}Umeå Centre for Molecular Pathogenesis, Umeå University, S-901 87 Umeå, Sweden

Received for publication, December 18, 2003 , and in revised form, February 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G+) bacteria activate the Toll/Dif pathway and that Gram-negative (G-) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G+ and G- bacteria. In Relish mutants, the Cecropin A gene was induced by the G+ bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G+ or G- bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G+ bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G+ bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G+ bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interest in innate immunity has increased dramatically in recent years after the discovery of Toll-like receptors (TLRs)1 in mammals (1–3). Mammalian TLRs are considered pattern recognition receptors (PRRs) involved in immune responses triggered by interaction of the TLR and/or coreceptor with specific microbial macromolecules such as lipopolysaccharide (LPS), peptidoglycan (PG), unmethylated CpG DNA, and double-stranded RNA.

First described in a screen for mutations affecting development in Drosophila melanogaster (4), the Toll receptor was later shown to be an important component of the Drosophila immune response (5, 6). Drosophila has proven to be an excellent model system in which to study innate immunity (7–10). Unlike mammalian TLRs, none of the nine Drosophila TLRs described to date appear to bind directly to microbial macromolecules. Instead, a group of at least 13 proteins similar to the peptidoglycan recognition proteins (PGRPs) of Bombyx mori, Trichoplusia ni, rats, and humans are present in Drosophila (11–16). These PGRPs, along with Gram-negative (G-) bacteria-binding protein and scavenger receptor class C, type I, are thought to represent the PRRs of Drosophila. Some of these Drosophila PRRs interact directly with bacterial macromolecules (17, 18).

PGRP-SA, PGRP-LC, and PGRP-LE have been shown to regulate the induction of peptides in Drosophila through one of two signal transduction pathways (19–22). One of these pathways, the Toll pathway, is involved in the induction of the antifungal peptide Drosomycin. Dif (Dorsal-related immunity factor) is a downstream transcription factor activated by Toll signaling that leads to Drosomycin expression, as was demonstrated in unchallenged adult flies carrying a constitutively active form of Toll (Toll10B) (23, 24). The second pathway, the Imd (immune deficiency) pathway, including the transcription factor Relish, is involved in the induction of the antibacterial peptides Diptericin and Cecropin A (25, 26). Early work suggested that the pattern recognition and induction of antimicrobial peptides in Drosophila involve the selective activation of either the Toll/Dif or Imd/Relish pathway depending on the Gram type of bacteria used for induction (9). It was proposed that G- bacteria stimulate the Imd/Relish pathway, whereas Gram-positive (G+) bacteria and fungi stimulate the Toll/Dif pathway. However, it was subsequently demonstrated that both pathways are activated upon exposure to G+ bacteria (27).2

Previously, we reported that flies lacking Relish are sensitive to bacterial infection and typically die within 17 h after injection with the G- bacterium Enterobacter cloacae (26). The sensitivity to this bacterial infection is likely due to the observed deficiency in antimicrobial peptide production, particularly Cecropin A and Diptericin A. The induction of these genes was not detectable by Northern blot analysis of Relish mutant flies following infection with E. cloacae. However, when injected with the G+ bacterium Micrococcus luteus, these Relish-deficient flies survived, and Cecropin A was induced (28).2 In this study, we sought to identify the putative pathogen-associated molecular pattern (PAMP) of M. luteus responsible for this Relish-independent induction of Cecropin A. Furthermore, we broadened the study by analyzing the expression of Cecropin A and other antimicrobial peptides in Relish and/or Dif mutants to examine the specific induction of the two signal transduction pathways by various bacteria, fungi, and microbial components.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purified Components—Escherichia coli 055:B5 LPS, poly(I)·poly(C), and Laminaria digitata laminarin were purchased from Sigma. Lipoteichoic acid from Staphylococcus aureus was from Biotrend (Cologne, Germany). The adjuvant S-[2,3-bis(palmitoyloxy)-(2R,2S)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH trihydrochloride (N-lipoprotein) was purchased from Roche Applied Science.

Drosophila Stocks and Microbes—Canton-S (29), RelishE20 (26), Dif2 (24), and Dif2/RelishE20 double mutant and pA10 Cecropin A1-lacZ reporter transformant (30) strains of D. melanogaster were maintained at 25 °C. The Dif2/RelishE20 double mutant strain was generated by standard methods using balancer strains w[*];If/CyO and w[*]; TM3,Sb(1)Ser(1)/TM6B,Tb (strain 2537; Bloomington Stock Center, Indiana University). The Gram- bacteria used were E. cloacae {beta}12 (31); E. coli D21 (31, 32); Erwinia caratovora CC101, CC301, and XVII (gifts from Robin Ludy, Oregon State University); and Pseudomonas aeruginosa OT97 (31). The Gram+ bacteria used were M. luteus 11 (33), Bacillus megaterium 11 (34), Bacillus subtilis 11 (31, 35), Bacillus thuringiensis 5 (31), Bacillus cereus 11 (36), S. aureus SA113 (gift from Dr. Dennis Hruby, Oregon State University), Lactobacillus acidophilus NCK1070 (gift from Dr. Todd Klaenhammer, North Carolina State University, Raleigh, NC), Lactobacillus reuteri 4020 (Biogaia, Inc., Raleigh), Lactococcus lactis C2 (37), and teichuronic acid (TUA)-deficient M. luteus AH-149 (38). The fungi Metarhizium anisopliae (strain 464.70), Beauvaria bassiana (strain 118.30), and Geotrichum candidum (strain 606.85) were obtained from the Centraalbureau voor Schimmel-cultures (Baarn, The Netherlands). Saccharomyces cerevisiae (strain W308) was a gift from Dr. Stefan Åström (Umeå University, Umeå, Sweden).

