Hijacking of the Pleiotropic Cytokine Interferon-γ by the Type III Secretion System of Yersinia pestis
et al. (2010) Hijacking of the Pleiotropic Cytokine Interferon-c by the Type III Secretion
System of Yersinia pestis. PLoS ONE 5(12): e15242. doi:10.1371/journal.pone.0015242
Hijacking of the Pleiotropic Cytokine Interferon-c by the Type III Secretion System of Yersinia pestis
Claire Gendrin 0
Ste phane Sarrazin 0
David Bonnaffe 0
Jean-Michel Jault 0
Hugues Lortat-Jacob 0
Andre a Dessen 0
Deepak Kaushal, Tulane University, United States of America
0 1 Institut de Biologie Structurale, UMR 5075 (Comissariat a` l'Ene rgie Atomique/Centre National de la Recherche Scientifique/Universite Grenoble I) , Grenoble , France , 2 Laboratoire de Chimie Organique Multifonctionnelle, Institut de Chimie Mole culaire et des Mate riaux d'Orsay, UMR 8182, Universite Paris-Sud 11 , Orsay , France
Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNc. In addition, we generate specific IFNc mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNc. Lastly, we show that the LcrV-IFNc interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.
Funding: CG was the recipient of a postdoctoral fellowship from the Comissariat a lEnergie Atomique (CEA) and SS was supported by a grant from the Mizutani
Foundation for Glycoscience. This work was supported by DGA grant number 06.70.151.00.470.75.96 (to AD). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Yersinia pestis is the etiologic agent of plague, a devastating,
acute infectious disease responsible for the death of 200 million
people throughout the course of three worldwide pandemics .
The infectious mechanism of Y. pestis is strictly dependent on its
type III secretion system (T3SS), a complex macromolecular
structure present on the bacterial surface. The system serves as a
conduit to inject T3SS-specific toxins directly into the cytosol of
target cells. It is composed of over twenty macromolecules that
associate into a basal structure spanning both bacterial
membranes, and is terminated by a hollow needle through which toxins
are believed to travel in semi-unfolded state . Toxin entry
into the eukaryotic cytoplasm requires the formation in the target
membrane of a structure called translocon, which is composed
of two T3SS-encoded membrane proteins and one hydrophilic
partner (the V antigen) [4, 57]. Similarities between proteins
from microorganisms carrying T3SS within the same family can
also be observed at the structural level . However, secreted
toxins are specific to each pathogen. In Y. pestis, genes that encode
proteins required for the formation of the T3SS are present on a
mobile genetic element (pCD1), which also carries genes for a
set of secreted antihost proteins (Yops), as well as the V antigen
(LcrV), all of which are absolutely necessary for infection
Bacterial invasion of a mammalian host usually results in a
prompt inflammatory response, generating the upregulation of
major proinflammatory cytokines, which corresponds to the first
level of innate immune defense. If this process on its own is not
capable of destroying invading organisms, at least it allows the host
to perform clonal selection and amplification of T-lymphocytes,
mediators of specific immunity . It has been shown that all
three pathogenic Yersinia species (Y. pestis and the
entheropathogenic species Y. pseudotuberculosis and Y. enterocolitica) are able to
down-regulate inflammation mechanisms. In the case of Y. pestis,
this down-regulation is strictly dependent on a functional T3SS
, and most specifically, on the presence of the V-antigen LcrV.
In addition to being detected in secreted form in the extracellular
milieu upon activation of the T3SS, LcrV forms a distinct
structure at the tip of the type III secretion needle and is associated
to YscF, the needle-forming subunit [15,16].
