Functional Diversity of the Drosophila PGRP-LC Gene Cluster in the Response to Lipopolysaccharide and Peptidoglycan*
Thomas Werner
,
Karin Borge-Renberg
,
Peter Mellroth
,
Håkan Steiner
and
Dan Hultmark
¶
From the
Umeå Centre for Molecular
Pathogenesis, Umeå University, S-901 87 Umeå, Sweden and the
Department of Microbiology, Stockholm
University, S-106 91 Stockholm, Sweden
Received for publication, April 30, 2003
, and in revised form, May 25, 2003.
 |
ABSTRACT
|
---|
The peptidoglycan recognition protein PGRP-LC is a major activator of the
imd/Relish pathway in the Drosophila immune response. Three
transcripts are generated by alternative splicing of the complex
PGRP-LC gene. The encoded transmembrane proteins share an identical
intracellular part, but each has a separate extracellular PGRP-domain: x,
y, or a. Here we show that two of these isoforms play unique
roles in the response to different microorganisms. Using RNA interference in
Drosophila mbn-2 cells, we found that PGRP-LCx is the only isoform
required to mediate signals from Gram-positive bacteria and purified bacterial
peptidoglycan. By contrast, the recognition of Gram-negative bacteria and
bacterial lipopolysaccharide requires both PGRP-LCa and LCx. The third
isoform, LCy, is expressed at lower levels and may be partially redundant. Two
additional PGRP domains in the gene cluster, z and w, are
both included in a single transcript of a separate gene, PGRP-LF.
Suppression of this transcript does not block the response to any of the
microorganisms tested.
 |
INTRODUCTION
|
---|
Insects and mammals use similar mechanisms and molecular pathways to
recognize and eliminate invading microorganisms. The best studied example is
the humoral immune response of the fruit fly Drosophila melanogaster,
where two major immune pathways signal the presence of microbes and mediate
the production of antimicrobial peptides, the imd-Relish pathway and
the Toll/Dif pathway
(14).
Members of the peptidoglycan recognition protein
(PGRP)1 family were
recently found to play key roles in the activation of these pathways
(59).
PGRPs have been found in many organisms, including vertebrates, and the family
includes peptidoglycan-binding pattern recognition molecules as well as
peptidoglycan-degrading amidases
(1013).
Several PGRP genes are found in the Drosophila genome
(14). One of them,
PGRP-LC, is required for the activation of the
imd/Relish pathway and is likely to encode a pattern
recognition molecule for the humoral immune response
(68).
Surprisingly, we found that PGRP-LC mediates the induction by bacterial
lipopolysaccharide (LPS) as well as peptidoglycan
(6), although the effect of LPS
was recently questioned (15).
In the present study we show that two different isoforms of PGRP-LC display
distinct functions in the response to LPS and peptidoglycan.
 |
EXPERIMENTAL PROCEDURES
|
---|
cDNA Library ScreenA ZAP Express cDNA expression library
made from induced l(3)mbn-1 larvae
(16) was screened using
Hybond-NX membranes (Amersham Biosciences) and radioactive probes against the
PGRP domains x, y, w, and z. We screened between 3 and 11
x 105 plaques with the different probes, corresponding to
0.62.2 times the estimated complexity of the library. The probes were
made as described for Northern blot detection. Positive pBK-CMV phagemid
clones derived from the ZAP Express vector (Stratagene) were obtained by
in vivo excision using the ExAssist helper phage (Stratagene). EST
clones of PGRP-LF were obtained from the Berkeley Drosophila Genome
Project/Howard Hughes Medical Institute EST Project. The cDNA clones were
sequenced using the Dyenamic ET terminator sequencing kit (Amersham
Biosciences) and 17- to 18-base-long primers from Cybergene AB (Huddinge,
Sweden).