Preparation of Bacterial Cell Wall Components—Cell walls from G+ bacteria were extracted in boiling SDS as described previously (39). Insoluble PGs prepared from M. luteus, B. megaterium, and B. subtilis as described (40) were a gift from Thomas Werner (Umeå University). TUA was purified as described previously (41). M. luteus genomic DNA was extracted from a 10-ml overnight culture according to Flamm et al. (42). A supernatant from M. luteus overnight cultures was collected by centrifugation. The absence of bacteria was verified by plating supernatants on agar plates.

Preparation of Heat-killed and Formaldehyde-fixed Cells—Three 1-ml samples from an overnight culture of M. luteus were centrifuged at 13,000 x g for 1 min, and the pellet was suspended in 1 ml of water. One sample was autoclaved. Another sample was brought to 4% formaldehyde and incubated for 1 h at room temperature. The third sample was untreated. The cells were collected by centrifugation as described above and suspended in 1 ml of water. Prior to injection, samples were collected by centrifugation and then suspended in 10 ml of Tübingen and Düsseldorf Drosophila Ringer's solution (43).

Lysozyme and Proteinase K Treatment—Cells from 10 ml of an overnight culture were collected by centrifugation, washed once with 1.5 ml of phosphate-buffered saline (PBS) containing 10% sucrose (PBS+S), and resuspended in 1.5 ml of PBS+S. Lysozyme (3 mg) was added to one 0.5-ml aliquot. Proteinase K (32 µg) was added to a second 0.5-ml aliquot. The third was untreated. All aliquots were incubated at 37 °C for 30 min. Pellets were collected by centrifugation, washed twice with 1.5 ml of PBS+S, and suspended in 1.5 ml of PBS+S. Prior to injection, samples were collected by centrifugation and then suspended in 10 ml of Ringer's solution.

Immune Challenge and Expression Studies—Injections were done as described previously (26). Bacterial M. luteus and L. lactis strains were grown at 30 °C in Luria broth and M17 broth, respectively. The Lactobacillus strains were grown anaerobically in MRS broth at 37 °C. All other bacterial strains were grown at 37 °C in Luria broth. Harvested bacteria and fungi were washed with Ringer's solution prior to injection. Extracted material and other test compounds were diluted to 10 µg/ml in sterile Ringer's solution before injection. LPS was pretreated by heating at 62 °C for 1 h before injection. Uninjected and injected adult flies were kept at room temperature (22–24 °C) for the indicated times before being frozen in liquid N2 or on dry ice. Total RNA was extracted with TRIzol (Invitrogen). For Northern analysis, 6–15 µg of total RNA was resolved on a MOPS/formaldehyde-agarose (1%) gel, blotted onto a nylon membrane (Roche Applied Science) or Hybond-XL filter (Amersham Biosciences), and hybridized under stringent conditions according to the manufacturers' recommendation or to conventional protocols (44, 45). Probes were derived from DNA clones (46–51) using digoxigenin or [{alpha}-32P]dCTP and the DIG RNA labeling kit (Roche Applied Science) or the rediprime DNA labeling system (Amersham Biosciences), respectively. For reverse transcription (RT)-PCR analysis, the SuperScript One-Step RT-PCR Platinum Taq kit (Invitrogen) was used with gene-specific primers for Cecropins A1 and A2. RT-PCR was performed on 1 µg of DNase I-treated total RNA prepared using a DNA-free kit (Ambion Inc.). {beta}-Galactosidase staining was performed as described previously (52).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two of Eight G+ Bacteria Induce Cecropin A in Relish Mutants—To assess the correlation between Gram stain and the induction of different immune response pathways in Drosophila, we injected various G+ or G- bacteria into Relish mutant flies and examined the induction of four antimicrobial peptide genes, Cecropin A, Diptericin A, Attacin A, and Drosomycin (Fig. 1). Uninjected flies served as a control for basal expression. Injection with sterile Ringer's solution served as a control for wounding and the injection procedure. As a Relish+ control, we used Canton-S flies and found a small increase in the levels of Cecropin A, Diptericin A, and Attacin A upon injection of sterile Ringer's solution. All bacteria, regardless of Gram stain, induced Cecropin A, Diptericin A, and Attacin A above the levels observed in the Ringer's control in Canton-S flies. Some bacteria, including M. luteus and S. aureus, induced Drosomycin above the level observed in the Ringer's control. In Relish mutants, only the G+ bacteria M. luteus and S. aureus elicited a substantial induction of Cecropin A above that in the Ringer's control. In a separate experiment, Lactobacillus strains 1070 and 4020 failed to elicit an induction of Cecropin A in Relish mutants (data not shown). As shown in Fig. 1, none of the G+ or G- bacteria induced Diptericin A mRNA in Relish mutant flies. Expression of Attacin A and Drosomycin was induced above that in the Ringer's control by M. luteus and S. aureus (but not by the other bacteria) in Relish mutants.



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  		FIG. 1.
Effect of various G+ and G- bacteria on the gene expression of four antimicrobial peptides. Shown are the results from Northern blot analysis of the expression of Cecropin A (CecA), Diptericin A (DptA), Attacin A (AttA), and Drosomycin (Drs) in wild-type (Canton-S) and Relish mutant (RelE20) adult flies 3 h after injection with bacteria. Uninjected flies served as a control for base-line expression. Flies injected with sterile Ringer's solution served as a control for wounding and the injection procedure. RpL32 was included as a loading control. The Gram type of bacteria are indicated.

 
Relish-independent Cecropin A Induction Is Abolished in a Dif/Relish Double Mutant—The induction of Cecropin A by M. luteus and S. aureus in Relish mutant flies indicated that a transcription factor other than Relish is involved in the response to these bacteria. Because Dif is a transcription factor in the Toll pathway, we generated a Dif2/RelishE20 double mutant line deficient in both transcription factors to examine the relative contributions of each pathway. Cecropin A mRNA was analyzed by Northern blotting following the injection of Dif mutant, Relish mutant, or Dif/Relish double mutant flies with E. cloacae, M. luteus, or S. aureus (Fig. 2A). Substantial induction of Cecropin A was observed in Canton-S and Dif mutant flies injected with the G- bacterium E. cloacae compared with the Ringer's control. However, Cecropin A was not induced by this bacterium in Relish mutant and Dif/Relish double mutant flies. The G+ bacteria M. luteus and S. aureus induced Cecropin A in Canton-S, Relish mutant, and Dif mutant flies, but not in Dif/Relish double mutant flies.



