Download Bovine Peptidoglycan Recognition Protein

The Journal of Immunology
Bovine Peptidoglycan Recognition Protein-S: Antimicrobial
Activity, Localization, Secretion, and Binding Properties1
C. Chace Tydell,2* Jun Yuan,* Patti Tran,* and Michael E. Selsted*†‡
I
nnate immunity provides the first line of antimicrobial defense in animals (1), and many components of this system
are evolutionarily conserved from insects to humans (2).
Pattern recognition molecules, encoded in the germ line, are
essential components of innate immunity, because they distinguish invading pathogens based on the recognition of microbial
surface molecules not present on host cells (3). The recently
characterized peptidoglycan (PGN)3 recognition proteins
(PGRPs), constitute an important family of pattern recognition
molecules (4 –11), some members of which exhibit antimicrobial activity in vitro (6, 10) and in vivo (12). PGRPs have been
classified into three categories based on transcript length: short
(PGRP-S), short (PGRP-I), and long (PGRP-L). Formerly
known as bovine oligosaccharide binding protein (bOBP), bovine PGRP-S (bPGRP-S) is an 18.7-kDa ortholog of human and
murine PGRP-S.
PGRPs, first discovered in insects, were named for their ability
to bind to PGN and Gram-positive bacteria (13, 14). Recent studies
*Department of Pathology and Laboratory Medicine, †Department of Microbiology
and Molecular Genetics, and ‡Center for Immunology, University of California, Irvine, CA 92697
Received for publication September 8, 2005. Accepted for publication October
28, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants A122931 and
AI54699-01 and the Hewitt Foundation.
2
Address correspondence and reprint requests to Dr. C. Chace Tydell, Division of
Biology, California Institute of Technology, Pasadena, CA 91125. E-mail address:
[email protected]
3
Abbreviations used in this paper: PGN, peptidoglycan; PGRP, peptidoglycan recognition protein; bOBP, bovine oligosaccharide binding protein; bPGRP, bovine
PGRP; LTA, lipoteichoic acid; MDP, muramyl dipeptide; PGRP-I, intermediate
PGRP; PGRP-L, long PGRP; PGRP-S, short PGRP; rb, recombinant bovine; HOAc,
acetic acid.
Copyright © 2006 by The American Association of Immunologists, Inc.
suggest that microbial recognition by PGRPs may not depend on
PGN exclusively. Bovine PGRP-S has been shown to kill microorganisms in which PGN is either buried (Gram-negative bacteria)
or absent (Cryptococcus neoformans) (10), and Holotrichia
PGRP-S has been shown to trigger an insect immune response by
specifically binding 1,3-␤-glucan (15). In addition, soluble murine
PGRP-L can mediate macrophage responses to LPS (16). Moreover, PGRPs mediate Drosophila immune responses to both
Gram-negative and Gram-positive bacteria (4, 5, 9, 11, 17), even
though PGN is only surface exposed on certain Gram-positive organisms. Although the roles of PGRPs in insects are well documented, less is known about the functions of these proteins in
mammalian systems.
To date, mammalian PGRPs have been identified in humans (6,
7, 13), rats (18), mice (13), cattle (10), camels (19), and pigs.
PGRPs have structural homology to bacteriophage T7 lysozyme,
and several PGRPs are reported to digest PGN (16, 20 –24). Analysis of the human genome predicts the existence of four PGRPs of
varying lengths (7, 17, 25). Although primary sequence data led to
predictions that human PGRP-Ia and -b and PGRP-L were membrane bound, recent studies suggest that they are probably soluble,
secreted proteins (16, 26). Crystallographic studies of PGRP-I revealed that the putative transmembrane domain may actually form
a second PGN binding domain (26). In mammals, PGRP proteins
have been found in peripheral white blood cells (10, 12) and Peyer’s patch tissue (27) and the mRNA is present in liver, esophagus
(7), and oral epithelium (28).
A clear understanding of PGRP action in vivo requires knowledge of the locations where the proteins are stored and where they
act. Our previous studies (10) demonstrated that PGRP-S is found
in neutrophils and eosinophils and has the same immune staining
pattern as ␤-defensin (29), which is a secreted protein found in
bovine dense/large granules. We therefore evaluated PGRP-S in
secretion studies and also definitively localized PGRP-S in both
0022-1767/06/$02.00
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Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from
insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are
essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found
to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand.
Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating
physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and
lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of
bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the
dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the
neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S
when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of
bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules
challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have
a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and
activity. The Journal of Immunology, 2006, 176: 1154 –1162.
The Journal of Immunology
Materials and Methods
Bovine granulocytes
Granulocytes were purified from fresh citrated bovine blood as described
previously (10, 30). Preparations contained an average of 1 ⫻ 109 granulocyte-enriched leukocytes/l whole blood, of which 93 ⫾ 3% were neutrophils and 4 ⫾ 1% were eosinophils.
Purification of bovine PGRP-S
Bovine PGRP-S was purified from 10% acetic acid extracts of bovine
granulocytes by a modification of our previous method (10). Acid extracts
of 3 ⫻ 108 cell equivalents of bovine leukocytes were loaded onto a 10 ⫻
25-cm Delta Pak reverse phase HPLC C18 cartridge (Waters) equilibrated
in 0.1% trifluoroacetic acid (solvent A) at a flow rate of 15 ml/min. A linear
gradient of acetonitrile containing 0.1% trifluoroacetic acid (solvent B) was
applied at 2.33%/min from 0 –35%, then held at 35% B for 10 min, followed by 0.2%/min from 35– 42%. The PGRP-S-containing fraction was
determined by Western blotting, and purity of ⬎99% was confirmed by gel
electrophoresis and reverse phase HPLC as previously described (10). Purified PGRP-S was lyophilized and resuspended in 0.01% acetic acid
(HOAc) for storage at ⫺70°C. The protein concentration was quantified
spectrophotometrically (1 mg/ml ⫽ 1.28A280) (10).