The numerous studies undertaken on LcrV from the various
Yersinia species have shown that LcrV plays a major role at the
interface with the immune system of the host, being the major
protective antigen against the different forms of Yersinia infection
[17,18] and inducing the expression of the anti-inflammatory
cytokine IL-10, which prevents upregulation of the
pro-inflammatory cytokines TNF-a and IFNc [19,20]. IFNc breakdown has
been observed in the spleens of infected mice, and active
immunization with an LcrV-derived fusion protein restores a
normal level of synthesis . IFNc is a pleiotropic cytokine
secreted by natural killer cells and T cells during innate immune
processes. It interacts with a specific ubiquitous membrane
receptor (IFNcR), which triggers expression of a variety of
proteins . The IFNc chain is 143-amino acids long and is
active as a homodimer; its structure reveals two intertwined
polypeptides carrying globular N-terminal domains and a flexible
C-terminal region . Receptor recognition involves amino acids
1826 and 108124 of the N-terminal region [23,24], as well as a
segment of the C-terminal flexible tail (residues 125134, 25). This
C-terminal region includes residues KRKR128131, which are also
involved in binding to heparan-sulfates (HS), highly sulfated
glycosaminoglycans present in the extracellular matrix and at the
cell surface . The competitive binding of IFNc to HS allows
regulation of the cytokine activity in vivo [25,27].
Recently, it has been proposed that LcrV from Y. pestis exhibits
IFNc-dependent binding to specific monocytic cell lines . In
addition, inhibition of TNF-a synthesis and induction of IL-10
expression were observed as a consequence of this interaction.
This study raised the question of a direct inhibition of IFNc by
LcrV, in addition to its role as an inhibitor of cytokine expression.
We thus set out to characterize a potential LcrV-IFNc complex
using biophysical methods and investigate the potential for its
disruption by small molecule inhibitors. In the present study, we
demonstrate the formation of an LcrV-IFNc complex, whose
association occurs with nanomolar affinity even in the absence of
IFNcR. By employing a panoply of IFNc mutants, we mapped the
LcrV-IFNc interaction region and found that it overlaps the HS
binding site of the cytokine. Based on this observation, we also
demonstrate that HS-like inhibitors are capable of blocking this
specific association. The molecules which demonstrate the highest
affinity constitute interesting candidates for the development of
novel antibacterials which could potentially interfere with Yersinias
immune system subversion mechanism.
Materials and Methods
Protein expression and purification
Expression of GST-LcrV , of PcrV  and of IFNc 
was performed in the E. coli BL21 DE3 strain (T7 express,
Biolabs) grown in 1 L of Luria broth medium. Expression was
induced with 1 mM isopropyl 1-thio-b-D-galactopyranoside at an
A600 of 0.7. For GST-LcrV and His-PcrV, growth was carried out
for 3 hours at 37uC at 180 rpm. GST-LcrV expressing cells were
harvested and lysed by sonication in buffer A (50 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 1 mM EDTA). The cell lysate was
cleared by centrifugation and the supernatant was applied onto a
GST-trap column (GE Healthcare) pre-equilibrated in buffer A.
The protein was eluted with buffer A containing 10 mM of
reduced glutathione. Digestion of the GST tag was performed with
50 units of PreScission proteaseTM (Amersham Pharmacia Biotech
Inc) for 3 h at room temperature. The digestion products were
reloaded onto the GST-column to allow separation of proteolyzed
LcrV from the GST domain. Fractions containing LcrV were
pooled and applied to a gel filtration column (Hiload 16/60
SuperdexTM200, GE Healthcare) equilibrated in 10 mM HEPES,
pH 7.4, 150 mM NaCl, 1 mM EDTA (buffer B).
Cells expressing His-PcrV were also lysed by sonication in
25 mM Tris-HCl, pH 8.0, 200 mM NaCl, 2% glycerol. The
cleared lysate was loaded onto a Ni-Sepharose column (GE
Healthcare). PcrV was eluted with a step imidazole gradient and
dialyzed to remove imidazole. Thrombin digestion allowed
cleavage of the His tag, which was retained on the Ni-column
upon reloading of the cleaved material. His-tag free PcrV was then
submitted to gel filtration using a Hiload 16/60 SuperdexTM200
column (GE Healthcare) in Tris 25 mM, pH 8.0, 100 mM NaCl,
1 mM EDTA.