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  		FIG. 2.
Role of the transcription factors Relish and Dif in the expression of Cecropin A. A, Northern blot analysis of the expression of Cecropin A (CecA) 3 h after the injection of Canton-S (wild-type control), Dif2 mutant, RelishE20 (RelE20) mutant, and Dif2/RelishE20 (Dif2/RelE20) double mutant adult flies with E. cloacae (G-), M. luteus (G+), or S. aureus (G+). B, expression of Cecropin A in unchallenged or challenged Toll gain-of-function (Toll10B (Tl10b)) flies. Flies were injected with G- E. cloacae or G+ M. luteus. Uninjected flies served as a control for base-line expression. Flies injected with sterile Ringer's solution served as a control for wounding and the injection procedure. rRNA stained with ethidium bromide was used as a loading control.

 
Since Dif is the transcription factor involved in Toll-activated Drosomycin expression, we analyzed Cecropin A expression in flies carrying a gain-of-function form of Toll (Toll10B). Cecropin A was not constitutively expressed in the absence of challenge in the Toll10B mutant, in contrast to Drosomycin (Fig. 2B) (6). However, Cecropin A expression was strongly induced by both E. cloacae and M. luteus.

The Inducing Factor from M. luteus Is a Component of the Cell Wall—To characterize the relative contribution of various components of the M. luteus bacterium in the induction of Cecropin A, we injected Relish mutants with genomic DNA, cell wall extracted with SDS, growth medium supernatant, or formaldehyde-fixed or autoclaved M. luteus and then assessed Cecropin A mRNA induction relative to the Ringer's control. As shown in Fig. 3A, the inducing factor for Cecropin A was present in cell walls and in autoclaved or formaldehyde-fixed M. luteus, but not in growth medium or genomic DNA.



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  		FIG. 3.
Identification of the inducing component of M. luteus. A, Northern blot analysis of Cecropin A (CecA) expression in Canton-S and RelishE20 (RelE20) mutant flies injected with one of the following derived from M. luteus: genomic DNA (gDNA), SDS-treated cell walls, growth medium supernatant (sup), whole cells, and formaldehyde-fixed or heat-killed cells. B, Northern blot analysis of Cecropin A expression in Canton-S or RelishE20 mutant flies injected with M. luteus cells, M. luteus cells treated with lysozyme or proteinase K, or a TUA-deficient strain of M. luteus (M. l AH-149). In both experiments, total RNA was extracted 3 h after injection. Uninjected flies and flies injected with Ringer's solution, Luria broth (LB), enzyme (Enz.) buffer, or Gram- E. cloacae were used as controls. RpL32 was included as a loading control.

 
To further differentiate the role of various components in the Relish-independent induction of Cecropin A, M. luteus cells were treated with lysozyme or proteinase K. Lysozyme hydrolyzes the polymeric backbone of PG. Proteinase K is a nonspecific protease. Treatment with lysozyme abolished Cecropin A induction in both wild-type and Relish mutant flies, whereas proteinase K had no effect (Fig. 3B).

Specific Recognition of M. luteus Peptidoglycan—Injection of cell wall or PG extracts from various G+ bacteria induced Cecropin A in Canton-S flies (Fig. 4A) relative to the Ringer's control. Only whole cells, cell wall, or PG from M. luteus induced Cecropin A in Relish mutants. By contrast, cell wall from G+ B. megaterium and PGs from B. megaterium and B. subtilis did not induce Cecropin A in Relish mutant flies. These results are consistent with the response to intact bacteria (Fig. 1).



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  		FIG. 4.
Identification of the inducing component. A, Northern blot analysis of Cecropin A (CecA) expression in Canton-S or RelishE20 (RelE20) mutant flies injected with cell walls (CW) and/or PGs extracted from G+ bacteria M. luteus, B. megaterium, and B. subtilis. B, Northern blot analysis of Cecropin A expression in Canton-S or RelishE20 mutant adult flies injected with TUA, lipoteichoic acid (LTA), genomic DNA (gDNA), poly(I)·poly(C) (poly I:C), LPS, laminarin, or a hexapeptide derived from the N terminus of bacterial lipoproteins (N-lipoprotein). In both sets of experiments, total RNA was extracted 3 h after injection. Uninjected flies served as a control for base-line expression. Flies injected with sterile Ringer's solution served as a control for wounding and the injection procedure.

 
We also tested the ability of other purified bacterial cell wall components to induce Cecropin A in wild-type and Relish mutants. These substances included purified TUA and genomic DNA from M. luteus, lipoteichoic acid from S. aureus, LPS from E. coli, laminarin from L. digitata, a lipohexapeptide derived from the immunologically active N terminus of bacterial lipoproteins (N-lipoprotein), and poly(I)·poly(C). Of the substances tested in Canton-S flies, LPS elicited the strongest induction of Cecropin A. A small induction above that in the Ringer's control was observed upon injection of TUA from M. luteus and N-lipoprotein. None of the substances stimulated Cecropin A expression above the Ringer's control levels in the Relish mutant flies (Fig. 4B). To eliminate the possibility that TUA has to be covalently linked to the cell wall to induce Cecropin A in Relish mutants, we injected Relish mutant flies with a strain of M. luteus (AH-149) that lacks TUA. As shown in Fig. 3B, these bacteria induced Cecropin A in Relish mutant flies.

