REVIEWS

Peptidoglycan recognition proteins:

modulators of the microbiome and
inflammation
Julien Royet*, Dipika Gupta‡ and Roman Dziarski‡
Abstract | All animals, including humans, live in symbiotic association with microorganisms.
The immune system accommodates host colonization by the microbiota, maintains
microbiota–host homeostasis and defends against pathogens. This Review analyses
how one family of antibacterial pattern recognition molecules — the peptidoglycan
recognition proteins — has evolved a fascinating variety of mechanisms to control host
interactions with mutualistic, commensal and parasitic microorganisms to benefit
both invertebrate and vertebrate hosts.

Eukaryotes
Organisms (such as animals,
plants and fungi) whose cells
contain genetic material
contained within a
membrane-enclosed nucleus.
Symbiotic
Symbiosis is a physical
association between two
organisms and includes
mutualism, commensalism
and parasitism. In common
(non-scientific) usage it is often
assumed to be beneficial to
both organisms.
*Institut de Biologie du
Développement de Marseille-
Luminy, UMR6216 CNRS et
Aix-Marseille Universites,
Parc Scientifique de Luminy,
Marseille, France.
‡
Indiana University School of
Medicine – Northwest,
Gary, Indiana 46408, USA.
Correspondence to J.R.  Genetic and environmental factors can result in
and R.D.
e-mails:
julien.royet@univmed.fr;
rdziar@iun.edu
doi:10.1038/nri3089
Published online
11 November 2011
All multicellular eukaryotes live in symbiotic (mutualistic or
commensal) association with microorganisms, which often
form complex communities on and/or in the body of the
host. These microorganisms make up the host microbiome.
All eukaryotes evolved in the presence of prokaryotes, and
thus eukaryotes and prokaryotes have often co-evolved
to use each other to their advantage. Eukaryotes acquired
prokaryotes to form two of their organelles: mitochondria
and chloroplasts. Multicellular eukaryotes have developed
long-lasting and usually mutually beneficial relationships
with specific microorganisms, which carry out essen-
tial functions needed by the multicellular eukaryotes in
exchange for a stable environment with a constant supply
of nutrients1–3. The most complex microbial communi-
ties have developed in the guts of mammals, in particular
omnivores and herbivores1. For example, the human gut
microbiome has ten times more microorganisms than the
number of cells in the human body, 100 times more genes
than the human genome, and a metabolic capacity equiva-
lent to the human liver 4. The gut microbiota provides the
host with essential nutrients (such as vitamins), breaks
down indigestible compounds (such as complex carbo-
hydrates), defends against colonization by pathogens, and
is required for the proper development of not only the
immune system1,2,4, but also the brain5 and gut4.
changes in mammalian microbiomes that might predis-
pose the host to various diseases. For example, obesity,
inflammatory bowel disease and colon cancer are asso-
ciated with (and probably, in part, caused by) changes
in the gut microbiota4,6,7. Changes in the gut microbiota
affect sensitivity not only to intestinal diseases, but also to
systemic diseases such as arthritis, asthma and diabetes8,9;
these effects are mediated by microbial products such as
short-chain fatty acids9 or ATP10. Thus, any component
of the host that influences the composition of the micro-
biota can have a profound effect on the development and
well-being of the host.
It has recently been recognized that the immune
system developed not only to defend the host against
pathogens and to react to tissue damage, but also to
accommodate host colonization by symbiotic micro­
organisms and to maintain microbiota–host homeo-
stasis11. As the health of the host relies on the proper
formation and maintenance of the microbiota, there
are multiple mechanisms that control the complex
interactions between the microbiota and the host.
In this Review, we focus on one family of pattern
recognition molecules of the innate immune system,
peptido­glycan recognition proteins (PGRPs), which are
conserved from insects to mammals and have diverse
functions in antimicrobial defence12. We discuss how both
invertebrate and vertebrate members of the PGRP fam-
ily have developed an amazing variety of mechanisms to
coordinate the host response to mutualistic, commensal
and parasitic microorganisms.
Peptidoglycan recognition proteins
Genes and proteins. PGRPs were first discovered in the
haemolymph of a silkworm (Bombyx mori) as proteins
that bind to bacterial peptidoglycan and activate the
prophenoloxidase cascade, an antimicrobial host defence
mechanism in insects13. Cloning of PGRP genes from the
fruitfly (Drosophila melanogaster)14 led to the discovery