PGRP-S secretion
Granulocyte-enriched populations of bovine peripheral leukocytes were
purified as described above and suspended to 1.2 ⫻ 107 cells/ml in HBSS
(137 mM NaCl, 5.6 mM glucose, 5 mM KCl, 4 mM NaHCO3 1 mM CaCl2,
0.5 mM MgCl2, 0.4 mM KH2PO4, 0.4 mM Na2HPO4, and 0.4 mM MgSO4
(pH 7.4)). Aliquots of the cell suspension were incubated for 60 min at
37°C in a final volume of 500 ␮l containing one of the following stimulants: 100 nM PMA, 20 ␮g/ml lipoteichoic acid (LTA) from Bacillus subtilis, 160 ␮g/ml muramyl dipeptide (MDP) (Sigma-Aldrich), 100 ␮g/ml
LPS from Staphylococcus typhimurium (List Biological Laboratories), 110
␮g/ml butyric acid, or 5.4 ⫻ 108 CFU/ml nonopsonized bacteria (Staphylococcus aureus 502a or Staphylococcus typhimurium 10428 PhoP⫺). The
viability of the leukocytes was ⱖ94% for each condition, as determined by
trypan blue exclusion at the beginning and the end of the incubation. Bacteria were separated from the cell pellet by centrifugation at 50 ⫻ g for 5
min at 4°C. The resulting bacterial suspensions, verified to be free of contaminating leukocytes by light microscopy, were then sedimented at
22,500 ⫻ g. Supernatants were acidified by addition of acetic acid to a final
concentration of 10% (v/v). Both leukocyte and bacterial pellets were extracted with 10% acetic acid for 36 h with rotation at 4°C. Aliquots of each
pellet extract or supernatant (4 ⫻ 104 cell equivalents) were lyophilized,
boiled in SDS-tricine sample buffer for 10 min, separated by tricine-SDS
PAGE and analyzed by Western blotting using anti-bPGRP-S IgG as previously described (10).
Immunogold labeling
Leukocytes exposed to serum-opsonized bacteria and naive leukocytes
were evaluated by electron microscopy. S. aureus and S. typhimurium were
opsonized with a 50/50 mixture of HBSS and autologous bovine serum for
30 min on ice before an equal volume of the bacterial suspension was
added to the leukocytes in HBSS. Phagocytosis was allowed to proceed for
60 min at 37°C. The mixture was then centrifuged, washed, and pelleted.
White blood cells were fixed with 2% glutaraldehyde in cacodylate buffer
(0.1 M sodium cacodylate, 1 mM MgSO4, and 3 mM CaCl2 (pH 7.4)),
washed, then dehydrated and embedded in LR White (London Resin)
according to manufacturer’s instructions. Sections (750 –900 Å) were
prepared using an Ultracut E microtome (Reichert). Primary staining
was performed using 1/32 anti-bPGRP-S IgG for 2 h, then washed for
10 min. Secondary staining was performed by an additional 1-h incubation with a 1/75 dilution of goat anti-rabbit IgG conjugated to 15-nm
gold beads (BB International) and washed again. Sections were stained
with 2% uranyl acetate and Reynold’s lead citrate before evaluation on
a Philips EM 201 transmission electron microscope. A negative control
staining was performed by substituting preimmune rabbit IgG in the
primary incubation.
Microbicidal assays
Listeria monocytogenes 967 and S. typhimurium 10428 PhoP⫺, suspended in 10 mM Tris-HCl and 5 mM glucose (pH 7.4), were used as
target organisms in microbicidal suspension assays as previously described (30, 31). The final concentration of bPGRP-S was 100 mg/ml
for S. typhimurium and 50 mg/ml for L. monocytogenes. The effects of
ionic strength, divalent cations, and osmolality on microbicidal activity
were evaluated by the addition of increasing concentrations of one of
the following: NaCl or KCl (0 –150 mM), MgCl2 or CaCl2 (0 –2 mM),
or sucrose (0 –250 mM).
The effects of bacterial cell envelope constituents on bactericidal activity
were analyzed by preincubating bPGRP-S with one of the following before
the protein was added to the suspension of target bacteria: 0 –240 ␮g/ml
smooth LPS from S. typhimurium (List Biological Laboratories) and LTA
from Bacillus subtilis or MDP (Sigma-Aldrich). Alternatively, 0 – 4.5
mg/ml PGN from S. aureus (Fluka) was added.
Microbial binding by PGRP-S
One-hundred microliter aliquots of log-phase organisms, suspended in
buffers that optimize their survival, were incubated with 200 ␮g/ml
bPGRP-S for 60 min at 37°C with continuous agitation. Candida albicans
16820, Cryptococcus neoformans 271a, S. aureus 502a, and Escherichia
coli ML35 were suspended in 10 mM PIPES and 5 mM glucose (pH 7.4).
Similar incubations were conducted with S. typhimurium 10428 PhoP⫺ and
Listeria monocytogenes 967 in 10 mM Tris-HCl and 5 mM glucose (pH
7.4). The organisms, either untreated or opsonized, were suspended in
buffer alone or in buffer supplemented with 2 mM MgCl2 or 150 mM NaCl.
Opsonization was accomplished with 50 or 100% fresh, autologous, bovine
serum on ice for 30 min before centrifugation and removal of the supernatant. Additional binding assays were performed in HBSS with or without
50% fresh bovine serum or in 100% serum.