Cecropin A2 Is Induced by M. luteus in Flies Lacking Relish—Cecropin A peptides are encoded by two separate genes in Drosophila. The two genes, Cecropins A1 and A2, are under the control of different promoters (46). Standard Northern blot analysis does not distinguish between Cecropin A1 and A2 gene expression. Previous studies that demonstrated that Cecropin A is induced by both the Imd/Relish and Toll/Dif pathways were in reality examining the collective expression of both genes (6, 26). To examine the specific induction of Cecropin A1 in Relish mutants after infection by M. luteus, a Cecropin A1-lacZ reporter construct (30) was crossed into a Relish mutant background. These flies were injected with G+ M. luteus. Uninjected flies (data not shown) and flies injected with Ringer's solution were used as controls. Expression of {beta}-galactosidase was detected in the Cecropin A1-lacZ reporter in Relish+ flies following injection with M. luteus (Fig. 5A). However, {beta}-galactosidase activity was not observed in Relish mutants containing the Cecropin A1-lacZ reporter injected with M. luteus. This suggests that M. luteus may induce Cecropin A2 in Relish mutants. To test this hypothesis, we performed non-quantitative RT-PCR with gene-specific primers for Cecropin A1 or A2. Cecropin A2 (but not Cecropin A1) was detected in Relish mutants following M. luteus infection (Fig. 5B).



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  		FIG. 5.
Differential expression of Cecropins A1 and A2. A, Cecropin A1 (CecA1)-lacZ reporter-containing transgenic flies with or without the RelishE20 (RelE20) mutation were injected with Ringer's solution or M. luteus. The abdomens were cut off, and the {beta}-galactosidase expression in the reporter construct was visualized with 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal). Upper panels, mid-abdominal segment, with the anterior up and dorsal midline to the right; lower panels, extruded fat body from the same animal. B, shown are the results from RT-PCR analysis of total RNA isolated 3 h after injection of Canton-S or RelishE20 mutant adult flies with E. cloacae or M. luteus. RT-PCR was performed on DNase-digested total RNA extracts using primers specific for Cecropin A1 or A2. RNAs from uninjected flies and from flies injected with Ringer's solution were used as controls. Genomic DNA (gDNA) extracted from Canton-S flies was used as a positive PCR control. The PCR product was visualized with ethidium bromide.

 
Are There Pathways Other than Toll/Dif and Imd/Relish Involved in the Antimicrobial Response?—To determine whether other pathways can be activated by M. luteus, S. aureus, and E. cloacae, we examined the expression levels of Diptericin B, Attacin C, and Drosomycin after challenging Canton-S, Relish mutant, or Dif/Relish double mutant flies with these bacteria (Fig. 6). Like Cecropin A, Diptericin B was induced by M. luteus and S. aureus in a Relish mutant, but was not expressed in the Dif/Relish double mutant. This was in contrast to the expression of Diptericin A (Fig. 1) and suggests that there are differences in the induction mechanisms for Diptericins A and B. In contrast to Cecropin A and Diptericin B, some induction of Attacin C above the Ringer's control levels was observed in the Dif/Relish double mutant. As also shown in Fig. 1, some basal expression of Drosomycin was seen in uninjected and Ringer's solution-injected Canton-S flies. However, a clear induction of Drosomycin was detected in these flies after injection with G- E. cloacae or G+ M. luteus or S. aureus. This induction was reduced in Relish single and Dif/Relish double mutants.



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  		FIG. 6.
Expression of antimicrobial peptide genes in flies mutant for the transcription factors Relish and/or Dif. Canton-S (wild-type control), Dif 2 mutant, RelishE20 (RelE20) mutant, and Dif 2/RelishE20 (dif 2/RelE20) double mutant adult flies were injected with G- E. cloacae or G+ M. luteus or S. aureus. Total RNA was isolated for Northern analysis 3 h after the injections. Uninjected flies served as a control for base-line expression. Flies injected with sterile Ringer's solution served as a control for wounding and the injection procedure. CecA, Cecropin A; DptB, Diptericin B; AttC, Attacin C; Drs, Drosomycin.

 
Specific Pathways for Antifungal Defense—Only the Toll pathway has been reported to respond to fungi (9). However, we previously reported that flies lacking Relish are more sensitive to fungi than flies expressing Relish (26). To investigate the relative importance of Dif and Relish in the response to fungi, we injected various fungi into Relish mutant, Dif mutant, and Dif/Relish double mutant flies and then examined the induction of Cecropin A, Diptericin A, Attacin A, and Drosomycin relative to the expression after injection with Ringer's solution (Fig. 7). Whereas all four antimicrobial peptide genes were induced in Canton-S flies by all fungi tested, the induction of individual peptides appeared to differentially involve the activity of Relish and Dif. Most striking was the G. candidum injection, where Cecropin A and Diptericin A were not induced in either the Relish mutant or the Dif/Relish double mutant, but were unaffected in the Dif mutant. In both transcription factor mutants, Cecropin A induction was severely reduced or abolished after infection with M. anisopliae, B. bassiana, or S. cerevisiae. None of the fungi induced Diptericin A in Relish single or Dif/Relish double mutants. In Dif mutants, Diptericin A induction was observed following injection with B. bassiana, G. candidum, or S. cerevisiae, but not after injection with M. anisopliae. Only B. bassiana and G. candidum induced Attacin A in a Relish or Dif mutant, respectively. The induction of Attacin A was suppressed in Relish or Dif mutants after injection with the other fungi. A clear induction of Drosomycin was visible in Canton-S flies despite the levels of Drosomycin in the Ringer's control. This induction was reduced in Dif mutants following injection with S. cerevisiae, B. bassiana, or M. anisopliae and possibly in Relish mutants after injection with G. candidum or S. cerevisiae. For the four antimicrobial peptides, induction was reduced or abolished after injection with any of the four fungi in the Dif/Relish double mutant.