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 837

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
Mutualistic
A form of symbiosis that is
beneficial to both organisms
involved.
Commensal
A form of symbiosis in which
one organism benefits and the
other derives neither benefit
nor harm.
Microbiome
The microbiome is the entire
community of microorganisms
that live in or on the body of
a multicellular eukaryotic
organism. Because in many
cases these microorganisms
can only be characterized by
genetic methods, the term
microbiome is often used to
refer to the collective genomes
present in all microorganisms
in a given microbial community.
Prokaryotes
Simple organisms (such as
bacteria) whose cells lack a
membrane-enclosed nucleus
and membrane-enclosed
organelles.
Pattern recognition
molecules
Proteins that recognize
molecular patterns that are
characteristic of microbial
molecules but not present
in the host, such as
peptidoglycan (which is found
in the cell walls of all bacteria)
or lipopolysaccharide (which
is found in the cell walls of
Gram-negative bacteria).
Some pattern recognition
molecules are found on the
surface of host cells and
function as pattern recognition
receptors to activate host cells,
whereas others are secreted
and function to trigger
pro-inflammatory cascades
or help to remove
microorganisms.
Parasitic
A form of symbiosis that is
beneficial to one organism
and detrimental to the other.
Prophenoloxidase cascade
A cascade of enzymatic
reactions that generates
quinones, which are toxic to
microorganisms, and melanin
pigments, which restrict the
spreading of microorganisms
within the host. It is an
important component of the
innate immune response in
invertebrates.
838 | DECEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
of families of PGRP genes in insects and other inverte-
brates, such as molluscs and echinoderms, as well as
in vertebrates, including fish, amphibians, birds and
mammals12,15. However, PGRPs have not been found so
far in plants or in lower metazoa, such as nematodes (for
example, Caenorhabditis elegans).
Insects usually have many PGRP genes. For example,
D. melanogaster has 13 PGRP genes that are transcribed
into 19 proteins14,16, and a mosquito (Anopheles gambiae)
has seven PGRP genes that are transcribed into nine pro-
teins17. Based on the size of the mRNA transcript, these
genes are usually classified into short and long forms,
known as S and L, respectively (FIG. 1). Mammals have
four PGRP genes, PGLYRP1, PGLYRP2, PGLYRP3 and
PGLYRP4, which were initially named PGRP-S, PGRP-L,
PGRP-Iα and PGRP-Iβ (on the basis of their short (S),
long (L) and intermediate (I) transcript lengths) by
analogy to insect PGRPs18.
Structure and specificity. All PGRPs have at least one
PGRP domain, which is ~165 amino acids long and
is structurally similar to bacteriophage and bacterial
type 2 amidases (which catalyse the hydrolysis of amide
bonds)12,14–18; the PGRP domain constitutes most of
the sequence of short PGRPs (FIG. 1). Some PGRPs (for
example, D. melanogaster PGRP-LF and mammalian
PGLYRP3 and PGLYRP4) have two PGRP domains,
which are homologous but not identical. Long PGRPs
usually have one PGRP domain and an additional
unique amino-terminal sequence (FIG. 1). Many PGRPs
are secreted, and some (such as mammalian PGLYRP1,
PGLYRP3 and PGLYRP4) form disulphide-linked
homodimers or heterodimers (for example, PGLYRP3–
PGLYRP4). Some PGRPs (such as D. melanogaster
PGRP-LC and PGRP-LF) are transmembrane recep-
tors, and some others are intracellular proteins (such as
D. melanogaster full-length PGRP-LE).
The general structural design of the PGRP domain
is similar to that of bacteriophage type 2 amidases in
that it has three peripheral α‑helices and several central
β‑strands12,15,19,20. The front face of the molecule has a cleft
that forms a peptidoglycan-binding groove, and the back
of the molecule has a PGRP-specific segment that is not
present in bacteriophage amidases (FIG. 1a); this region is
often hydrophobic and is diverse among various PGRPs,
and it might bind additional ligands or other molecules19.
In some PGRPs (such as D. melanogaster PGRP-LCa
and PGRP-LF), the peptidoglycan-binding groove is dis-
rupted, which prevents direct recognition of peptidoglycan
fragments by these PGRP family members21,22.
The peptidoglycan-binding groove of PGRPs typi-
cally binds the muramyl pentapeptide or tetrapeptide
fragment of peptidoglycan with the highest affinity
(FIG. 1b), and this binding usually induces a structural
change in the PGRP domain or an interaction with
another PGRP molecule. Both of these changes lock
the muramyl peptide in the binding site. Many PGRPs
can also discriminate between different amino acids in
the peptide or between N‑acetylmuramic acid and its
anhydro form within peptidoglycan 12,20, and this is
discussed below in more detail.
© 2011 Macmillan Publishers Limited. All rights reserved
Although most PGRPs bind to peptidoglycan12,15,20,
some PGRPs also bind with high affinity to other
microbial molecules, such as lipopolysaccharide (LPS)
and lipoteichoic acid; these molecules usually bind
outside the peptidoglycan-binding groove23–25. In addi-
tion, certain fungus-derived molecules can be bound by
several PGRPs (including bovine PGLYRP1 and human
PGLYRP3 and PGLYRP4)24,26.
Control of the microbiota by insect PGRPs
To detect and control microorganisms, insects use a
relatively small number of pattern recognition mol-
ecules. Work carried out mainly using D. melanogaster
has highlighted the central role of the members of the
PGRP family in bacterial recognition and control in
insects; indeed, PGRPs are the largest and most versatile
family of pattern recognition molecules for microbial
products in these organisms. By contrast, vertebrates
have fewer PGRPs with a much narrower range of func-
tions because, in addition to expressing PGRPs, they have
evolved a vast array of other pattern recognition receptors
(PRRs). These include Toll-like receptors (TLRs), NOD-
like receptors (NLRs), C‑type lectin receptors (CLRs),
RIG-I-like receptors (RLRs), scavenger receptors and the
mannose receptor. Moreover, they possess many secreted
recognition molecules, such as complement, collectins,
pentraxins and C‑reactive protein.
PGRPs are upstream PRRs of D. melanogaster immune
cascades. The functions of D. melanogaster PGRPs as
PRRs were initially discovered by investigating the
role of these proteins as sentinel receptors upstream
of the pathways that regulate the systemic immune
response27–30. In flies, the Toll pathway is mainly acti-
vated by Gram-positive bacteria and induces the pro-
duction of antimicrobial peptides (AMPs) by cells of the
fat body; AMPs are then released into the circulating
haemolymph, where they kill infectious bacteria2 (FIG. 2).
Two PGRP family members (PGRP-SA and PGRP-SD)
and the misnamed Gram-negative binding protein 1
(GNBP1) form a circulating PRR complex upstream of
the Toll pathway 27,31–33. By binding to peptidoglycan from
the cell walls of Gram-positive bacteria, PGRP-SA trig-
gers a protease cascade that ultimately transforms the
inactive form of Spätzle into an active ligand, which then
induces Toll dimerization and downstream signalling to
nuclear factor-κB (NF-κB). D. melanogaster mutants that
have genetic defects in Toll pathway components or in
the upstream PRRs are, as a result, highly susceptible to
infection with Gram-positive bacteria27,34.
The D. melanogaster immune system can discrimi-
nate between various types of bacteria based on their
peptidoglycan composition12,35,36. Whereas bacteria that
have a lysine residue in the third position of the peptido­
glycan stem peptide (Lys-type peptidoglycan, which is
present in most Gram-positive bacteria) elicit the Toll
pathway, bacteria with meso-diaminopimelic acid in this
position (DAP-type peptidoglycan, which is present in
most Gram-negative bacteria and in bacilli) activate
another signalling cascade upstream of NF‑κB known
as the IMD pathway.
 REVIEWS

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Figure 1 | The structure of PGRPs. a | The ribbon diagrams show the
conservation of structure between a Drosophila melanogaster
amidase (peptidoglycan recognition protein-LB (PGRP-LB)) and an
ancestral type 2 amidase (bacteriophage T7 lysozyme). A Zn2+ ion
(green sphere), which is required for amidase activity, is shown in the
peptidoglycan (PGN)-binding groove. The PGRP-unique segment is
shown in yellow. Images are reproduced, with permission, from REF. 19
© (2003) Macmillan Publishers Ltd. All rights reserved. b | The PGRP
domain of D. melanogaster PGRP-LCx is shown in complex with a PGN
fragment, tracheal cytotoxin (TCT), the structure of which is shown
0CVWTG4GXKGYU^+OOWPQNQI[
on the right. Images are reproduced, with permission, from REF. 40
© (2006) The American Association for the Advancement of Science.
c | The figure shows diagrams of the domain structure and the main
functions of D. melanogaster, Euprymna scolopes, Danio rerio and
mammalian PGRPs. GPI, glycosylphosphatidylinositol; PRR, pattern
recognition receptor; RHIM, RIP homotypic interaction motif.