For each incubation condition, bacteria were centrifuged after 60 min at
22,500 ⫻ g for 10 min at 4°C, washed once with buffer, pelleted again,
resuspended in HBSS, and transferred to a new microcentrifuge tube. The
pellet resulting from a third centrifugation was boiled for 5 min in SDStricine sample buffer, vortexed for 30 s, and boiled for another 5 min.
Solubilized samples were resolved by tricine-SDS PAGE (15% acrylamide), and the amount of bPGRP-S present was estimated by determining
the signal (MultiImage Light Cabinet; Alpha Innotech) obtained by Western blots with anti-bPGRP-S IgG (10), using purified bPGRP-S as
standard.
PGRP-S interaction with LPS
Biosynthetically tritiated rough LPS (80 dpm/pmol recombinant bovine
LPS (rbLPS)) from an rb strain of E. coli (List Biological Laboratories)
was suspended according to the manufacturer’s directions in 10 mM TrisHCl buffer (pH 7.4) at concentrations from 10 to 100 ␮M. To minimize
nonspecific binding of tritiated LPS, microcentrifuge tubes were coated
with a 200 ␮g/ml solution of unlabeled, smooth LPS (List Biological Laboratories) in 10 mM Tris-HCl for 1 h with rotation. The coating solution
was removed immediately before the tubes were used. Thirty-milliliter
aliquots of LPS were added to equal volumes of 0 – 6.7 ␮M bPGRP-S in
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naive and phagocytic neutrophils by Immunogold electron
microscopy.
An antimicrobial role for mammalian PGRPs has been shown in
studies of PGRP-S knockout mice, which exhibit both increased
susceptibility to bacterial infection and deficiency in killing of
phagocytosed bacteria by neutrophils (12). However, bPGRP-S is
the first PGRP shown to be microbicidal in vitro (10), acting
against a range of organisms. Purification of PGRP-S from bovine
blood provides milligram quantities of natural protein for study,
avoiding the use of recombinant material with multiple histidines
at the terminus. To further elucidate the role of PGRP-S in innate
immunity, we analyzed the binding and microbicidal activities of
bovine PGRP-S under conditions approximating the extracellular
milieu by modulating cations, ionic strength, osmolality, and serum concentration.
The complete set of molecular targets for PGRPs has yet to be
elucidated. Our previous results provided evidence that bPGRP-S
could act in a PGN-independent manner. In this study we report
the results of competition assays that identify additional bacterial
target molecules and show that they are recognized with higher
affinity than PGN. Using a direct precipitation assay, we demonstrate that the interaction between bPGRP-S and LPS, one of these
alternate molecules, is both dose dependent and saturable. Finally,
bPGRP-S was shown to lyse heat-killed bacteria; however, bacterial killing is independent of this function.
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0.01% HOAc. Samples were prepared in triplicate. After the mixtures were
incubated at room temperature for 2 h, each sample received 140 ␮l 10 mM
Tris-HCl and was centrifuged at 22,500 ⫻ g for 15 min, and 190 ␮l of
supernatant was removed. Tritiated LPS in each supernatant and residual
fraction were quantified by scintillation counting (LS9000; Beckman
Coulter) using Packard Ultima Gold scintillation mixture (Sigma-Aldrich).
Precipitated LPS was quantified by measuring tritiated LPS counts in the
residual 10-␮l aliquot of bPGRP-S-containing samples and subtracting tritiated LPS counts in 10-␮l control aliquots lacking PGRP-S. At the highest
LPS concentration tested (50 ␮M), spontaneous sedimentation accounted
for 6 –16% of the input material.
Bacterial cell wall lysis
Results
Secretion of PGRP-S by neutrophils
It has not been clear whether mammalian PGRPs are secreted to
act in the extracellular milieu, or they function exclusively in an
intracellular compartment. We previously demonstrated (10) that
bovine PGRP-S has the same immunohistochemical staining pattern as ␤-defensin, a secreted protein stored in the large/dense
granules of bovine neutrophils (29). To address the possibility that
PGRP-S may be mobilized for secretion by activated leukocytes,
we analyzed bPGRP-S secretion by neutrophils stimulated with
100 nM PMA, 20 mg/ml LTA, 160 ␮g/ml MDP, 100 mg/ml LPS,
110 mg/ml butyric acid, or ⬃45 nonopsonized bacteria/leukocyte
(S. typhimurium or S. aureus). Opsonized bacteria were not used in
this assay because this would have contaminated the leukocyte
fraction with phagocytosed bacteria. Leukocytes, bacteria, and the
resulting supernatants were evaluated by Western blotting. PMA
induced a nearly complete release of PGRP-S from leukocytes
(Fig. 1). This could not be accounted for by cell lysis, because the
treated cells were 98% viable as determined by trypan blue exclusion. Stimulation of granulocytes with nonopsonized S. aureus or
S. typhimurium also induced measurable secretion (Fig. 1). In contrast, PGRP-S secretion was not detected in supernatants of granulocytes treated with purified butyric acid, LPS, MDP, or LTA
(data not shown). Therefore, PGRP-S appears to be secreted in
response to specific extracellular stimuli. Under these experimental
conditions, bPGRP-S was not found to bind to bacterial pellets.
FIGURE 1. Secretion of PGRP-S from neutrophils. Aliquots of 3 ⫻ 106
granulocyte-enriched bovine leukocytes were incubated for 1 h in buffer
alone (sample marked 60 min) with 100 nM PMA or nonopsonized bacteria before separating the leukocytes from the supernatant and bacteria.