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  		FIG. 7.
Expression of antimicrobial peptides after injection with different fungi. Canton-S (wild-type control), Dif 2 mutant, RelishE20 (RelE20) mutant, and Dif 2/RelishE20 (Dif 2/RelE20) double mutant adult flies were injected with M. anisopliae, B. bassiana, G. candidum, or S. cerevisiae. Total RNA was isolated for Northern analysis 3 h after injection. Uninjected flies served as a control for base-line expression. Flies injected with sterile Ringer's solution served as a control for wounding and the injection procedure. CecA, Cecropin A; DptA, Diptericin A; AttA, Attacin A; Drs, Drosomycin. RpL32 was included as a loading control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here reveal a greater level of complexity in the Drosophila immune system than previously reported. It has been evident that activation of different immune pathways in Drosophila is dependent upon the type of infecting microorganism. For example, it has been speculated that fungi and G+ bacteria activate the Toll/Dif pathway, whereas G- bacteria activate the Imd/Relish pathway (8, 9). In this study, we have demonstrated that Cecropin A induction is compromised in Relish mutants following injection with G- bacteria, as expected. Of the G+ bacteria tested, only M. luteus and S. aureus induced Cecropin A in Relish mutants (Fig. 1). The results depicted in Figs. 1, 2, and 4A indicate that the G+ bacteria B. megaterium and B. subtilis induce Cecropin A expression via activation of the Imd/Relish pathway, whereas M. luteus appears to utilize both the Imd/Relish and Toll/Dif pathways (Fig. 8A). Interestingly, all three bacteria are G+. These results indicate that some G+ bacteria activate the Imd/Relish pathway and that subsets of PRRs that can distinguish between various G+ bacteria exist in Drosophila.



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  		FIG. 8.
Abbreviated model of the selective immune activation in adult Drosophila by various bacteria and fungi. Various PAMPs on bacteria and fungi are recognized by PRRs in the Drosophila immune system, leading to induction of antimicrobial peptides via differential activation of transcription factors. The Toll/Dif and Imd/Relish pathways are stimulated by different PAMPs, leading to differential induction of antimicrobial peptides. We have not ruled out that additional factors and pathways exist for induction of antimicrobial peptides. A, immune activation by bacteria; B, immune activation by fungi. See "Discussion" for details. TF indicates an unknown transcription factor that can be Relish, Dif, or another transcription factor. The question mark indicates a possible interaction. CecA1 and CecA2, Cecropins A1 and A2, respectively; Drs, Drosomycin; AttA and AttC, Attacins A and C, respectively; DptA and DptB, Diptericins A and B, respectively.

 
Our findings demonstrate that some component from the cell envelope of G+ bacteria is needed to induce the immune response. Fixed or heat-killed M. luteus cells stimulated Relish-independent induction of Cecropin A, whereas the supernatants from M. luteus cell cultures had no effect (Fig. 3A). Furthermore, treatment of M. luteus cells with lysozyme abolished the induction of Cecropin A in wild-type flies (Fig. 3B). This indicates that intact PG polysaccharide is necessary for recognition by the fly immune system. Indeed, insoluble PGs purified from M. luteus, B. megaterium, and B. subtilis induced Cecropin A in wild-type flies (Fig. 4A). However, of these PGs, only M. luteus PG could induce Cecropin A in a Relish mutant.

The chemical composition of the polysaccharide backbone of PG is nearly invariant among all bacteria (53). However, TUA, which is covalently linked to the PG backbone, varies in composition in M. luteus, B. megaterium, and B. subtilis (39); yet M. luteus mutants lacking TUA induced Cecropin A in Relish mutants (Fig. 3B), whereas an extract containing TUA from M. luteus did not induce Cecropin A in these mutants (Fig. 4B). These results suggest that TUA is not responsible for the Cecropin A induction in Relish mutant flies after injection with M. luteus. Another variation of the PGs of B. megaterium, S. aureus, and M. luteus is the PG peptide and cross-bridge (53). However, it is possible that the peptide moiety itself is not the recognized PAMP, but instead may specify an overall architecture of the PG required for recognition by the immune system of Drosophila. A comparison of the crystal structure of Drosophila PGRP-LB with models of other PGRPs from the same species reveals a putative PG-binding site containing varying residues on the surface (54). If the binding site on the PGRP has an inflexible defined geometry, as has been described for a tunicate C-type lectin (55), the varying surface residues on the PGRP could account for the distinctive binding of different PGs. Interestingly, the crystal structure also reveals a conserved putative protein-binding site on the PGRP molecule. Some mammalian cytokines have carbohydrate binding properties through their carbohydrate recognition domain located on the opposite side of the receptor-binding domain (56). It is possible that the function of some PGRPs mimics the function of the carbohydrate recognition domain-containing cytokines or vice versa.

Our observation that Cecropin A2 (but not Diptericin A) was induced in a Relish mutant (Figs. 1 and 5) indicates a redundancy in the immune recognition of M. luteus by Drosophila PRRs. PGRP-SA is a PRR that binds with high affinity to insoluble PG from M. luteus (15). As indicated by the model in Fig. 8A, previous studies have revealed that PGRP-SA is a component of the Toll/Dif pathway. For example, mutants of PGRP-SA (Semmelweis) affect the induction of Drosomycin after an M. luteus infection (19), suggesting that M. luteus activates the Toll/Dif pathway. However, the observation that Diptericin A was not induced in a Relish mutant suggests that M. luteus also activates the Imd/Relish pathway. Diptericin A induction is reduced in a PGRP-LC mutant (ird7) after injection with M. luteus (20), and epistasis experiments placed PGRP-LC upstream of Imd (21). These data suggest that activation of the Imd/Relish pathway by M. luteus is via PGRP-LC (Fig. 8A). Since reports about Diptericin A induction by M. luteus PG are conflicting (20, 57), it is possible that a molecule other than PG from M. luteus is responsible for activation of the Imd/Relish pathway. Taken together, these results indicate that both PGRP-SA and PGRP-LC are involved in immune activation by M. luteus and possibly by its PG (Fig. 8A).