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 839

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

Toll pathway The receptor proteins of the IMD pathway are various specificities. Whereas PGRP-LCa and PGRP-
A signalling cascade in insects un­related to Toll and all belong to the PGRP family. The LCx pair to form a receptor for monomeric peptido­
that regulates the expression main transmembrane receptor is PGRP-LC, which func- glycan fragments, such as tracheal cytotoxin (TCT; also
of antimicrobial peptides tions both as a microbial binding protein (through its known as anhydromonomer)21,22,38–40 (FIG. 1b), PGRP-LCx
after infection by fungi or
Gram-positive bacteria.
extra­cellular PGRP domain) and as a signalling receptor homo­dimerizes to recognize polymeric peptidoglycan. By
Detection of fungi by GNBP3 (through its intracellular tail that interacts directly with forming inactive heterodimers with PGRP-LCx, another
and of bacteria by circulating IMD, an orthologue of mammalian RIP1)28–30,37 (FIGS 2,3). member of the family — PGRP-LF (a transmembrane
peptidoglycan recognition In this way, PGRP-LC is similar to mammalian TLRs, but receptor that lacks the intracellular signalling domain)
proteins (PGRPs) triggers the
unlike the D. melanogaster Toll protein. By alternative — prevents the formation of PGRP-LCa–PGRP-LCx
cleavage of the cytokine
Spätzle, which then activates
splicing, the gene encoding PGRP-LC generates three pro- heterodimers and, hence, functions as a negative regu-
the Toll receptor, and this tein isoforms (x, y and a) that have identical intra­cellular lator of the IMD pathway by decreasing downstream
initiates the activation of the domains but different extracellular PGRP domains with signalling 41,42 (FIG. 2). DAP-type peptidoglycan is also
NF‑κB-like transcription factors
Dif and Dorsal and their
translocation into the nucleus.
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Figure 2 | Insect PGRPs recognize peptidoglycan and regulate multiple defence pathways. Depending on their
peptidoglycan (PGN) composition, bacteria can activate either the Toll pathway or the IMD pathway in Drosophila
melanogaster cells. Extracellular bacteria that contain Lys-type PGN are recognized by peptidoglycan recognition
protein-SA (PGRP-SA), PGRP-SD and Gram-negative binding protein 1 (GNBP1). This induces a protease cascade that
ultimately cleaves pro-Spätzle into an active ligand for Toll, leading to nuclear factor-κB (NF‑κB)-dependent signalling and
antimicrobial peptide (AMP) production. Bacteria that contain DAP-type PGN elicit the IMD 0CVWTG4GXKGYU^+OOWPQNQI[
pathway in flies. Polymeric
PGN is recognized by both PGRP-LCx and an extracellular version of PGRP-LE that comprises only the PGRP domain
(PGRP-LEpg). Monomeric PGN (such as tracheal cytotoxin (TCT)) is recognized extracellularly by a PGRP-LCa–PGRP-LCx
heterodimer and intracellularly by a full-length cytoplasmic form of PGRP-LE (PGRP-LEfl). Both complexes activate AMP
production through NF‑κB signalling. It is unclear how TCT enters the cells. Extracellular IMD pathway activation is
inhibited by PGRPs with amidase activity (namely, PGRP-LB and PGRP-SC), which cleave PGN into inactive amino sugars
and peptides, and by PGRP-LF, which competes with PGRP-LCa for binding to PGRP-LCx and forms non-signalling
PGRP-LF–PGRP-LCx heterodimers. Some extracellular PGRPs also act as scavengers through enzymatic degradation of
PGN (in the case of PGRP-SB) or as opsonins for phagocytosis (in the case of PGRP-SC). Finally, PGRP-LEfl induces the
elimination of intracellular bacteria by promoting autophagy, a process that is independent of IMD.

840 | DECEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol

© 2011 Macmillan Publishers Limited. All rights reserved

 REVIEWS

Autophagy recognized in flies by PGRP-LE, which exists in two Insect PGRPs also participate in other antibacterial
Autophagocytosis that involves forms. A full-length isoform (PGRP-LEfl) detects intra- mechanisms. In D. melanogaster, full-length PGRP-LE
the degradation of the cell’s cellular peptidoglycan, and a cleaved form (PGRP-LEpg) is required to eliminate intracellular bacteria (such as
own components through the enhances PGRP-LCx-mediated cell-surface recognition Listeria monocytogenes) by promoting autophagy43 (FIG. 2),
lysosomal machinery. This
process has a role in normal
of polymeric peptidoglycan43,44 (FIG. 2). which has a protective role against intracellular patho-
cell growth and development, In contrast to systemic immune responses, which are gens (in addition to its catabolic role in the degradation
and also has a protective role controlled by both the Toll and IMD pathways, AMP pro- of the cell’s own components). Some extracellular PGRPs
against infection with duction locally in the respiratory and intestinal epithelia also function as bactericidal proteins (for example,
intracellular pathogens.
depends on the IMD pathway only. This indicates that PGRP-SB) or as opsonins (for example, PGRP-SC)12,15,45.
Gram-negative bacteria are probably the main inducers In various insects, several extracellular PGRPs trigger the
of these epithelial immune responses in D. melanogaster. prophenoloxidase cascade, which results in the formation
of melanin and of other antimicrobial compounds that
have been less well defined12,13,15,46.
&TQUQRJKNC
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PGRPs regulate the gut microbiota in D. melanogaster.
)WV AMPs are constantly produced at basal levels in the
uninfected gut of D. melanogaster, but their transcrip-
tion is highly upregulated following feeding on food
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415
shown genetically that IMD-dependent AMP produc-
tion in the gut is essential for flies to resist infection by
numerous ingested pathogens, such as Serratia marc-
2)425%QT2)42.$ escens or Pseudomonas entomophila 47,48. Although it
2)42.%
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&71:
gut response (FIG. 3).
In addition to regulating the induction of AMP pro-
+/& duction following infection, the IMD pathway has an
important role in shaping the gut response to the endog-
enous microbiota. The commensal bacterial community
R +-- in the intestinal tract of D. melanogaster is much simpler
than in humans and consists of ~3 × 105 microbial cells of
10 to 20 different species (mainly of the Acetobacter and
4GNKUJ Lactobacillus genera)49–52. Although the D. melanogaster
microbiota is a new area of research, it is already clear
4GNKUJ 2KTM
that, as shown for vertebrates, these microorganisms
#6(
influence the physiology of the host fly. The gut micro-
&71:
4GNKUJ 2)42U biota promotes larval growth by modulating hormonal
signals53 and even triggers sexual selection preference in
D. melanogaster populations by altering the production
0WENGWU %CWFCN 4GNKUJ #/2U of sex pheromones54. This implies that a tight qualita-
tive and quantitative control of the gut microbiota is
required for the optimal adaptation of D. melanogaster
to its environment.
/KFIWVGRKVJGNKCNEGNN The constant contact between bacteria and the gut
Figure 3 | Drosophila melanogaster PGRPs protect the gut from infections and mucosal epithelium raises the question of how the epi-
maintain gut microbiome homeostasis. DAP-type peptidoglycan (PGN) from thelium manages this continuous input of immune-
0CVWTG4GXKGYU^+OOWPQNQI[
intestinal bacteria is sensed by peptidoglycan recognition protein-LC (PGRP-LC), which activating signals derived from commensal micro­
triggers the IMD and p38 mitogen-activated protein kinase pathways. The activation organisms. Recent evidence points to the important
of p38 enhances the transactivating function of ATF2, which in turn activates the role of PGRP family members in the tolerance of gut
transcription of dual oxidase (Duox). This leads to the production of reactive oxygen epithelia towards the endogenous microbiota. Of the
species (ROS) in the intestinal lumen, where they control endogenous and infectious 19 D. melanogaster PGRP proteins, seven have amidase
bacteria. PGN recognition by PGRP-LC also triggers (through the IMD pathway) the activity, which allows them to cleave pro-inflammatory
cleavage and nuclear translocation of the nuclear factor‑κB family member Relish, which peptidoglycan into non-stimulatory muropeptides55,56.
then induces increased transcription of antimicrobial peptide (AMP) genes. However, in
These seven proteins are PGRP-LB‑A, PGRP-LB‑B,
the gut epithelium, the IMD pathway is under tight negative control. Two of the genes
that are most responsive to IMD activation encode PGRP-LB, which cleaves PGN and PGRP-LB‑C, PGRP‑SB1, PGRP‑SB2, PGRP‑SC1 and
therefore blocks activation of the IMD pathway, and Pirk, which interferes with the plasma PGRP‑SC2. Through their amidase activity, PGRP-SC
membrane localization of PGRP-LC. Finally, in some gut cells, the endogenous microbiota and PGRP-LB synergize to sustain a low basal level of
causes nuclear translocation of an inhibitor (Caudal), which prevents constant AMP peptidoglycan in the gut, a mechanism that prevents
production and the destruction of the normal microbiota. IKK, IκB kinase. spontaneous overactivation of the IMD pathway by the