IFNc-expressing cells were induced 5 h at 37uC, and lysed by
two passages through a French press in lysis buffer (50 mM Tris,
pH 7.2, NaCl 100 mM) Purification from inclusion bodies was
performed as previously described  with slight modifications.
Briefly, inclusion bodies were solubilized in 6 M guanidine HCl,
and the protein was refolded in a 12.5 volume of phosphate buffer,
pH 7.5. IFNc was purified by ion exchange on a home-packed
5 mL source S column (resin bought from GE Healthcare) and gel
filtration (Hiload 16/60 SuperdexTM200, GE Healthcare) in
buffer B. The integrity of all three purified proteins was checked by
Mutagenesis was performed using the QuickChange II
SiteDirected Mutagenesis Kit (Stratagene) following manufacturers
instructions. The vector pET11a containing the wild-type human
IFNc cDNA was used as the DNA template. Complementary
primers were designed as to convert the selected basic amino acid
into a serine residue. Amino acids were mutated one by one, each
mutated vector being used as a template for the following
Biacore-based binding assays
Binding analyses were performed using a Biacore 3000 system.
Flow cells of a CM3 sensor chip were activated with 0.2 M
Nethyl-N-(diethylaminopropyl)-carbodiimide and 0.05 M
N-hydroxysuccinimide (EDC/NHS). The flow cell destined to recieve
LcrV was then submitted to the injection of 80 mM
2-2(pyridinyldithio)ethaneaminehydrochloride (PDEA thiol coupling
reagent) in 0.1 M borate buffer, pH 8.5, which allows quantitative
activation of NHS esters on the surface. Following PDEA
injection, 35 mL of LcrV at 50 mg/mL in 10 mM sodium acetate
buffer, pH 4.1, were injected, which typically allowed coupling of
12002000 resonance units (RU) of the protein on the surface.
Excess reactive groups were deactivated by injecting 50 mM
Lcysteine in formate buffer, pH 4.3, containing 1 M NaCl. For
comparison of LcrV and PcrV properties, proteins were bound
directly after activation by injecting 50 mL of the purified protein
at the concentration of 100 mg/mL. The remaining activated
groups were blocked with 1 M ethanolamine, pH 8.5. For all
experiments, a control flow cell was prepared by injection of
10 mM sodium acetate buffer, pH 4.1, directly after activation,
followed by blocking with 1 M ethanolamine, pH 8.5. For IFNcR
binding experiments, a flow cell of a CM4 sensor chip was EDC/
NHS activated as described above, after which 50 mL of
Streptavidin (Sigma-Aldrich) at 200 mg/mL in 10 mM sodium
acetate buffer, pH 4.1, were injected. Biotinylated IFNcR
(0.3 mg/mL) was then captured to a level of 1600 RU on this
For binding assays, IFNc and its derivatives were simultaneously
injected over the surface presenting the tested protein and the
negative control. Surfaces were regenerated using 12 mL pulses of
10 mM HCl. To determine the equilibrium constant (Kd), the
binding responses at the end of the injection phase were extracted
from the sensorgrams, and plotted according to the Scatchard
representation (Req/C against Req, where Req is the steady state
value at equilibrium and C the concentration of the injected
cytokine. To test the potential inhibitors of the LcrV-IFNc
interaction, 0.5 equivalents of each inhibitor were incubated with a
120 nM solution of IFNc. The mix was injected over immobilized
LcrV, and the percentage of inhibition was calculated at the end of
the association phase. The inhibitory potential of 2O32 was further
investigated by preincubating increasing concentrations of 2O32
(0350 nM) with IFNc (120 nM), and injecting the complexes
over immobilized LcrV. Fitting of the data was performed using
the Origin 8.1 software (OriginLab). During Biacore studies, the
chips were submitted to continuous flow of HBS-P buffer (10 mM
Hepes, 0.15 M NaCl, and 0.05% P20 detergent, pH 7.4), and all
injected material were diluted in the same buffer. Except when
indicated, reagents used for these studies are from GE Healthcare.