In contrast to PG from M. luteus, PGs from B. megaterium and B. subtilis failed to induce Cecropin A in a Relish mutant (Fig. 4A). Takehana et al. (27) reported that PGRP-LE binds to the diaminopimelic acid-type (A1{gamma}) PG, but not to the lysine-type (A2) PG. Our data support their findings and indicate that PGRP-LE acts upstream of Relish and is responsible for binding PG from B. megaterium or B. subtilis (Fig. 8A). Therefore, both PGRP-LC and PGRP-LE can regulate Relish. In light of other experiments (19–22, 27), our experiments suggest that PGRP-SA and PGRP-LC are involved in activation of the Toll/Dif and Imd/Relish pathways, respectively, by M. luteus. In addition, it is likely that PGRP-LE recognizes B. megaterium and B. subtilis upstream of the Imd/Relish pathway (Fig. 8A). Analysis of a loss-of-function mutant of PGRP-LE would provide further support for this model.

Our experiments suggest that the Imd/Relish pathway regulates Cecropin A1 expression and that the Toll/Dif pathway regulates Cecropin A2 expression (Fig. 8A). The induction of Cecropin A1 by all bacteria tested required the participation of Relish, whereas the induction of Cecropin A2 by M. luteus and S. aureus required the participation of at least Dif when tested in a Relish mutant background (Figs. 1, 2, and 5). However, it is possible either that activation of Dif by M. luteus or S. aureus is independent of Toll or that a second Toll-independent signal is required for the induction of the Cecropin A2 gene (Fig. 8A). Although Dif was shown to be the transcription factor involved in Toll-induced Drosomycin expression in the Toll10B mutant (24), our work (Fig. 2B) and the work of others (6) have shown that unchallenged adult flies carrying a constitutively active form of Toll (Toll10B) do not express Cecropin A. These results suggest that some factor in addition to Dif is required for the induction of the Cecropin A2 gene (Fig. 8A).

Our observation that Attacin C (but not Cecropin A and Diptericin B) could still be induced in a Dif/Relish double mutant by E. cloacae and M. luteus (Fig. 6) suggests that there may be a pathway for Attacin C induction independent of Relish and Dif (Fig. 8A). One possible constituent of a third pathway is 18-wheeler. This TLR has been shown to regulate the induction of Attacins A and C, but has yet to be placed in a pathway (49, 58). Genetic screening experiments involving 18-wheeler and/or Attacin C could be useful in determining the role of 18-wheeler in Attacin C induction as well as identifying other components of such a pathway.

Dnup88 is another protein that is known to regulate antimicrobial peptide expression and that has yet to be placed in a pathway. Interestingly, a mutant of this nuclear pore complex protein affects the induction of Diptericin A (59), although Relish can still translocate to the nucleus in this mutant,3 suggesting that a factor in addition to Relish is important for Diptericin A induction (Fig. 8A).

Although fungi were originally reported as activating the Toll pathway (6), immune stimulation by fungi is likely to be more complex. Previously, we reported that Relish mutants show reduced survival when injected with fungi (26). As shown in Fig. 7, it appears that both Relish and Dif play important roles in the Drosophila immune response to fungi, although to varying degrees depending upon the infecting fungus. G. candidum appears to primarily activate the Relish pathway, whereas B. bassiana appears to activate the Dif pathway. M. anisopliae and S. cerevisiae appear to utilize both pathways for the induction of the antimicrobial peptides Cecropin A and Attacin A. However, only Dif appears to be important for the induction of Drosomycin by these two fungi. Taken as a whole, the data from Fig. 7 indicate that recognition of fungi occurs upstream of both Dif and Relish and hence leads to activation of either or both transcription factors (Fig. 8B). We postulate that, as with bacteria, Drosophila must have multiple PRRs for recognition of different fungi and that these PRRs can distinguish between at least G. candidum and B. bassiana. Whether these PRRs are the same PRRs that recognize bacteria remains to be examined. However, given the lack of similarity between bacterial PG and fungal cell wall structure (53, 60–62), it is likely that bacteria and fungi are recognized by different PRRs.

In this study, we have demonstrated that G+ bacteria and fungi do not all activate the same immune pathway in Drosophila, suggesting that there must be an additional complexity in the recognition of various microbes. We have shown that these differences involve specific recognition of different PG species. Many different PGs have been structurally characterized and placed in different groups and subgroups (53); yet it is not clear if each of these PG groups is recognized by a distinct PRR. Different combinations of homo- and/or heterodimers of PRRs could change the specificity for different PAMPs as has been shown for TLRs in mammals (63). Alternatively, differential splicing of exons might generate a diversity of responses as demonstrated by the Drosophila PGRP-LC gene (16). We have demonstrated that PGs from G+ bacteria can induce antimicrobial peptides through the Imd/Relish and Toll/Dif pathways. It is possible that PGs from G- bacteria stimulate the Imd/Relish pathway, as reported by Leulier et al. (57). However, to examine the response of PGs from various G- bacteria, LPS must first be removed since it might induce the Drosophila immune system (Fig. 4B) (16).

Although PGRPs have been shown to recognize PG, it is possible that more than one PAMP on each microorganism is recognized. Our results demonstrate that M. luteus can stimulate more than one pathway, probably by binding to different PRRs. Further studies involving the stimulation of various combinations of different immune-related mutants with specific microbial components may lead to a deeper understanding of the specificity of the Drosophila immune response to different microorganisms.


   FOOTNOTES
 
* This work was supported by Everett Boaz Comer. A preliminary account of this work was presented at the 43rd Annual Drosophila Research Conference, April 10–14, 2002, San Diego, CA (Suppl. 1038C, Flybase ID FBrf0146714). 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. Back

§§ Present address: Boyce Thompson Institute, Tower Rd., Ithaca, NY 14853. Back

To whom correspondence should be addressed: Dept. of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331. Tel.: 541-737-2260; Fax: 541-737-0496; E-mail: hedengrm{at}science.oregonstate.edu.