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 841

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
Reactive oxygen species
(ROS). Aerobic organisms
derive their energy from the
reduction of oxygen. The
metabolism of oxygen, and
in particular its reduction
through the mitochondrial
electron-transfer chain,
generates by-products such
as superoxide (O2–), hydrogen
peroxide (H2O2) and hydroxyl
radicals (•OH). These three
species and the unstable
intermediates that are formed
by lipid peroxidation are
known as ROS. ROS can
damage important intracellular
targets such as DNA,
carbohydrates and proteins.
842 | DECEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
gut microbiota (B. Lemaitre, personal communication)
(FIG. 3). These enzymes also prevent excessive transfer
across the epithelial barrier (and into the haemolymph)
of immunostimulatory peptidoglycan fragments that
are released from gut bacteria. This function is revealed
by the overactivation of the systemic inflammatory
response in the fat body cells of flies that have inactivated
catalytic PGRPs in the gut 57.
Another mechanism that flies use to render their
endogenous microbiota invisible to their immune sys-
tem is the modulation of PGRP-LC localization in the
epithelial cell membrane. This function is mediated
by the intracellular protein Pirk (poor IMD response
upon knock-in; also known as Rudra or Pims), which,
by sequestering PGRP-LC in the cytoplasm, prevents
it from accessing extracellular peptidoglycan and thus
decreases IMD signalling 58–60 (FIG. 3). Accordingly, loss-
of-function mutations in the genes encoding PGRP-SC,
PGRP-LB and Pirk are associated with an increased
constitutive production of AMPs in the gut, and this
disappears when flies are grown in axenic conditions
(free of all microorganisms). Because these genes are
also transcriptional targets of the IMD pathway, their
protein products are constitutively produced at basal
levels in the gut in response to the microbiota. This
establishes a negative feedback loop that prevents hyper-
activation of the IMD target genes by peptidoglycan
derived from commensal bacteria (FIG. 3). It is expected,
although not yet demonstrated, that the dysregulated
immune activation observed in flies with mutant forms
of PGRP-SC, PGRP-LB and Pirk will induce a shift in
the composition of the gut microbiota. Similar results
have been obtained by analysing the effects of another
IMD pathway regulator, Caudal, on the composition of
the microbiota. This transcription factor directly sup-
presses IMD-dependent NF‑κB-mediated expression
of AMPs in the gut by blocking AMP gene promoters52.
The absence of such a control, as observed by inhibition
of Caudal expression, results in changes in the com-
mensal microbiota52. The baseline repression of the
IMD pathway mediated by these various proteins is also
essential for maintaining the fitness of the flies. The
absence of PGRP-LB, PGRP-SC, Pirk or Caudal dis-
rupts gut homeostasis, provokes epithelial cell renewal
and decreases the lifespan of D.  melanogaster 52,61
(B. Lemaitre, personal communication).
Control of the microbiota through regulation of ROS
production in D. melanogaster. AMP production by the
gut epithelium downstream of the IMD pathway is not
the only effector function of the intestinal antimicrobial
immune response. Consistently, flies lacking gut AMP
expression are resistant to infection by many bacterial
species and, as discussed earlier, IMD pathway activa-
tion is under strong negative regulation in the gut to
preserve the commensal microbiota and host health.
Recent studies have shown that reactive oxygen species
(ROS) are important microbicidal effectors in the fly
gut 62. Although ROS and AMPs are directed towards
the same microbial targets, their production in the
D. melanogaster gut is regulated by different mechanisms.
© 2011 Macmillan Publishers Limited. All rights reserved
ROS are produced at basal levels by dual oxidase
(DUOX), which is associated with the gut membrane.
Following infection, increases in both Duox gene activa-
tion and DUOX protein expression are required to coun-
ter the increased bacterial burden in the gut 63. DUOX
enzymatic activation requires an increase in intra­cellular
calcium levels and probably depends on signalling
through an as yet unidentified upstream G protein-
coupled receptor (and is therefore independent of
PGRPs). By contrast, Duox transcriptional upregula-
tion is triggered by both peptidoglycan-dependent and
-independent pathways. In the peptidoglycan-dependent
pathway, peptidoglycan recognition by PGRP-LC acti-
vates the transcription of Duox through IMD, MEKK1,
p38 and ATF2. This results in increased levels of DUOX
in the epithelial cell membrane and thus enhances the
release of ROS into the gut lumen63 (FIG. 3).
The above findings show that D. melanogaster gut
innate immune responses, which are Toll independent,
rely on a finely tuned balance between the IMD and the
DUOX pathways, which are both activated by upstream
PGRP molecules. Activation or repression of these path-
ways allows the host to adapt its intestinal antimicrobial
response to the gut microbial load. Phenotypic analy-
ses of flies with mutations in genes that are involved in
these pathways demonstrate that controlling the quantity
and the quality of the intestinal microbiota is crucial for
the flies, and that PGRP family members are important
players in these defence and homeostatic mechanisms.
Control of symbiotic bacteria and parasites in other
insects. Haematophagous insects frequently encoun-
ter blood-borne infectious agents, including para-
sites and viruses. For example, female Anopheles spp.
mosquitoes become infected with malaria parasites
(Plasmodium spp.) during feeding on infected humans
and can then transmit the parasites to new hosts dur-
ing subsequent bites. Blood-meal ingestion is associated
with an increase in the size of the microbial commu-
nity in the gut of the female mosquito, and this triggers
PGRP-LC-mediated NF‑κB signalling, which induces
AMP production to control the microbial burden64
(FIG. 4a). In mosquitoes in which PGRP-LC levels have
been downregulated by RNA interference, gut bacterial
loads are strongly increased compared with those in con-
trol mosquitoes. Interestingly, this PGRP-LC-mediated
response not only affects gut bacteria but also seems
to decrease the infection efficiency of protozoal para-
sites. Silencing of the gene encoding PGRP-LC results
in an increased number of Plasmodium spp. oocysts in
infected mosquitoes (FIG. 4a). Because this effect is no
longer observed in mosquitoes in which the gut micro-
biota has been eliminated by antibiotic treatment, these
results indicate that PGRP-LC-mediated antibacte-
rial defence also has an indirect antiparasitic effect in
Anopheles gambiae65. It is probable that, in addition to
controlling the gut microbiota, some of the bacterium-
induced defence molecules (such as AMPs and ROS)
might interfere with the normal development of malaria
parasites during the midgut stages of their life cycle,
although this has not yet been clearly shown65.
 REVIEWS