RT-PCRTotal RNA from induced flies, induced mbn-2 cells,
and untreated mbn-2 cells was used for the superscript one-step RT-PCR with
platinum taq (Invitrogen) reactions with one common 5'
PGRP-LC primer and one y-specific 3' primer. The
reaction was run at 50 °C 30 min and 94 °C 2 min, followed by 30
cycles (94 °C 15 s, 50 °C 30 s, 72 °C 1 min), and ended by 72
°C 7 min. The product was cloned into the pCR2.1Topo vector using Topo TA
cloning (Invitogen) and sequenced.
Cells, Microorganisms, and Cell Wall IsolatesThe mbn-2 cell
line (17) was grown at 25
°C in Schneider's medium with 10% fetal calf serum. The bacteria
Micrococcus luteus Ml11, Bacillus megaterium Bm11, and
Enterobacter cloacae
12 were originally obtained from Hans G.
Boman (see Ref. 18 and
references therein). The Erwinia carotovora carotovora SSC3193 wild
type strain was obtained from Kenneth Söderhäll (Uppsala University,
Uppsala, Sweden), and Escherichia coli O55:B5 was received from the
CCUG (Culture Collection, University of Göteborg, Göteborg, Sweden).
The fungus Dipodascopsis uninucleata var. wickerhamii was obtained
from the Fungal Biodiversity Center (Utrecht, The Netherlands).
Insoluble peptidoglycan was prepared from B. megaterium Bm11, as
described in Ref. 19. LPS from
E. coli O55:B5 was obtained from Fluka and taken up in distilled
water. Before use, the stock solution was pretreated for 1 h at 62 °C.
Peptidoglycan contaminants were removed by incubating LPS preparations (2
mg/ml) with PGRP-SC1B (20 µg/ml) at 25 °C for 12 h. The activity of the
enzyme preparation was confirmed on purified peptidoglycan as described in
Ref. 13. In a separate
experiment it was shown that LPS (2 mg/ml) did not inhibit the PGRP-SC1B
degradation of peptidoglycan (data not shown). To make a fungal cell fragment
preparation, 5 ml of D. uninucleata cell pellet was washed and
resuspended in 50 ml of sterile Ringer's solution. The cells were fragmented
in an ice-chilled Bead Beater chamber in 45 1-min runs, with 50 ml of 0.5-mm
glass beads in a total volume of 100 ml.
RNAiWe amplified the PGRP regions of interest from cDNA or
genomic DNA by PCR with primers containing the T7 sequence. The fragments were
subcloned into pCR2.1Topo by using the Topo TA Cloning kit (Invitrogen). As
templates for in vitro transcription (Ribomax large scale RNA
production system T7 kit, Invitrogen), we used 10 µg of linear plasmid DNA
containing the PGRP sequences, flanked by a T7-promoter on each side.
The dsRNA for the different genes and domains corresponded to the following
parts and lengths: PGRP-LC, 861 bp from the common exons 2 and 3;
LCa domain, 523 bp; LCx domain, 354 bp; LCy domain,
584 bp; and PGRP-LF, 738 bp covering both domains. For RNAi, we
plated 5 ml of mbn-2 cell suspension, 1 million cells/ml, in Schneider's
medium with 10% fetal calf serum. The cells were kept at 25 °C and
transfected 1 day later with 10 µg of dsRNA. Three days after transfection,
the mbn-2 cells were induced with washed live bacteria, homogenized fungal
cells, insoluble peptidoglycan, LPS, or sterile Ringer's solution as control
for 6 h (2 h for LPS). The pellets of bacterial overnight cultures were washed
and resuspended 1:100 in sterile Ringer's solution, and 15 µl were used per
plate. The quantity of fungal cell fragments used for induction corresponds to
1.5 µl of cell pellet per plate. The final concentration of peptidoglycan
and LPS was 1 µg/ml cell culture. The mbn-2 cells were harvested on ice
after 6 h, centrifuged for 10 min at 500 x g, and total RNA was
extracted.