1 The abbreviations used are: TLR, Toll-like receptor; PRR, pattern recognition receptor; LPS, lipopolysaccharide; PG, peptidoglycan; PGRP, peptidoglycan recognition protein; G-, Gram-negative; G+ Gram-positive; PAMP, pathogen-associated molecular pattern; teichuronic acid; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription. Back

2 M. Hedengren-Olcott and S. Ekengren, unpublished data. Back

3 C. Samakovlis, personal communication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Toshihiko Monodane, Dr. Dennis Hruby, Robin Ludy, and Dr. Todd Klaenhammer for bacterial strains; Dr. Stefan Åström for a yeast strain; Drs. Ylva Engström and Dan Hulmark for Drosophila stocks; and Thomas Werner for PG preparations. We also thank Drs. Christopher Bayne and Christopher K. Mathews for critical review of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Medzhitov, R., Preston, H. P., and Janeway, C. J. (1997) Nature 388, 394-397[CrossRef][Medline] [Order article via Infotrieve]
  2. Akira, S. (2001) Adv. Immunol. 78, 1-56[Medline] [Order article via Infotrieve]
  3. Janeway, C. A., Jr., and Medzhitov, R. (2002) Annu. Rev. Immunol. 20, 197-216[CrossRef][Medline] [Order article via Infotrieve]
  4. Belvin, M. P., and Anderson, K. V. (1996) Annu. Rev. Cell Dev. Biol. 12, 393-416[CrossRef][Medline] [Order article via Infotrieve]
  5. Hashimoto, C., Hudson, K. L., and Anderson, K. V. (1988) Cell 52, 269-279[CrossRef][Medline] [Order article via Infotrieve]
  6. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A. (1996) Cell 86, 973-983[CrossRef][Medline] [Order article via Infotrieve]
  7. Tzou, P., De Gregorio, E., and Lemaitre, B. (2002) Curr. Opin. Microbiol. 5, 102-110[CrossRef][Medline] [Order article via Infotrieve]
  8. Hoffmann, J. A., and Reichhart, J. M. (2002) Nat. Immunol. 3, 121-126[CrossRef][Medline] [Order article via Infotrieve]
  9. Hoffmann, J. A. (2003) Nature 426, 33-38[CrossRef][Medline] [Order article via Infotrieve]
  10. Hultmark, D. (2003) Curr. Opin. Immunol. 15, 12-19[CrossRef][Medline] [Order article via Infotrieve]
  11. Liu, C., Xu, Z., Gupta, D., and Dziarski, R. (2001) J. Biol. Chem. 276, 34686-34694[Abstract/Free Full Text]
  12. Rehman, A., Taishi, P., Fang, J., Majde, J. A., and Krueger, J. M. (2001) Cytokine 13, 8-17[CrossRef][Medline] [Order article via Infotrieve]
  13. Kang, D., Liu, G., Lundstrom, A., Gelius, E., and Steiner, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10078-10082[Abstract/Free Full Text]
  14. Yoshida, H., Kinoshita, K., and Ashida, M. (1996) J. Biol. Chem. 271, 13854-13860[Abstract/Free Full Text]
  15. Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., and Hultmark, D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13772-13777[Abstract/Free Full Text]
  16. Werner, T., Borge-Renberg, K., Mellroth, P., Steiner, H., and Hultmark, D. (2003) J. Biol. Chem. 278, 26319-26322[Abstract/Free Full Text]
  17. Kim, Y. S., Ryu, J. H., Han, S. J., Choi, K. H., Nam, K. B., Jang, I. H., Lemaitre, B., Brey, P. T., and Lee, W. J. (2000) J. Biol. Chem. 275, 32721-32727[Abstract/Free Full Text]
  18. Ramet, M., Pearson, A., Manfruelli, P., Li, X., Koziel, H., Gobel, V., Chung, E., Krieger, M., and Ezekowitz, R. A. (2001) Immunity 15, 1027-1038[CrossRef][Medline] [Order article via Infotrieve]
  19. Michel, T., Reichhart, J. M., Hoffmann, J. A., and Royet, J. (2001) Nature 414, 756-759[CrossRef][Medline] [Order article via Infotrieve]
  20. Choe, K. M., Werner, T., Stoven, S., Hultmark, D., and Anderson, K. V. (2002) Science 296, 359-362[Abstract/Free Full Text]
  21. Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D., and Royet, J. (2002) Nature 416, 640-644[CrossRef][Medline] [Order article via Infotrieve]
  22. Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., and Ezekowitz, R. A. (2002) Nature 416, 644-648[CrossRef][Medline] [Order article via Infotrieve]
  23. Ip, Y. T., Reach, M., Engstrom, Y., Kadalayil, L., Cai, H., Gonzalez-Crespo, S., Tatei, K., and Levine, M. (1993) Cell 75, 753-763[CrossRef][Medline] [Order article via Infotrieve]
  24. Rutschmann, S., Jung, A. C., Hetru, C., Reichhart, J. M., Hoffmann, J. A., and Ferrandon, D. (2000) Immunity 12, 569-580[CrossRef][Medline] [Order article via Infotrieve]
  25. Dushay, M. S., Asling, B., and Hultmark, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10343-10347[Abstract/Free Full Text]
  26. Hedengren, M., Asling, B., Dushay, M. S., Ando, I., Ekengren, S., Wihlborg, M., and Hultmark, D. (1999) Mol. Cell 4, 827-837[CrossRef][Medline] [Order article via Infotrieve]
  27. Takehana, A., Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T., and Kurata, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13705-13710[Abstract/Free Full Text]
  28. Vidal, S., Khush, R. S., Leulier, F., Tzou, P., Nakamura, M., and Lemaitre, B. (2001) Genes Dev. 15, 1900-1912[Abstract/Free Full Text]