Leukocyte and bacterial pellets were acid extracted, and the resulting supernatants were acidified before aliquots (6 ⫻ 104 CE) were analyzed by
Western blotting. Sample in buffer alone was collected at time zero as a
control. The samples are as follows: C, purified bPGRP-S standard; P,
leukocyte pellet; S, reaction supernatant; B, bacterial pellet.
Cytoplasmic localization of PGRP-S in neutrophils
Evidence that PGRP-S is secreted by neutrophils does not, by itself, identify the intracellular location of the protein. Identification
of the granule type in which bPGRP-S is stored would, however,
offer insight into how the neutrophil uses the protein. We sought to
determine the intracellular address of PGRP-S using Immunogold
transmission electron microscopy with anti-bPGRP-S IgG. As
shown in Fig. 2, A and B, gold particles were concentrated over the
unique large granules (also known as dense granules or tertiary
granules) observed in neutrophils of cattle (33). A small amount of
gold present in the cytosol may be background or may indicate the
presence of PGRP-S in small secretory vesicles. The immunostaining pattern is virtually identical with that observed in the Immunogold localization of bovine neutrophil ␤-defensin-12 (29). Neutrophils that were incubated with S. aureus or S. typhimurium
(opsonized and suspended in a 50/50 mixture of serum and HBSS)
contained numerous ingested bacteria and showed marked degranulation of PGRP-S-containing dense granules (Fig. 2C). PGRP-S
was associated with S. aureus bacteria in phagolysosomes (Fig. 2,
C and D), but it is not clear whether PGRP-S was deposited into
the phagolysosome directly or whether secreted PGRP-S bound
to bacteria before engulfment. Conversely, although the neutrophils that phagocytosed S. typhimurium had similar depletion
of large/dense granules and cellular PGRP-S (Fig. 2E), the protein was not associated with the bacteria, and PGRP-S was not
detected inside these phagolysosomes (Fig. 2F). The few remaining large granules in these cells were positive for PGRP-S.
In the control, preimmune IgG did not bind to the dense granules or to S. aureus inside the phagolysosome (Fig. 2, G and H).
These experiments provide additional evidence that PGRP-S is
secreted from neutrophils upon stimulation by either Gram-negative or -positive bacteria. The data also suggest that bovine
PGRP may enter the phagolysosome after binding to bacteria in
the extracellular milieu.
Solute modulation of PGRP-S-mediated microbial killing
To date, bovine PGRP-S is the only PGRP reported to kill microorganisms in vitro (10). As a secreted protein, it may perform this
function in the extracellular milieu. Previous microbicidal studies
were performed in low ionic strength medium that may not reflect
the in vivo conditions. We therefore analyzed PGRP-S-mediated
killing of bacteria in a range of buffer conditions reflective of the
extracellular milieu to determine the effects of increasing ionic
strength and divalent cations on PGRP-S-mediated killing. As
shown in Fig. 3, A and B, increasing concentrations of NaCl or KCl
markedly reduced, but did not ablate, PGRP-S-mediated killing of
bacteria. Killing of L. monocytogenes was reduced from 4 logs
(99.99% killing) to slightly ⬎1 log by 30 mM NaCl or 60 mM
KCl, but bovine PGRP-S continued to kill ⬎90% of the bacteria
at salt concentrations up to 150 mM. Similarly, killing of S.
typhimurium by PGRP-S was reduced from nearly 5 to 2 logs by
30 mM NaCl, but ⬎1 log PGRP-S-mediated killing occurred
even at physiologic concentrations of NaCl (150 mM). The effect of KCl on PGRP-S-mediated killing of S. typhimurium
could not be determined because KCl concentrations ⬎60 mM
were toxic to the bacteria. Incubation buffers modified with sucrose had no impact on PGRP-S-mediated killing (Fig. 3, E and
F), indicating that NaCl and KCl interfered with killing by
changing the ionic environment, rather than by their effects on
solvent osmolality.
In contrast, the addition of MgCl2 or CaCl2, ablated PGRPS-mediated killing of S. typhimurium (Fig. 3D) in a dose-dependent manner, while having no impact on bovine PGRP-S’s
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Dissolution of bacterial cell walls was measured by the lysoplate method of
Osserman and Lawlor (32), except that 10 mM HEPES or Tris-HCl (pH
7.4.) with 0 –154 mM NaCl was used in place of PBS for preparation of the
buffered 1% agarose. Each sample well of the lysoplate received 15 ml of
lysozyme standard or bPGRP-S. After incubation for 20 h at room temperature, the radius of each zone of lysis was measured to the nearest 0.5
mm. Lysozyme solutions were prepared in distilled water, and bPGRP-S
solutions were prepared in 0.01% HOAc. HOAc (0.01%) was used as a
negative control. To verify the enzymatic nature of the lysis, we tested
protein solutions that had been boiled for 10 min and rapidly cooled on ice.