RNA Preparation, Northern Blot, and HybridizationTotal RNA
was prepared with TRIzol (Invitrogen) and dissolved in 50 µl RNase free
water. For Northern blots, 5 µl (
15 µg) of total RNA per lane was
run on a 1% agarose gel containing formaldehyde. Hybridization was performed
under high stringency conditions (50% formamide, 42 °C). Inserts from the
cDNA clones pAttA for Attacin A, k-7 for Cecropin A, and
BSJM108 for Diptericin
(2022)
were cut out with EcoRI and used as templates for probes. The probes
were 32P-labeled with the Rediprime II kit from Amersham
Biosciences. Radioactivity was monitored using a PhosphorImager (Amersham
Biosciences).
 |
RESULTS AND DISCUSSION
|
---|
Alternative Transcripts in the Complex PGRP-LC Locus The
PGRP-LC locus contains five PGRP homology domains
(14)
(Fig. 1). Two of them are
expressed in the alternatively spliced PGRP-LCa and PGRP-LCx
transcripts (6,
14). To identify transcripts
that express the neighboring y, z and w PGRP domains, we
screened a hemocyte-enriched cDNA library. We found 8 clones out of 450,000
screened plaques for PGRP-LCx, but only one for LCy out of
1.1 million. This single y clone corresponds to an aberrantly
terminated PGRP-LCx transcript, in which the y domain was
part of the 3'-untranslated region. We therefore resorted to RT-PCR and
could amplify a functional PGRP-LCy-specific band with RNA from mbn-2
cells and immunostimulated adult flies. This PGRP-LCy transcript is
completely analogous to PGRP-LCx and uses the same splice donor site
in the third exon, 57 bp upstream of the one used for the PGRP-LCa
transcript. As a result, the linker between the PGRP domain and the predicted
transmembrane region is 19 amino acid residues shorter in PGRP-LCx and LCy
than in LCa.

View larger version (7K):
[in this window]
[in a new window]
|
FIG. 1. The Drosophila PGRP-LC gene cluster. A,
organization of the transcription units. Open reading frames are shown as
boxes, and the PGRP domain is shown in black. The direction
of transcription is indicated by arrows. Three PGRP domains, a,
x, and y, are alternatively spliced to the common exon 3 of the
PGRP-LC gene. The PGRP-LF transcript encodes two PGRP
domains, z and w. B, predicted membrane topology of the
PGRP-LC and LF proteins. The PGRP domains are in black.
|
|
Eighteen out of 300,000 clones were positive for the w domain. At
least 12, possibly all, of the w-containing clones also hybridized to
the z probe, suggesting that both domains are included in a single
transcript. This was confirmed by sequencing two of the cDNA clones and is
also consistent with one 5' EST sequence and one fully sequenced
transcript from the genome project. It does not overlap with the
PGRP-LC transcripts and therefore defines a unique PGRP
gene, PGRP-LF. The encoded 369-amino acid PGRP-LF protein includes
the z domain in exons 2 and 3 and the w domain in exon 4
(Fig. 1A). Membrane
topology prediction suggests that the extracellular PGRP domains are
immediately preceded by a transmembrane region and a very short intracellular
domain of 23 residues (Fig.
1B).
Alternative Splice Forms of PGRP-LC with Different
SpecificityThe ability of the PGRP-LC gene to express
three alternative PGRP domains, x, y, and a, suggests that
the PGRP-LC isoforms display different recognition capabilities to various
microbial patterns. To investigate the functions of the three PGRP-LC isoforms
and of PGRP-LF, we used transcript-specific RNAi in mbn-2 cells. Control
experiments show that double-stranded RNA directed against the unique exons
suppress the corresponding transcript levels by at least 90%, without
affecting the other alternative transcripts
(Fig. 2, CF).