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Figure 4 | In blood-sucking insects, PGRPs maintain the balance between symbiotic bacteria and protozoal
parasites. a | Peptidoglycan recognition protein-LC (PGRP-LC) controls intestinal symbiotic and infectious bacteria in
Anopheles gambiae by inducing the production of antimicrobial peptides (AMPs). This inhibits 0CVWTG4GXKGYU^+OOWPQNQI[
the proliferation of gut
bacteria after a mosquito blood meal. PGRP-LC activation also strongly decreases the number of Plasmodium spp.
oocysts in the A. gambiae gut after a blood meal. The tripartite interaction between symbiotic bacteria, invading
parasites and the mosquito immune system is important for the balance of mosquito susceptibility and resistance to
malaria and perpetuates disease transmission. b | The tsetse fly (Glossina spp.) is the sole African vector for trypanosomes
and has co-evolved with the mutualistic endosymbiont Wigglesworthia glossinidia. W. glossinidia induces the expression
of PGRP-LB by infected midgut cells (bacteriocytes), and PGRP-LB cleaves peptidoglycan (PGN) and decreases the
damage to the host associated with the permanent presence of the endosymbiont. PGRP-LB also maintains low levels of
trypanosomes by an unknown mechanism. Elimination of W. glossinidia renders tsetse flies sterile and increases their
susceptibility to trypanosome infection.

PGRP family members also regulate interactions
between some insects and their microbial endosymbi-
onts. Adult tsetse flies (Glossina spp.) feed exclusively on
vertebrate blood, a very unbalanced meal lacking vital
components, such as many vitamins. To compensate
for this nutritionally restricted diet, tsetse flies carry
the obligate endosymbiont Wigglesworthia glossinidia,
the presence of which is required for adequate diges-
tion and nutrition in the flies and for their successful
reproduction. The Gram-negative W. glossinidia bacteria

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 843

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
reside in differentiated midgut cells, where they form an
organ known as a ‘bacteriome’. W. glossinidia elimina-
tion results in sterility of tsetse flies and increases their
susceptibility to trypanosome infection66, demonstrating
the essential role of this endosymbiont in the life cycle of
tsetse flies (FIG. 4b).
The symbiosis between the tsetse fly and W. glossi-
nidia raises two questions: how does the tsetse fly
immune system tolerate the constant presence of its
gut symbiont, and what is the mechanism by which
W. glossinidia mediates its antitrypanosome effect?
Part of the answer was obtained by studying the func-
tion of the DAP-type peptidoglycan-cleaving enzyme
PGRP-LB in tsetse flies. It has been shown that W. glossi-
nidia triggers the expression of tsetse fly PGRP-LB in
its bacteriome67 (FIG. 4b). It is thought that by scavenging
W. glossinidia peptidoglycan, PGRP-LB prevents consti-
tutive IMD pathway activation and, hence, establishes
tolerance towards this symbiont. Consistently, decreas-
ing the in vivo PGRP-LB level induces an increase in the
level of AMPs and decreases the number of W. glossi-
nidia in the tsetse fly gut. Unexpectedly, depletion of
PGRP-LB also increases the susceptibility of tsetse flies
to trypanosome parasites (FIG. 4b), and this indicates that
PGRP-LB might have an antiprotozoal activity that con-
fers a resistance to these parasites67. Bacteriomes contain-
ing symbiotic bacteria are common in other insects, and
the mutualistic interactions between the insects and their
endosymbionts are probably also controlled by PGRPs;
for example, this has been suggested by the high level of
expression of PGRP-LB in the bacteriome of the weevil
Sitophilus zeamais 68.
Thus, in insects that do not belong to the Drosophilidae
family, PGRPs and the host innate immune response
interact to fine-tune the homeostatic balance between
tolerance to indispensable mutualistic microorganisms
and resistance to pathogenic microorganisms. It is possi-
ble that this tripartite interaction between the gut micro-
biome of insects, PGRP-mediated antibacterial defences
and parasites such as Plasmodium spp. or trypanosomes
could be exploited in future interventions aiming to
control disease transmission.
Control of luminescent bacteria by squid PGRPs
PGRPs are also present in other invertebrates. In
molluscs — such as oysters69, scallops70,71, squid72–76
and snails77 — PGRPs are expressed in the mantle,
gills, haemocytes, digestive diverticula, kidneys and
gonads69–72,77. Some of the mollusc PGRPs are predicted
or proven to be peptidoglycan hydrolases, whereas oth-
ers have defensin-like domains, indicating that they
might have a direct bactericidal function69–72,77. These
mollusc PGRPs probably have a role in defence against
infections, because their expression is increased in
response to bacterial pathogens, such as Echinostoma
paraensei 70.
In the Hawaiian bobtail squid, Euprymna scol-
opes, PGRPs are involved in highly specialized host–
microorganism interactions that result in the establish-
ment and maintenance of the symbiotic luminescent
bacterium Vibrio fischeri within the squid light organ72.
844 | DECEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
© 2011 Macmillan Publishers Limited. All rights reserved
These squid are nocturnal animals, and light produced
by the symbiotic bacteria provides counter-illumination
to moonlight and makes the squid less visible to
predators.
E. scolopes are not associated with any bacteria
at birth; however, within a few hours after hatching,
the light organ becomes populated with V. fischeri in
an association that is highly specific for this bacterial
species75,76. The nascent light organ has a pair of pro-
truding ciliated appendages on the opposing sides of
three pores (FIG. 5). In response to bacteria in the sur-
rounding water, epithelial cells of the appendages
secrete mucus that covers the pores and appendages74–76.
The beating of the cilia concentrates bacteria from the
surrounding water into the mucus, which starts the
‘winnowing’ of V. fischeri from hundreds of other bac-
terial species present in the water, so that only V. fis-
cheri enters through the pores into the ducts of the light
organ. These bacteria then swim into the deep crypts,
where they lose their flagella and permanently colonize
the host 74–76. Entry of V. fischeri into the ducts induces
apoptosis and morphogenesis in the light organ; this
involves the loss of ciliated epithelium and the shorten-
ing and eventual loss of the appendages, which prevents
the entry of other bacteria into the light organ73–76.
PGRPs have been proposed to participate in the
winnowing and maintenance of V. fischeri in the light
organ72–76, but the basis for the selectivity of this win-
nowing is still poorly understood. E. scolopes has four
PGRPs, and they are all expressed in the light organ72.
Two PGRPs have been proposed to function as recep-
tors on the surface of light organ cells; these are PGRP3,
which has a predicted glycosylphosphatidylinositol
(GPI) membrane anchor and amidase activity, and
PGRP4, which is predicted to have two transmembrane
domains, an extracellular PGRP domain and no amidase
activity 72 (FIG. 5D). Light organ cells also express a TLR
and an LPS-binding protein (LBP)72. It is proposed that
bacterial products, such as peptidoglycan and LPS, acti-
vate the transcription factors NF‑κB, ETS and Krüppel
through PGRP3, PGRP4 and TLR. These transcrip-
tion factors then induce the synthesis of antibacterial
factors — such as mucin, nitric oxide synthase (NOS)
and LBP — and also of PGRP1 and PGRP2 by the
ciliated epithelial cells72 (FIG. 5D).
PGRP2 is secreted with the mucus and is an ami-
dase that hydrolyses pro-inflammatory peptido­
glycan fragments74. PGRP2 is also secreted into the
lumen of the crypts74, where it helps to maintain the
homeostatic balance between E. scolopes and the per-
manently present V. fischeri. PGRP2 accomplishes
this by hydrolysing peptidoglycan fragments that are
constantly released from V. fischeri and thus prevent-
ing the inflammatory effects of these peptidoglycan
fragments, which allows beneficial coexistence of the
symbiont with the host 74. PGRP2 might also have a low
level of antibacterial activity that helps in the winnow-
ing function of the appendage-derived mucus and in
maintaining the proper numbers of V. fischeri in the
crypts, although this antibacterial activity has not yet
been experimentally proven.
 REVIEWS