  29. Lindsley, D. L., and Zimm, G. G. (1992) The Genome of Drosophila melanogaster, Academic Press, Inc., San Diego, CA
  30. Roos, E., Bjorklund, G., and Engstrom, Y. (1998) Insect Mol. Biol. 7, 51-62[CrossRef][Medline] [Order article via Infotrieve]
  31. Boman, H. G., Nilsson-Faye, I., Paul, K., and Rasmuson, T., Jr. (1974) Infect. Immun. 10, 136-145[Abstract/Free Full Text]
  32. Boman, H. G., Eriksson-Grennberg, K. G., Normark, S., and Matsson, E. (1968) Genet. Res. 12, 169-185[Medline] [Order article via Infotrieve]
  33. Hultmark, D., Engstrom, A., Bennich, H., Kapur, R., and Boman, H. G. (1982) Eur. J. Biochem. 127, 207-217[Medline] [Order article via Infotrieve]
  34. Rasmuson, T., and Boman, H. G. (1977) in Developmental Immunobiology (Solomon, J. B., and Horton, J. D., eds) Elsevier North-Holland Biomedical Press, Amsterdam
  35. Faye, I., Pye, A., Rasmuson, T., Boman, H. G., and Boman, I. A. (1975) Infect. Immun. 12, 1426-1438[Abstract/Free Full Text]
  36. Edlund, T., Siden, I., and Boman, H. G. (1976) Infect. Immun. 14, 934-941[Abstract/Free Full Text]
  37. Geller, B. L., Ivey, R. G., Trempy, J. E., and Hettinger-Smith, B. (1993) J. Bacteriol. 175, 5510-5519[Abstract/Free Full Text]
  38. Yamada, M., Hirose, A., and Matsuhashi, M. (1975) J. Bacteriol. 123, 678-686[Abstract/Free Full Text]
  39. Hancock, I. C., and Poxton, I. R. (1988) Bacterial Cell Surface Techniques, John Wiley & Sons Ltd., Chichester, United Kingdom
  40. Morishima, I. (1998) in Techniques in Insect Immunology (Wiesner, A., Dunphy, G. B., Marmaras, V. J., Morishima, I., Sugumaran, M., and Yamakawa, M., eds) pp. 59-64, SOS Publications, Fair Haven, NY
  41. Johnson, S. D., Lacher, K. P., and Anderson, J. S. (1981) Biochemistry 20, 4781-4785[CrossRef][Medline] [Order article via Infotrieve]
  42. Flamm, R. K., Hinrichs, D. J., and Thomashow, M. F. (1984) Infect. Immun. 44, 157-161[Abstract/Free Full Text]
  43. Ashburner, M. (1989) Drosophila: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  44. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular biology, Elsevier Science Inc., New York
  45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  46. Kylsten, P., Samakovlis, C., and Hultmark, D. (1990) EMBO J. 9, 217-224[Medline] [Order article via Infotrieve]
  47. Wicker, C., Reichhart, J. M., Hoffmann, D., Hultmark, D., Samakovlis, C., and Hoffmann, J. A. (1990) J. Biol. Chem. 265, 22493-22498[Abstract/Free Full Text]
  48. Asling, B., Dushay, M. S., and Hultmark, D. (1995) Insect Biochem. Mol. Biol. 25, 511-518[CrossRef][Medline] [Order article via Infotrieve]
  49. Hedengren, M., Borge, K., and Hultmark, D. (2000) Biochem. Biophys. Res. Commun. 279, 574-581[CrossRef][Medline] [Order article via Infotrieve]
  50. Fehlbaum, P., Bulet, P., Michaut, L., Lagueux, M., Broekaert, W. F., Hetru, C., and Hoffmann, J. A. (1994) J. Biol. Chem. 269, 33159-33163[Abstract/Free Full Text]
  51. O'Connell, P. O., and Rosbash, M. (1984) Nucleic Acids Res. 12, 5495-5513[Abstract/Free Full Text]
  52. Engström, Y., Schneuwly, S., and Gehring, W. J. (1992) Roux's Arch. Dev. Biol. 201, 65-80[CrossRef]
  53. Schleifer, K. H., and Kandler, O. (1972) Bacteriol. Rev. 36, 407-477[Free Full Text]
  54. Kim, M. S., Byun, M., and Oh, B. H. (2003) Nat. Immunol. 4, 787-793[CrossRef][Medline] [Order article via Infotrieve]
  55. Poget, S. F., Freund, S. M., Howard, M. J., and Bycroft, M. (2001) Biochemistry 40, 10966-10972[CrossRef][Medline] [Order article via Infotrieve]
  56. Cebo, C., Vergoten, G., and Zanetta, J. P. (2002) Biochim. Biophys. Acta 1572, 422-434[Medline] [Order article via Infotrieve]
  57. Leulier, F., Parquet, C., Pili-Floury, S., Ryu, J. H., Caroff, M., Lee, W. J., Mengin-Lecreulx, D., and Lemaitre, B. (2003) Nat. Immunol. 4, 478-484[CrossRef][Medline] [Order article via Infotrieve]
  58. Williams, M. J., Rodriguez, A., Kimbrell, D. A., and Eldon, E. D. (1997) EMBO J. 16, 6120-6130[CrossRef][Medline] [Order article via Infotrieve]
  59. Uv, A. E., Roth, P., Xylourgidis, N., Wickberg, A., Cantera, R., and Samakovlis, C. (2000) Genes Dev. 14, 1945-1957[Abstract/Free Full Text]
  60. Labischinski, H., and Maidhof, H. (1994) in Bacterial Cell Wall (Ghuysen, J.-M., and Hackenbeck, R., eds) Elsevier Science B. V., Amsterdam
  61. de Nobel, H., Sietsma, J. H., van den Ende, H., and Klis, F. M. (2001) in The Mycota: Biology of the Fungal Cell (Howard, R. J., and Gow, N. A. R., eds) Vol. VII, Springer-Verlag, Berlin
  62. Peberdy, J. F. (1990) in Biochemistry of Cell Walls and Membrane of Fungi (Kuhn, P. J., Trinci, A. P. J., Jung, M. J., Goosey, M. W., and Copping, L. G., eds) Springer-Verlag, Berlin
  63. Takeda, K., Kaisho, T., and Akira, S. (2003) Annu. Rev. Immunol. 21, 335-376[CrossRef][Medline] [Order article via Infotrieve]

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