We then tested the cells for their ability to respond to the Gram-positive
bacteria M. luteus and B. megaterium, the Gram-negative
bacteria E. cloacae, E. carotovora, and E. coli, and one
fungus, D. uninucleata. We monitored the immune response by Northern
blot, using probes for the antimicrobial Diptericin, Attacin A, and
Cecropin A genes, which are regulated by the
imd/Relish pathway in this cell line. Our results support
the suggestion that the PGRP-LC isoforms display distinct recognition
abilities. PGRP-LCx is absolutely required for induction by all
tested bacteria, and suppression of this transcript has the same effect as
suppression of all PGRP-LC transcripts with dsRNA from the common
exons 2 and 3 (Fig. 2, A and
B). By contrast, the removal of PGRP-LCa
specifically blocks the inducibility of the imd/Relish
pathway by Gram-negative bacteria, without affecting the induction by
Gram-positive bacteria. This clearly demonstrates a functional difference
between PGRP-LCa and LCx.

View larger version (83K):
[in this window]
[in a new window]
|
FIG. 2. The role of PGRP-LC and PGRP-LF transcripts in the response to different
microorganisms. A and B, two separate experiments, in
which the different PGRP-LC and PGRP-LF transcripts in mbn-2
cells were suppressed by transfection of dsRNA. The cells were treated with
dsRNA and induced for 6 h with Gram-positive bacteria (M. luteus
(M. lut), B. megaterium (B. mega)), Gram-negative
bacteria (E. cloacae (E. clo.), E. coli (E.
coli), E. carotovora (E. caro.)), and cell fragments of
the fungus D. uninucleata (D. uni.). We used sterile
Ringer's solution as a negative control for induction and dsRNA against
bacterial -galactosidase (LacZ) as a negative control for RNAi.
PGRP-LCx-suppressed cells show an abolished immune response to all
tested microorganisms, whereas dsRNA against PGRP-LCa only effects
the inducibility by Gram-negative bacteria. The PGRP-LC and
PGRP-LCx phenotypes are identical. dsRNA against PGRP-LF
does not abolish, but increases, the immune response to a variable extent,
while dsRNA against PGRP-LCy has no effect. Cell fragments of the
fungus D. uninucleata did not induce the antimicrobial peptide gene
transcription above the normal background level. CE, RNAi acts
specifically on the different PGRP-LC splice forms. Expression of the
different transcripts in the mbn-2 cells was analyzed by RT-PCR (35 amplifying
cycles), using 0.3 or 0.03 µg of total RNA as indicated. The cells had been
treated with dsRNA for PGRP-LCa (C), LCx
(D), or LCy (C). F, dsRNA for
PGRP-LF decreases the transcript level (25 amplifying cycles).
|
|
A Specific Role of PGRP-LCa in the Response to LPSThe
specific requirement of PGRP-LCa for induction by Gram-negative bacteria
strongly suggested that this isoform could be involved in the response to LPS.
Since LPS preparations are likely to be contaminated with peptidoglycan, we
pretreated E. coli LPS with purified PGRP-SC1B, an amidase that
degrades peptidoglycan and removes its immunostimulatory effect on
Drosophila cells
(13).
Fig. 3 shows that a
peptidoglycan-free LPS preparation induces the mbn-2 cells in a
PGRP-LCa-dependent way, while crude LPS signals via PGRP-LCx in a
PGRP-LCa-independent manner. This changed pattern confirms the effectiveness
of the peptidoglycan removal. The chemical structure of peptidoglycan is
identical in E. coli and B. megaterium
(23), and induction by
peptidoglycan from the latter species does not require PGRP-LCa
(Fig. 3). The
PGRP-LCa-dependent induction by LPS can therefore not be due to contaminating
peptidoglycan, as suggested by Leulier et al.
(15). It must be caused by
some other factor in the preparation, most likely LPS itself. Thus, we
conclude that whereas PGRP-LCx is the only isoform needed for induction by
peptidoglycan, the response to LPS or Gram-negative bacteria requires both
PGRP-LCa and LCx. This suggests that LPS is indeed the major inducing factor
in the Gram-negative bacterial envelope.

View larger version (62K):
[in this window]
[in a new window]
|
FIG. 3. PGRP-LCa is required for induction by LPS but not by peptidoglycan.