Chemical modification by N-Bromosuccinimide (NBS)
A 10 mM solution of LcrV was prepared in 50 mM
Trisacetate, pH 5.0, 20% glycerol, 1 mM EDTA.
N-Bromosuccinimide (NBS, Sigma) was resuspended in the same buffer and an
80fold molar excess was added to the LcrV solution. After a 10 min
incubation at 30uC in the dark, unreacted NBS was neutralized by
an equal amount of free L-tryptophan, and precipitated material
was eliminated by centrifugation. Modified LcrV was equilibrated
in buffer B by gel filtration in PD-10 desalting columns (GE
Intrinsic fluorescence measurements
Tryptophan fluorescence emission spectra were measured on a
PTI quanta master 4 (Photon Technology International, London,
ON, Canada) at 25uC. Emission spectra were recorded from 310
to 370 nm using an excitation wavelength of 295 nm, with a 2 nm
excitation and a 4 nm emission band pass. The cuvette contained
1 mL of 1 mM IFNc, and increasing concentrations of oxidized
LcrV were added. The values were corrected for dilution and for
LcrVox inner-filter effect using another cuvette containing 4 mM
of N-acetyltryptophanamide (NATA), as previously described
. Data were analyzed using the GraFit Erithacus 5.0 software.
The experimental data were fit to the equation:
where F corresponds to the intensities of the various spectra
analyzed, p is the concentration of IFNc in the cuvette, L is the
concentration of injected LcrVox, and K is the dissociation
LcrV interacts directly with IFNc
LcrV was shown to bind to human U937 monocytic leukemia
cells and human alveolar macrophages, provided that human
IFNc was present; in addition, it was suggested that cellular
recognition occurred through interaction of LcrV with the
IFNcRIFNc complex . In order to further characterize the
immunoregulatory function of LcrV, we explored the LcrV-IFNc
interaction (in the presence or absence of IFNcR) using a
Biacore-based approach. We took advantage of the fact that LcrV
possesses a single cysteine residue to immobilize it in an oriented
fashion on a CM3 sensorchip, as described in the methods section.
Injection of IFNc over the surface produced a significant binding
that demonstrated the existence of an IFNc-LcrV complex
(Figure 1A) IFNc was found to bind immobilized LcrV in a
concentration-dependent manner, and a Scatchard analysis of the
binding responses obtained at equilibrium produced a dissociation
constant (Kd) of 32 nM (Figure 1A). PcrV, the LcrV homolog from
P. aeruginosa, was also immobilized on a CM3 sensorchip and tested
for its ability to bind IFNc (Figure 1B). Notably, almost no binding
was observed even at the highest concentration of 5 mg/mL IFNc.
LcrV was also tested for its ability to interact with IFNcR-IFNc
(Figure 1C and 1D). Interestingly, the signal obtained upon
injection of the complex IFNc-IFNcR was of lower value than that
observed when we injected the same concentration of purified
IFNc, which suggests that LcrV may not bind to the IFNcR-IFNc
complex as well as to IFNc alone. Subsequently, biotinylated
IFNcR was immobilized on a streptavidin-coated surface, and
IFNc alone, or a mixture of LcrV and IFNc, were injected over
the surface (Figure 1D). The binding of IFNc to its receptor was
not modified in the presence of LcrV, which strongly suggests that
the interaction between LcrV and IFNc does not prevent the
association of IFNc with its receptor. Thus, these data indicate
that LcrV binds IFNc with nanomolar affinity, even in the absence
of the IFNc receptor.