BOVINE PGRP-S
The Journal of Immunology
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Binding of PGRP-S to microorganisms
FIGURE 2. Immunogold localization of PGRP-S. A, Immunogold labeling of a bovine neutrophil incubated with anti-bPGRP-S IgG reveals
specific labeling of the abundant dense granules. B, Enlargement of the
boxed area of A. C, Immunogold labeling, with anti-bPGRP-S IgG, of a
neutrophil that has phagocytosed S. aureus reveals depletion of dense granules and bPGRP-S from the cytoplasm, but label is found inside phagolysosome associated with bacterial cells. D, Enlargement of the boxed area of
C, showing phagosome. E, Immunogold labeling, with anti-bPGRP-S IgG,
of a neutrophil that has phagocytosed S. typhimurium, showing loss of
dense granules and PGRP-S from the cell. The phagolysosome containing
the bacterial cells shows no PGRP-S. F, Enlargement of the boxed area of
E, showing phagosome. G, Immunogold labeling, with preimmune IgG, of
a neutrophil that has phagocytosed S. aureus shows the absence of nonspecific Immunogold labeling. H, Enlargement of the boxed area of G,
showing phagosome. Phagosomes are indicated by filled arrows. C and D,
Electron-lucent areas in cells (open arrows; A and C) are LR White embedding artifacts.
activity against L. monocytogenes (Fig. 3C). These data are
consistent with the hypothesis that divalent cations interfere
with PGRP-S-mediated killing through stabilization of the
Gram-negative outer membrane (34), rather than by interacting
directly with the protein.
We previously reported evidence that bovine PGRP-S can kill bacteria even in the absence of exposed PGN. Therefore, we investigated the prerequisites for PGRP-S binding to target cells using a
direct binding assay. PGRP-S was seen to bind not only to Grampositive bacteria (S. aureus and L. monocytogenes), which express
exposed PGN, but also to Gram-negative bacteria (E. coli and S.
typhimurium), on which PGN is buried beneath an LPS bilayer,
and to fungi (C. neoformans), which lack PGN altogether (Fig. 4).
The specificity of this binding was indicated by the fact that C.
FIGURE 4. Binding of PGRP-S to microorganisms. Aliquots of bacteria or fungi were incubated with bPGRP-S in the presence or the absence
of 150 mM NaCl or 2 mM MgCl2 in buffers optimal for the survival of the
microorganism. Tris buffer is 10 mM Tris-HCl and 5 mM glucose (pH 7.4);
PIPES denotes 10 mM PIPES and 5 mM glucose (pH 7.4). Bacterial pellets
were analyzed by Western blotting. Microorganisms incubated without
bPGRP-S did not show a positive signal on Western blots (not shown).
Aliquots represent 1 ⫻ 106 CFU Salmonella or Listeria, 1 ⫻ 107 CFU E.
coli, 2 ⫻ 106 CFU S. aureus, or 4 ⫻ 105 CFU Cryptococcus or Candida.
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FIGURE 3. Solute modulation of PGRP-S-mediated bactericidal activity. Log-phase bacteria (L. monocytogenes and S. typhimurium) were incubated with bPGRP-S in buffer at the indicated concentrations of NaCl or
KCl (A and B), MgCl2 or CaCl2 (C and D), or sucrose (E and F) for 2 h at
37°C before plating. Microbicidal activity was determined by colony
counting of plates after 16 –18 h of incubation at 37°C. Hand-plating of
undiluted aliquots of the suspension allowed quantitation of killing to 5
logs.
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FIGURE 5. Effect of serum on PGRP-S binding. Aliquots (4 ⫻ 106
CFU each) of nonopsonized or serum-opsonized S. aureus or S. typhimurium were incubated with bPGRP-S (1 ␮g) in HBSS, a 50/50 mixture of
HBSS and serum, or 100% serum. Bacterial pellets were analyzed by
Western blot with anti-bPGRP-S IgG. The purified protein standard is 100
ng of bPGRP-S.
Antagonism of microbicidal activities by bacterial molecules
The ability of bovine PGRP-S to bind to Gram-negative bacteria and fungi suggests that PGRP-S might recognize microbial
FIGURE 6. Inhibition of PGRP-S-mediated killing by
bacterial molecules. bPGRP-S was preincubated with
0 –25 mg/ml purified LPS, LTA, PGN, or MDP for 30
min. Preincubated bPGRP-S was added to log-phase organisms, Gram-positive L. monocytogenes in A and
Gram-negative S. typhimurium in B, and incubated for 2 h
at 37°C before plating. Microbicidal activity was determined by colony counting of plates after 16 –18 h of incubation at 37°C. Inhibition of activity was defined as the
percentage of CFU killed without preincubation minus the
percentage CFU killed with preincubation divided by the
percentage CFU killed without preincubation.
molecules other than PGN. To identify targets of PGRP-S binding, we determined whether prebinding of PGRP-S with purified bacterial molecules could competitively inhibit PGRP-Smediated killing of bacteria. To assess the specificity of any
competition, killing was assayed on Gram-negative as well as
Gram-positive bacteria. Before incubation with bacterial suspensions, PGRP-S was mixed with 0 –25 ␮g/ml LPS, LTA,
PGN, or MDP, then tested for its ability to kill bacteria (see
Materials and Methods). As shown in Fig. 6A, LPS and LTA
inhibited the killing of a Gram-positive organism (Listeria) by
PGRP-S in a dose-dependent manner. Surprisingly, PGN and
MDP had little effect; concentrations of PGN up to 448 ␮g/ml
decreased killing of L. monocytogenes by only 2% (data not
shown), whereas nearly complete inhibition of the listericidal
activity occurred if PGRP-S was preincubated with 12 ␮g/ml
LPS or 24 ␮g/ml LTA (Fig. 6A).
In contrast, killing of Gram-negative bacteria (S. typhimurium)
was not inhibited by preincubation of PGRP-S with LTA (Fig. 6B).
Preincubation with LPS showed less inhibition of PGRP-S-mediated killing in the Gram-negative assay than in the Gram-positive
assay. As shown in Fig. 6B, 12 ␮g/ml LPS reduced the PGRP-Smediated killing of S. typhimurium by ⬃50%, but at the highest
LPS concentration tested, killing was inhibited by ⬍60%. Under
these same conditions, 448 ␮g/ml PGN inhibited killing of S. typhimurium by only 10% (data not shown).