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Figure 5 | Euprymna scolopes PGRPs control colonization and maintenance of the luminescent bacterium Vibrio
0CVWTG4GXKGYU^+OOWPQNQI[
fischeri within the light organ of the squid. A | The location of the light organ in the body cavity of E. scolopes is shown.
B | The figure shows the colonization of the crypts in the light organ by V. fischeri. Seawater contains many different
bacteria, which stimulate epithelial cells of the light organ to secrete mucus containing peptidoglycan recognition
protein 2 (PGRP2) and probably other antibacterial factors that select V. fischeri (a). As a result, only Gram-negative
bacteria aggregate between the appendages of the light organ (b), and V. fischeri becomes dominant (c). V. fischeri then
enter the ducts (d) and permanently colonize the crypts (e). C | Successful establishment of V. fischeri in the crypts results
in the loss of ciliated appendages. D | Light organ cells express Toll-like receptor (TLR) and four PGRPs with different
cellular locations. PGRP1 is intracellular, PGRP2 is secreted, PGRP3 has a predicted glycosylphosphatidylinositol
membrane anchor and PGRP4 has two predicted transmembrane domains and an extracellular PGRP domain. It is
proposed that PGRP3 and PGRP4 function as cell surface receptors for bacterial products and, together with TLR, activate
the transcription factors nuclear factor‑κB (NF‑κB), ETS and Krüppel, which induce the synthesis of mucin, nitric oxide
synthase (NOS), lipopolysaccharide binding protein (LBP), PGRP1 and PGRP2 in ciliated epithelial cells, and PGRP2 in
crypt epithelial cells. Loss of PGRP1 expression in ciliated epithelial cells after colonization of the light organ by V. fischeri
leads to apoptosis and the loss of appendages. PGRP2 is secreted by epithelial cells, first in the appendages and then in
the crypts, where it degrades peptidoglycan fragments that are released by V. fischeri. This maintains homeostasis
between the host and the symbiont. Figure parts A–C are modified, with permission, from REF. 76 © (2004) Macmillan
Publishers Ltd. All rights reserved.