RNAi in mbn-2 cells was done as described in the legend to
Fig. 2. PGRP-LCa and PGRP-LCx
are required for the induction by peptidoglycan-free LPS, generated by removal
of peptidoglycan contaminants by PGRP-SC1B pretreatment. In contrast,
insoluble peptidoglycan (PG) from B. megaterium (B.
mega.) and crude LPS (Fluka) signal via PGRP-LCx in a
PGRP-LCa-independent manner.
|
|
Quantification of the Northern blot shows that stimulation by
peptidoglycan-free LPS is about 7-fold weaker than that of living
Gram-negative bacteria. This might be related to how the LPS molecules are
presented in the micelles of the purified sample. The colloidal state of LPS
is probably important and we obtain more reproducible induction when the stock
solution has been
heat-treated.2 Such
factors could explain why Leulier et al.
(15) were unable to detect any
effect of LPS in vivo. Besides, free LPS is probably short-lived in
hemolymph (24).
Unlike PGRP-LCa and PGRP-LCx, dsRNA against
PGRP-LCy and PGRP-LF did not suppress the immune response
(Fig. 2B). If
anything, the induction is even slightly increased by dsRNA against
PGRP-LF, although this could be an indirect effect. Trying to find
functions for PGRP-LCy and PGRP-LF, we expanded our experiments by using the
yeast-like fungus D. uninucleata, which is highly immunostimulatory
in adult flies, in a Relish-dependent manner
(25,
26). However, neither intact
cells (not shown) nor cell fragments of this microorganism induce any response
in the mbn-2 cells (Fig.
2B), most likely due to missing factors in the cell
culture system.
In conclusion, our experiments demonstrate that alternative splicing of
PGRP-LC contributes to the capacity of the system to respond to both LPS and
peptidoglycan. It is likely that PGRP-LCx acts as a direct pattern recognition
molecule for
peptidoglycans,3 but
we cannot exclude the possibility that other components are also involved. The
simultaneous requirement of two splice forms for the response to LPS suggests
that the PGRPs may act as heterodimers or perhaps as higher multimers. This
would further add to the flexibility of the system.
 |
FOOTNOTES
|
---|
* This work was supported by grants from the Swedish Research Council (to D.
H. and H. S.), the Wallenberg Consortium North (to D. H. and H. S.), and the
European Union Quality of Life and Management of Living Resources Program (to
H. S.). 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. 
¶
To whom correspondence should be addressed. Tel.: 46-90-785-67-78; Fax:
46-90-77-80-07; E-mail:
dan.hultmark{at}ucmp.umu.se.
1 The abbreviations used are: PGRP, peptidoglycan recognition protein; LPS,
lipopolysaccharide; EST, expressed sequence tag; RT, reverse transcriptase;
dsRNA, double-stranded RNA. 
2 S. Ekengren, personal communication. 
3 P. Mellroth and H. Steiner, unpublished data. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Sophia Ekengren for advice on LPS pretreatment and Svenja
Stöven, Tobias Hainzl, Michael Williams, Karin Johansson, István
Andó, and Magda-Lena Wiklund for helpful discussions.
 |
REFERENCES
|
---|
- Engström, Y. (1999) Dev. Comp.
Immunol. 23,
345358[CrossRef][Medline]
[Order article via Infotrieve]
- Hoffmann, J. A., and Reichhart, J. M. (2002)
Nat. Immunol. 3,
121126[CrossRef][Medline]
[Order article via Infotrieve]
- Tzou, P., De Gregorio, E., and Lemaitre, B. (2002)
Curr. Opin. Microbiol.
5,
102110[CrossRef][Medline]
[Order article via Infotrieve]
- Hultmark, D. (2003) Curr. Opin.
Immunol. 15,
1219[CrossRef][Medline]
[Order article via Infotrieve]
- Michel, T., Reichhart, J. M., Hoffmann, J. A., and Royet, J.