PGRP-S interaction with LPS
The ability of bovine PGRP-S to bind to Gram-negative bacteria and the marked inhibition of PGRP-S-mediated killing by
preincubation of the protein with LPS suggested that LPS and
PGRP-S might have a specific binding interaction. To clarify
this relationship, we determined the effect of PGRP-S on LPS
solubility. In three separate assays, triplicate samples of biosynthetically radiolabeled rbLPS (0 –50 ␮M) were incubated
with purified bPGRP-S (750 nM or 3.35 ␮M.). PGRP-S efficiently precipitated up to 84% of the tritiated LPS in a sample
(Fig. 7). Kd values for binding affinity could not be calculated
by the Scatchard method, because there was no binding plateau.
Suggesting specificity in the interaction between LPS and
PGRP-S, precipitation failed when the LPS/PGRP-S molar ratio
exceeded 3 at 3.35 ␮M PGRP-S or 7 at 750 nM PGRP-S (Fig.
7). The kinetics of PGRP-S-induced precipitation of LPS are
similar to the hook effect or prozone phenomenon, wherein
cross-linking of Ag by divalent Ab can be inhibited by excess
Ag. A higher concentration of PGRP-S shifts the precipitation
curve to the left (Fig. 7), reflecting more effective precipitation
of LPS. Recent crystallization data suggest that PGRPs may
function as multimers (21) or have tandem PGN-binding domains (26), which may explain PGRP-S-mediated precipitation
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albicans, a yeast that is neither inhibited nor killed by PGRP-S
(10), did not bind PGRP-S even in salt-free buffer.
The addition of 2 mM MgCl2 to the incubation buffer ablated
PGRP-S-mediated killing of S. typhimurium (see above), but did
not eliminate the binding of PGRP-S to L. monocytogenes or S.
typhimurium (Fig. 4). This suggests that binding of PGRP-S to
target organisms is not sufficient for bacterial killing. In contrast, 150 mM NaCl, which permitted 90 –95% killing of L.
monocytogenes or S. typhimurium, reduced PGRP-S binding to
levels undetectable by Western blotting (Fig. 4). This result
may indicate that very low concentrations of PGRP-S are required to kill bacteria or that bacterial death may release
PGRP-S under high salt, serum-free conditions. NaCl inhibited
binding of PGRP-S to each organism tested. Consistent with
these results, HBSS, a buffer that contains physiologic levels of
mM NaCl (137 mM), Mg2⫹ (0.9 mM) and Ca2⫹ (0.9 mM),
antagonized PGRP-S binding to both L. monocytogenes and S.
typhimurium (Fig. 4).
To assess the binding of PGRP-S to bacteria under conditions
most similar to the extracellular milieu, we analyzed the effects of
opsonization and serum on PGRP-S binding to target organisms.
Opsonized/washed and nonopsonized S. aureus or S. typhimurium
were incubated with bPGRP-S under three conditions: in fresh
bovine serum, in HBSS, or in 50/50 HBSS/serum. Opsonization of
S. aureus markedly increased PGRP-S binding to the bacteria, and
this effect was enhanced by the presence of serum in the incubation
buffer (Fig. 5). Binding of PGRP-S to S. typhimurium also increased under these conditions (Fig. 5). These results suggest that
binding of bPGRP-S to bacteria is likely to occur in the extracellular environment in vivo.
BOVINE PGRP-S
The Journal of Immunology
and the greater efficiency of interaction at higher PGRP-S
concentrations.
Bacterial cell wall lysis
Among the known PGRPs, there are some that have been reported to digest PGN (20, 22, 23). Other PGRPs, such as
bPGRP-S, lack a critical zinc-binding residue and were predicted to be incapable of enzymatic activity. The finding of
carboxypeptidase activity of Drosophila PGRP-SA (24), which
lacks the zinc-binding amino acid, led us to test bovine PGRP-S
for lytic activity. Bovine PGRP-S has been shown to be sensitive to buffer conditions in microbial binding and killing assays,
so we tested for bacterial cell wall lysis by the lysoplate method
(32) using buffer lacking NaCl. Assays using lysoplates made
with 10 mM HEPES (pH 7.4) demonstrated that lysozyme and
bPGRP-S have opposite salt requirements for optimal activity
(Fig. 8 and data not shown). The addition of 50 or 154 mM
NaCl to HEPES buffer improved the lytic activity of lysozyme
and interfered with bPGRP-S-mediated lysis of bacterial cell
walls (data not shown). Although lysoplates made with PBS
FIGURE 8. Bacterial cell wall lysis. The lysoplate method was used to
evaluate lysis of bacterial cell walls by bOBP. Lyophilized M lysodeikticus
was suspended in molten 1% agarose made with 10 mM HEPES (for bOBP
assays) or in 10 mM HEPES containing 154 mM NaCl (for lysozyme
assays) and poured into petri dishes. Wells cored into the cooled agarose
received 12 ␮l of either chicken egg white lysozyme or bOBP at the indicated concentrations. The radius of the lytic zone is plotted against the
log of the protein concentration.
appear to be the optimal substrate for egg white lysozyme activity, bPGRP-S was not able to digest Micrococcus lysodeikticus cell walls under these conditions (data not shown).
To substantiate that lysis of the cell wall preparation was the
result of enzymatic activity, both the lysozyme and bPGRP-S solutions were boiled for 10 min and rapidly iced before they were
used in the lysoplate assay. Boiling of the protein samples reduced
lysozyme’s ability to generate a lytic zone by 50-fold. The lytic
activity of bPGRP-S was reduced 20- to 30-fold (data not shown).