NATURE REVIEWS | IMMUNOLOGY VOLUME 11 | DECEMBER 2011 | 845

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
PGRP1 is a predicted amidase, but it does not have
a signal peptide and is located in the nuclei and the
cytoplasm of ciliated epithelial cells in the appendages.
The loss of PGRP1 expression by these cells (presum-
ably when the activating signals derived from bacteria
decrease) correlates with the initiation of their apopto-
sis73, but the exact mechanism of this process is unknown.
Apoptosis of epithelial cells is followed by the loss of cilia
and eventually appendages73, and this marks the com-
pleted colonization of the light organ with V. fischeri
(FIG. 5). PGRP1 is also expressed in the crypts, but its role
there is unknown.
PGRPs are also expressed by another group of
marine invertebrates, the echinoderms. In the European
starfish, Asterias rubens, PGRP‑S1a is expressed by the
epithelial cells lining the coelom, and PGRP‑S2a is
expressed by phagocytes. PGRP‑S2a hydrolyses pepti-
doglycan and increases the phagocytosis of Micrococcus
luteus, indicating that it has a role in the defence
against bacteria78.
Defence against infections by fish PGRPs
PGRPs have been identified in several species of fish,
including zebrafish 79, rockfish 80 and large yellow
croaker 81. Zebrafish have four PGRP genes, three of
which have been cloned and characterized (namely,
pglyrp2, pglyrp5 and pglyrp6) 79. These PGRPs are
expressed widely in different tissues of adult fish, includ-
ing in the skin, gills, liver, intestine and pancreas (FIG. 6a),
indicating that they function both in the defence against
bacteria that are present in the environment and during
systemic infections. The high-level expression of PGRPs
in the intestine also indicates a probable role of these
proteins in the modulation of the fish gut microbiome.
Zebrafish Pglyrp2, Pglyrp5 and Pglyrp6 are amidases
that cleave the bond between N‑acetylmuramic acid and
alanine in peptidoglycan79. They are also bactericidal
against both Gram-positive and Gram-negative bacte-
ria79 (FIG. 6a). Rockfish PGRPs also have both amidase
and bactericidal activities80.
Zebrafish Pglyrp2 is highly expressed in eggs and in
developing oocytes, and Pglyrp5 is induced during early
embryogenesis and is strongly expressed at 72 hours
post fertilization79. These proteins protect the develop-
ing embryo from infection by bacteria that are present in
the surrounding water (FIG. 6a). Knockdown of pglyrp5
expression during early embryogenesis (using a specific
morpholino) results in increased sensitivity to infections
with Salmonella enterica and Bacillus subtilis and a high
level of embryonic mortality 79. Similarly, inhibition of
Pglyrp6 expression increases the sensitivity and mortal-
ity of zebrafish embryos to Flavobacterium columnare
infection82. Therefore, PGRPs have an essential role in
the defence against bacteria during the early stages of fish
development, when adaptive immunity has not yet devel-
oped and thus, like in invertebrates, innate immunity is
the only defence mechanism.
Zebrafish Pglyrp5 and Pglyrp6 might also affect
multiple intracellular pathways. Inhibition of Pglyrp5
expression in the developing embryo using small inter-
fering RNA modified the expression of genes involved in
846 | DECEMBER 2011 | VOLUME 11 www.nature.com/reviews/immunol
© 2011 Macmillan Publishers Limited. All rights reserved
several pathways, including immune and inflammatory
responses, signalling pathways, transcription and metab-
olism82,83. Inhibition of Pglyrp5 increases the expression
of TLR2, TLR3, MAPK-interacting serine/threonine
kinase 2, the interleukin‑17 receptor and NF‑κB83 (FIG. 6a).
Therefore, PGRPs can directly or indirectly down­regulate
the immune response to bacteria in the surrounding
water, thus preventing a constant state of inflammation.
However, additional studies confirming these roles of
PGRPs are needed.
Protection against colitis by mammalian PGRPs
Mammals have four PGRPs, three of which (PGLYRP1,
PGLYRP3 and PGLYRP4) are bactericidal24,26,84,85 (BOX 1)
but have no amidase activity 26,85,86, which distinguishes
them from fish PGRPs, which have both activities79.
Mammalian PGLYRP2, like fish PGRPs, has both ami-
dase86,87 and antibacterial activities (R.D., S.‑Y. Wang and
D.G., unpublished observations). PGLYRP1 is mainly
expressed in the granules of neutrophils and eosino-
phils and contributes to the antibacterial activity of these
cells88–90. PGLYRP1 is also expressed to a lesser extent in
the salivary glands, tongue, throat, oesophagus, jejunum,
ileum and caecum. PGLYRP2 has the highest constitutive
level of expression in the liver, from where it is secreted
into the blood91,92, and PGLYRP2 expression is induced
by bacteria and cytokines (such as interleukin‑1β and
tumour necrosis factor) in the skin and in other epithe-
lial cells, including those in all segments of the gastro­
intestinal tract 26,93–95. PGLYRP3 and PGLYRP4 have
the highest level of expression in the skin and on the
mucous membranes, in particular in the mouth, throat,
oesophagus and salivary glands and, to a lesser extent,
in the stomach and the small and large intestines26,96,97.
Thus, mammalian PGRPs are strategically positioned
in all areas of the body that come into contact with
environmental microorganisms and that are inhabited
by commensal microorganisms, and it is thought that
through their bactericidal effects mammalian PGRPs
can influence the microbiota at these sites. PGRPs (in
particular, PGLYRP1 and PGLYRP4) are also expressed
in the mammary glands and are present in milk97,98, and
thus these PGRPs could influence the mouth and gut
microbiota as soon as newborns begin to drink milk.
All four mammalian PGRPs control the acquisition
and maintenance of the normal gut microbiota, which
protects the host from enhanced inflammation, tis-
sue damage and colitis97 (FIG. 6b). Pglyrp1–/–, Pglyrp2–/–,
Pglyrp3–/– and Pglyrp4–/– mice are all more sensitive
than wild-type mice to experimental ulcerative colitis
induced by dextran sulphate sodium (DSS), a com-
pound that damages the intestinal epithelium. The
probable sequence of events that makes PGRP-deficient
mice more sensitive to colitis is as follows (FIG. 6b). First,
deficiencies in individual PGRPs induce dysbiosis in
the colonic microbiota; this involves shifts in the levels
of various groups of bacteria in the colon, and these
alterations are unique for mice deficient in different
PGRPs. For example, there is a decrease in the numbers
of lactobacilli (which often have beneficial probiotic
effects) in Pglyrp2–/–, Pglyrp3–/– and Pglyrp4–/– mice.
 REVIEWS

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Figure 6 | Vertebrate PGRPs are bactericidal, defend against
infections, regulate intestinal microorganisms and protect
mammals against colitis. a | Danio rerio (zebrafish) peptidoglycan
recognition proteins (PGRPs) are expressed in the adults, eggs and
embryos and protect against bacterial infections during early
development. Zebrafish PGRPs have amidase, bactericidal and
immunomodulatory activities and are differentially expressed in various
organs, including the skin, gills, intestine, liver and gonads in the adult
fish. Zebrafish embryos develop in bacteria-contaminated water and
rely on innate immunity for host defence. Zebrafish eggs express
Pglyrp2 and the embryos express Pglyrp2, Pglyrp5 and Pglyrp6, and this
protects the eggs and embryos from bacterial infections. b | Mammalian
PGRPs promote beneficial intestinal microorganisms, which facilitate
healing following intestinal injury and thus protect against colitis. A lack
of PGRPs results in the overgrowth of harmful intestinal microorganisms
and, following intestinal injury with dextran sulphate sodium (DSS), this
results in increased production of natural killer (NK) cell-attracting and
0CVWTG4GXKGYU^+OOWPQNQI[
-activating chemokines (namely, CXCL9, CXCL10 and CXCL11). This
leads to the recruitment of NK cells to the colonic epithelium, the
production of interferon-γ (IFNγ) by NK cells, the activation of
IFN-inducible genes (which include NK cell-attracting chemokine
genes) and eventually colitis, which is manifested by hyperplasia of the
lamina propria, loss of colonic epithelium, ulceration, bleeding and
death. PGRPs promote beneficial gut microorganisms that do not
induce these chemokines and do not cause NK cell recruitment and
IFNγ production, and thus they protect against colitis. This paradigm has
been experimentally verified in a mouse model of colitis, but it probably
applies to all mammals, including humans. IL‑17R, interleukin‑17
receptor; MKNK2, MAPK-interacting serine/threonine kinase 2;
NF‑κB, nuclear factor‑κB; TLR, Toll-like receptor.