(2001) Nature
414,
756759[CrossRef][Medline]
[Order article via Infotrieve]
- Choe, K. M., Werner, T., Stöven, S., Hultmark, D., and
Anderson, K. V. (2002) Science
296,
359362[Abstract/Free Full Text]
- Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann,
J. A., Ferrandon, D., and Royet, J. (2002)
Nature 416,
640644[CrossRef][Medline]
[Order article via Infotrieve]
- Rämet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., and
Ezekowitz, R. A. (2002) Nature
416,
644648[CrossRef][Medline]
[Order article via Infotrieve]
- Takehana, A., Katsuyama, T., Yano, T., Oshima, Y., Takada, H.,
Aigaki, T., and Kurata, S. (2002) Proc. Natl. Acad.
Sci. U. S. A. 99,
1370513710[Abstract/Free Full Text]
- Yoshida, H., Ochiai, M., and Ashida, M. (1986)
Biochem. Biophys. Res. Commun.
141,
11771184[Medline]
[Order article via Infotrieve]
- Kang, D., Liu, G., Lundström, A., Gelius, E., and Steiner, H.
(1998) Proc. Natl. Acad. Sci. U. S. A.
95,
1007810082[Abstract/Free Full Text]
- Liu, C., Xu, Z., Gupta, D., and Dziarski, R. (2001)
J. Biol. Chem. 276,
3468634694[Abstract/Free Full Text]
- Mellroth, P., Karlsson, J., and Steiner, H. (2003)
J. Biol. Chem. 278,
70597064[Abstract/Free Full Text]
- Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., and
Hultmark, D. (2000) Proc. Natl. Acad. Sci. U. S.
A. 97,
1377213777[Abstract/Free Full Text]
- 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,
478484[CrossRef][Medline]
[Order article via Infotrieve]
- Kurucz, E., Zettervall, C.-J., Sinka, R., Vilmos, P., Pivarcsi, A.,
Ekengren, S., Hegedüs, Z., Ando, I., and Hultmark, D. (2003)
Proc. Natl. Acad. Sci. U. S. A.
100,
26222627[Abstract/Free Full Text]
- Gateff, E., Gissmann, L., Shrestha, R., Plus, N., Pfister, H.,
Schröder, J., and zur Hausen, H. (1980) in
Invertebrate Systems in Vitro (Kurstak, E.,
Maramorosch, K., and Dübendorfer, A., eds) pp.
517533, Elsevier/North-Holland Biomedical
Press, Amsterdam
- Hultmark, D., Engström, Å., Bennich, H., Kapur, R., and
Boman, H. G. (1982) Eur. J. Biochem.
127,
207217[Abstract]
- 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.
5964, SOS Publications, Fair Haven,
NJ
- Åsling, B., Dushay, M. S., and Hultmark, D.
(1995) Insect Biochem. Mol. Biol.
25,
511518[CrossRef][Medline]
[Order article via Infotrieve]
- Kylsten, P., Samakovlis, C., and Hultmark, D. (1990)
EMBO J. 9,
217224[Abstract]
- Wicker, C., Reichhart, J.-M., Hoffmann, D., Hultmark, D.,
Samakovlis, C., and Hoffmann, J. A. (1990) J. Biol.
Chem. 265,
2249322498[Abstract/Free Full Text]
- Schleifer, K. H., and Kandler, O. (1972)
Bacteriol. Rev. 36,
407477[Medline]
[Order article via Infotrieve]
- Kato, Y., Motoi, Y., Taniai, K., Kadonookuda, K., Hiramatsu, M.,
and Yamakawa, M. (1994) Insect Biochem. Mol.
Biol. 24,
539545
- Ekengren, S., and Hultmark, D. (1999)
Insect Biochem. Mol. Biol.
29,
965972[CrossRef][Medline]
[Order article via Infotrieve]
- Ekengren, S. (2001) Stress and Immune
Defense in Drosophila melanogaster. Ph.D. thesis, the Wenner-Gren
Institute, Stockholm University, Stockholm