Boiled bPGRP-S was tested in microbicidal assays to explore the
relationship between bacterial cell wall lysis and bPGRP-S-mediated killing. Although boiled bPGRP-S lost bacterial cell wall lytic
activity, it maintained 100% of its microbicidal activity against
Listeria (data not shown), indicating that PGRP-S-mediated killing
of bacteria is independent of this lytic activity.
Discussion
Bovine PGRP-S, an ortholog of human and murine PGRP-S, is
secreted from neutrophils, is stored in neutrophil large/dense granules, and is also found associated with bacteria in the phagolysosome. Purification of bPGRP-S (formerly termed bOBP) from bovine blood allows for the evaluation of natural PGRP-S in in vitro
assays. This protein is verified to bind to and kill a range of microorganisms in a PGN-independent manner in solutions approximating physiologic conditions. Among the bacterial components
tested, bPGRP-S demonstrated the highest affinity for LPS. This
PGRP showed significant affinity for LTA and little for PGN in
killing inhibition assays.
The first PGRP was named for its ability to bind to Grampositive bacteria and PGN. As the family of known PGRPs has
grown, investigations have shown that members of this protein
family recognize a range of microorganisms and cell envelope
constituents (4, 5, 9, 10, 17, 25), leading researchers to emphasize the discovery of specific molecular targets for each PGRP
analogous to the TLR family (35). However, bPGRP-S recognizes multiple microbial components, as do Holotrichia
PGRP-S (15) and Drosophila PGRP-LC (4), suggesting that
PGRP recognition of pathogens is less specific than that reported for TLRs. It may be that PGRPs bind common moieties
or secondary structures in their target molecules or, alternatively, that they have binding sites for more than one microbial
component. The report that Holotrichia PGRP-S binds laminaripentaose, a component of the fungal cell wall (15), is consistent with the hypothesis that PGRPs recognize diverse microbial ligands by conserved or similar oligosaccharide moieties.
Unlike the TLRs, mammalian PGRPs have not been shown to
act in signaling, although Drosophila PGRPs are involved in
both immune signaling cascades (4, 5, 9, 17, 36, 37).
A major question about PGRPs has been the anatomical site in
which they are active or stored. Our staining localizes bPGRP-S to
large granules, in agreement with differential centrifugation studies
of the murine ortholog (12). Large granules of bovine neutrophils,
equivalent to the tertiary/dense granules of humans and mice, contain the oxygen-independent bactericidal proteins of these cells
(33, 38). Electron microscopy of Immunogold-labeled neutrophils
that were exposed to opsonized bacteria in 50/50 HBSS/serum also
confirms our data showing that PGRP-S is secreted. Specifically,
neutrophils that have phagocytosed either Gram-negative or Grampositive bacteria show marked loss of their large granules and
nearly complete loss of PGRP-S, which is not primarily translocated into the phagolysosome. Neutrophils that phagocytosed bacteria had PGRP-S inside the phagolysosome associated with S.
aureus, but not S. typhimurium. The absence of PGRP-S from
phagolysosomes containing S. typhimurium is not consistent with
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FIGURE 7. Precipitation of LPS by PGRP-S. Biosynthetically radiolabeled rbLPS from E. coli was suspended in buffer at concentrations from
10 –100 ␮M. Aliquots (30 ␮l) of LPS were added to equal volumes of
0.01% HOAc (negative control) or bPGRP-S (1.5 or 6.7 ␮M) dissolved in
0.01% HOAc. Samples were performed in triplicate and had negligible
SDs. Bovine PGRP-S (750 nM) was used in two separate assay dates (E
and F). Results are graphed as the percentage of the total LPS in the
sample precipitated vs the LPS/bPGRP-S molar ratio.
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1160
sonization alone (Fig. 5). This verifies that bPGRP-S is likely to
bind to microorganisms in the extracellular milieu. The serumrich extracellular milieu contains many components that may be
responsible for enhancing target binding. Such factors might
also enhance bPGRP-S-mediated killing. One relevant serum
constituent may be PGRP-L, recently identified as a component
of normal mammalian serum (16). Indeed, other PGRPs, such as
PGRP-LE and PGRP-LC of Drosophila, have been found to act
synergistically (37).
Physiological levels of divalent salts may play another role in
PGRP-S interaction with target organisms. Concentrations of
MgCl2 or CaCl2 as low as 0.8 mM ablate bPGRP-S-mediated killing of S. typhimurium (Fig. 3D), but have no effect on killing of
Gram-positive L. monocytogenes at concentrations up to 2 mM
(Fig. 3C). The moderate reduction of bPGRP-S binding caused by
2 mM MgCl2 (Fig. 4) does not explain this effect, because binding
to L. monocytogenes and S. typhimurium is similarly affected. Protection of Gram-negative, but not Gram-positive, bacteria by
MgCl2 and CaCl2 suggests that these divalent cations do not act on
bPGRP-S directly, but, rather, exert an effect on the LPS outer
membrane. This protective effect may be related to the decrease in
LPS bilayer fluidity conferred by divalent cations (34). Complete
protection of S. typhimurium from the bactericidal activity of
bPGRP-S by MgCl2 concentrations that only moderately reduce
protein binding indicates that binding alone is not sufficient for
PGRP-S-mediated killing of bacteria.
The ability of bovine PGRP-S to bind to intact yeast and Gramnegative bacteria confirms that PGRP-S does not require PGN for
recognizing and binding to a range of microorganisms. Although
bPGRP-S binds to and kills C. neoformans (10), another fungus, C.
albicans, resists antimicrobial activity and is the only organism
tested that does not bind PGRP-S in salt-free buffer. This result
suggests that microorganisms may escape PGRP-S-mediated killing by avoiding binding.