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Box 1 | PGRPs kill bacteria by activating two-component systems

Peptidoglycan recognition proteins (PGRPs) kill bacteria by exploiting bacterial two-component stress response systems,
such as CssR–CssS in Bacillus subtilis and CpxA–CpxR in Escherichia coli85 (see the figure). These two-component systems
consist of a transmembrane sensor and a cytoplasmic regulator; they detect extracytoplasmic misfolded and aggregated
bacterial proteins, which are generated under stress, exported from the cell and degraded by proteases104,105.
In the case of Gram-positive bacteria, PGRPs bind to peptidoglycan in bacterial cell walls at the sites of hydrolysis
by LytE and LytF enzymes, which in B. subtilis separate daughter cells after cell division. In the case of Gram-negative
bacteria, PGRPs bind uniformly to the entire outer membrane85, which is composed of lipopolysaccharide (LPS) and
covers a thin peptidoglycan layer. In addition to binding peptidoglycan12,15,20, PGRPs bind to LPS and other molecules23–25
using binding sites outside the peptidoglycan-binding groove. The binding of PGRPs to peptidoglycan or LPS probably
induces PGRP oligomerization into ribbon-like structures106, which are then detected by the B. subtilis CssR–CssS or
E. coli CpxA–CpxR two-component systems85.
Activation of the protein-sensing two-component systems not only triggers the removal of the misfolded proteins,
but also signals into the cell to activate a repair and defence response, and induces membrane depolarization and the
production of toxic hydroxyl radicals (•OH). This is accompanied by the cessation of all major intracellular biosynthetic
reactions, probably because of the lack of energy generation through mechanisms that are dependent on the membrane
potential85,105. If stress is sustained or its level is too high, the bacteria die. This suicide mechanism might have evolved as
being advantageous to the entire bacterial population, because bacteria that are damaged beyond their ability to repair
themselves would die quickly and not use up scarce resources. The mammalian PGRPs (and probably other PGRPs)
trigger this bacterial defence/suicide mechanism to kill bacteria85. PGRPs use this mechanism both to defend against
pathogens79 and to control the levels of symbiotic microorganisms97; it is possible that symbiotic microorganisms
have a higher tolerance to PGRPs than other bacteria26.

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The stool microbiota from PGRP-deficient mice is
more pro-inflammatory and more damaging to the
colon than the stool microbiota from wild-type mice.
So, in vivo, after intestinal injury (for example, by DSS),
the more pro-inflammatory and damaging microbiota
in the colon of PGRP-deficient mice induces the pro-
duction of higher levels of chemokines that attract
innate immune cells, in particular natural killer (NK)
cells, to the site of injury. NK cells produce excessive
amounts of interferon‑γ, which results in the activa-
tion of interferon-inducible genes (including more NK
cell-attracting chemokine genes) that promote intesti-
nal damage and prevent healing. This insult leads to
hyperplasia of the colonic lamina propria (the layer of
cells underlying the colonic epithelium), loss of colonic
epithelium, ulceration, bleeding and eventually death97.
The role of dysregulation of the colonic microbiota
as the primary factor responsible for this sequence of
events in PGRP-deficient mice has been shown by the
0CVWTG4GXKGYU^+OOWPQNQI[
ability of stools from PGRP-deficient mice, but not stools
from wild-type mice, to transfer this increased sensitivity
to colitis to wild-type germ-free mice97. This scenario
is an experimental example of a genetic deficiency that
results in dysbiosis, with the clinical consequence of
increased sensitivity to colitis following damage to the
colonic epithelium. Nucleotide-binding oligomerization
domain 2 (Nod2) is another gene that, when mutated,
induces intestinal dysbiosis and increased sensitivity to
inflammatory bowel disease (IBD)99.
Thus, in wild-type animals, PGRPs promote the
acquisition and maintenance of beneficial gut micro-
organisms that, following intestinal injury, promote
intestinal healing and protect the animal from devel-
oping severe and usually lethal colitis (FIG. 6b). Although
not yet proven, PGRPs probably have a similar protec-
tive role in all mammals, including humans, through
the maintenance of beneficial microbial communities
within the gut.

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 REVIEWS

Conclusions and future directions
PGRPs control the levels of symbiotic microorganisms
and defend against other bacteria in all animals that have
so far been studied. In some animals, they have very spe-
cialized functions to control highly specialized symbi-
onts, whereas in other animals they control and defend
against a wide range of microorganisms, and even control
parasites. PGRPs accomplish these tasks both through
direct antibacterial activity and through indirect effects,
including the induction of antimicrobial peptides and the
modulation of inflammation and immune responses.
There are many questions regarding how PGRPs
accomplish these functions. PGRPs kill bacteria by acti-
vating their two-component systems (BOX 1). Future work
should define whether this activation occurs directly or
indirectly and how this activation induces bactericidal
mechanisms, such as membrane depolarization and the
inhibition of biosynthetic reactions.
Although we know that PGRPs induce changes in
the microbiota, future work should define these changes
more precisely and should determine how alterations in
the microbiota induce diseases or immunological imbal-
ance. It should also be determined whether the effects of
PGRPs on the gut microbial communities have systemic
consequences in other inflammatory diseases and, if so,
which microbial products mediate these effects. Although
it is possible that all the in vivo effects of PGRPs are based
on changes in the microbiota, it is likely that some effects
of PGRPs (for example, PGLYRP2‑dependent sensitivity
to experimental arthritis100) are based on other intrinsic
characteristics of PGRPs, and the mechanisms responsible
for these outcomes need to be defined.
Increased sensitivity to colitis in PGRP-deficient mice
raises the possibility that genetic defects in PGRPs might
predispose humans to IBD. Many genes are associated
with IBD101. The human PGLYRP2 gene is located at
position 19p13.12, within the IBD susceptibility locus
19p13, for which the susceptibility gene has not yet been
identified101–103. Therefore, PGLYRP2 could be one of
the human IBD susceptibility genes. Mutations in other
PGRP genes could also predispose patients to IBD, as
many previously unknown IBD susceptibility genes
are continuing to be discovered. These possibilities are
currently being tested by genotyping patients with IBD.
If human PGRPs are associated with IBD, it should be
determined whether this effect is mediated through
changes in the intestinal microbiota and whether this
predisposition could be treated by oral administration
of PGRPs.
Many other applications based on the interactions
of PGRPs with the microbiota can be envisioned. New
antimicrobial therapies could exploit the activation
of bacterial and fungal two-component systems by
PGRPs, PGRP fragments or PGRP mimetics. The role
of PGRPs in maintaining beneficial microorganisms in
the mouth and gut could be applied to develop toothpastes
and mouthwashes that promote this healthy microbiota.
Transgenic cows that express human PGRPs in milk could
be produced, and this milk could be used to reduce the
incidence of food-borne infections and to promote
the maintenance of beneficial intestinal micro­organisms.
Furthermore, PGRPs or PGRP mimetics could be used
to control insect-transmitted diseases, such as malaria
and trypanosomiasis. Parallel dissection of PGRP
functions in various invertebrates and vertebrates, and
a more intense focus on the functions of PGRPs in
mammals will shed more light on PGRP evolution and
lead to new approaches to control the microbiome and
to prevent and treat infections and other diseases that are
affected by the microbiome.

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Acknowledgements
Research in the authors’ laboratories is supported by the
National Institutes of Health (D.G. and R.D.) and the Action
Concertée Insitative, Fondation pour la Recherche Médicale,
Institut Universitaire de France, and Agence Nationale de la
Recherche (J.R.).
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
Julien Royet’s homepage: http://www.ibdml.univ-mrs.fr/
equipes/equipe_gb.php?id=63
Dipika Gupta’s homepage: http://iusm-nw.medicine.iu.edu/
faculty/dipika-gupta-ph-d/
Roman Dziarski’s homepage: http://iusm-nw.medicine.iu.
edu/faculty/roman-dziarski-ph-d/
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