The range of microbial cell envelope constituents that may
bind PGRP-S was explored by testing a variety of bacterial
components for their ability to inhibit bPGRP-S-mediated killing of Gram-positive and -negative bacteria. Not only was inhibition of killing not selective for PGN or its primary constituent, MDP, but PGN was found to be a poor inhibitor of killing.
In fact, LPS (S. typhimurium) and LTA (B. subtilis) were up to
100-fold more potent inhibitors of bacterial killing than PGN
from S. aureus on a molar basis. These findings suggest that
binding of PGRP-S to Gram-positive bacteria might be mediated as much by surface-exposed teichoic acids, which constitute 10 –50% of the mass of the Gram-positive cell wall (42), as
by PGN. The fact that LTA effectively inhibited killing of
Gram-positive L. monocytogenes yet had no effect in assays
with Gram-negative S. typhimurium suggests that bovine
PGRP-S has greater affinity for LPS than for LTA. The incomplete inhibition of bPGRP-S-mediated killing of S. typhimurium
by LPS, a molecule also present on the surface of these bacteria,
is expected in a competition assay.
The interaction between the PGRP binding domain(s) and microbial constituents are beginning to be elucidated. The affinity of
bPGRP-S for LPS, LTA, and an unidentified fungal constituent is
not in conflict with any published data, although the mechanism of
this interaction is not immediately apparent. The microbial molecules that interact with bPGRP-S share only limited oligosaccharide moieties, but may share significant structural similarities.
Analysis of the PGRP-LB crystal structure indicates that poor conservation of amino acids predicted to line the binding cleft would
generate widely varying specificities of individual PGRPs (21, 43).
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our analysis of microbial binding by PGRP-S, suggesting that either PGRP-S has been degraded in the phagolysosome or that the
Ab epitope has become unavailable, possibly through interaction
with LPS.
Evidence that bPGRP-S is secreted and also present in the
phagolysosome may indicate that the protein re-enters the cell
bound to susceptible microorganisms. Adding weight to this hypothesis is the fact that the killing capacity of neutrophils from
PGRP-S knockout mice was reconstituted to the level of wild-type
mice by the addition of exogenous PGRP-S to the incubation medium (12). Our data do not, however, exclude the possibility that
PGRP-S might be both secreted and translocated into the
phagosome.
Although other PGRPs have shown bacteriostatic activity (6,
12, 39), bPGRP-S is the only PGRP shown to be microbicidal.
Dziarski et al. (12) demonstrated that murine PGRP-S participates in the intracellular killing of bacteria in neutrophils. However, they found only inhibition of bacterial growth by protein
with a multiple His tag using the salt-free assay described in
this study. Recently, Cho et al. (39) have demonstrated that
recombinant human PGRP-S cooperates with lysozyme to inhibit bacterial growth. This difference between the measured
microbicidal activity of natural bPGRP-S protein and the recombinant human and murine PGRP-S may be due to the chemical modification of the cloned human and mouse PGRPs. A
terminal His tag or the salt precipitation method used to purify
the recombinant proteins may affect PGRP-S-mediated microbial killing. Microbicidal studies of natural, purified human and
mouse PGRPs would address this issue, although it is possible
that the primary sequence of the bovine protein (murine and
bovine PGRP-S are 64% identical) makes it unique among
PGRPs in this function.
Bovine PGRP-S has been demonstrated to kill Gram-positive
bacteria, Gram-negative bacteria, and also fungi in vitro (10). Additional microbicidal assays using a range of buffer conditions reveal that although the microbicidal activity of bPGRP-S does not
require a hypotonic milieu, killing is reduced by NaCl concentrations as low as 30 mM. Interestingly, even though 150 mM NaCl
reduced the binding of bPGRP-S to target bacteria below the level
of detection by Western blot analysis, bPGRP-S was still able to
kill 90 –99% of the bacteria at this salt concentration. This may
indicate that very little protein is required for efficient killing,
bacterial death causes the release of bound PGRP-S under highsalt, serum-free conditions, or this method of measuring binding is not correlative with killing. Sensitivity to ionic concentration is a quality that bPGRP-S shares with other microbicidal
proteins, such as defensins (40, 41). NaCl may interfere with
the interaction between bPGRP-S and cell envelope constituents through reducing the electrostatic interaction between the
negatively charged microbial surface and the positively charged
protein, pI 9.38 (10).
This reduction of bPGRP-S-mediated killing by NaCl could
suggest that the protein has another role in vivo, the remaining
bactericidal activity (90 –99% killing) is adequate for host defense, or the presence of serum ameliorates the salt effect. To
address this question we evaluated the binding activity of
PGRP-S in physiologic salt buffer with and without serum using
opsonized bacteria. We found that opsonization facilitated
PGRP-S binding to S. aureus, but not S. typhimurium (Fig. 5).
Additional studies of binding suggested that physiologic levels
of salts in simple buffers (Tris-HCl or HBSS) interfered with
PGRP-S binding to microorganisms, but when fresh serum was
used, binding of PGRP-S to S. aureus was enhanced over op-
BOVINE PGRP-S
The Journal of Immunology
Acknowledgments
We acknowledge the assistance of Patrick Koen and Jean Edens (Caltech
Electron Microscope Facility) in performing the electron microscopy. We
are most indebted to Ellen V. Rothenberg, Rochelle Diamond,
Marianne Bronner-Fraser, Mary Yui, and Jon Moore for critical reading of
the manuscript.
Disclosures
The authors have no financial conflict of interest.
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