Fluorescence analysis of the LcrV-IFNc complex
In order to further characterize the interaction between LcrV
and IFNc, we measured the change of intrinsic fluorescence of
IFNc upon complex formation. LcrV and IFNc each include one
single tryptophan residue, whose spectral property could
potentially be altered upon their interaction. To precisely dissect the
fluorescence events related to complex formation and pinpoint
uniquely the contribution of the tryptophan found in IFNc, we
treated LcrV with N-Bromosuccinimide (NBS) , in order to
chemically modify its Trp 113. This chemical oxidation produced
drastic changes on the LcrV emission spectrum, characterized by a
large decrease in intensity (Figure 2A).
Oxidized LcrV (LcrVox) was then introduced into a cuvette
containing 1 mM IFNc, and the variations of IFNc emission
spectra were recorded between 310 and 370 nm. Control
injections of LcrVox into a solution of N-acetyltryptophanamide
(NATA) allowed correction of the inner-filter effect of LcrVox.
The fitting of the data enabled the determination of a Kd value of
42 nM (Figure 2B), which is comparable to that obtained from the
Biacore studies (see above). These data lead to the conclusion that,
whether LcrV is immobilized or free in solution, it binds to IFNc
with strong affinity.
The two basic clusters of the IFNc C-terminal domain are
crucial for the interaction with LcrV
IFNc is a dimeric cytokine whose biological activity is related to
its unstructured C-terminal domain [reviewed in 21]. This domain
includes two basic patches, D1 and D2 (amino acids 125131 and
137140, respectively, see Figure 3A), both of which have been
reported to bind HS . Notably, the basic clusters of IFNc (and
specifically the GRRA138141 motif, which overlaps the second
basic cluster) play a role in LcrV recognition in cell culture
In order to precisely map the LcrV-IFNc interaction region, we
investigated whether the two basic clusters of IFNc were involved
in the interaction with immobilized LcrV by surface plasmon
resonance. We generated specific mutants of IFNc which display
serial mutations of R and K residues either within the D1 or D2
domain, allowing an independent assessment of the role of each of
these specific basic clusters (Figure 3A). When R131 of cluster D1
was substituted by a serine residue, the sensorgram reached a
maximum RU value of 175, compared to 300 RU for wild-type
IFNc. Further mutations within the D1 domain (mutants S2 to S5,
Figure 3B) strongly impaired binding, demonstrating the
importance of cluster D1 for the interaction to take place. An even more
pronounced phenomenon was observed when IFNc basic cluster
Figure 1. Free IFNc interacts with LcrV, but not with PcrV, in a surface plasmon resonance assay. (A) Scatchard analysis of IFNc binding
to LcrV immobilized on a CM3 sensorchip (1150 RU). Req, steady state value at equilibrium; C, concentration of injected IFNc. (B) 5.0 mg/mL IFNc was
injected over LcrV (2700 RU, black curve) and PcrV (1200 RU, grey curve) immobilized on two different lanes of the same sensorchip. (C) Injection over
immobilized LcrV (1150 RU) of 2 mg/mL IFNc (black curve) or of 2 mg/mL IFNc and an equimolar amount of IFNcR (grey curve). RU = resonance units.
(D) Injection of 1 mg/mL IFNc alone (black curve) or in combination with LcrV (1 mg/mL of each, grey curve) over immobilized IFNcR (1600 RU). For all
experiments, non-specific binding to the sensor chip was subtracted from the raw data.
D2 was modified, with a single mutation at position 140 almost
completely abolishing LcrV recognition. Binding to immobilized
LcrV decreased gradually for mutants S7 and S8, as compared to
wild-type IFNc. Our data thus indicate that both basic clusters of
IFNc are fundamental for the interaction with LcrV, with a strong
involvement of the most distal one (D2). Notably, the surface of
LcrV is highly acidic (Figure 3D, see below), providing a number
of possibilities for recognition of the basic patches on IFNc.
Interestingly, however, a peptide corresponding to the
Cterminal region of IFNc encompassing both clusters D1 and D2
could not be shown to interact with LcrV neither by Biacore nor
fluorescence techniques (not shown), suggesting that this region
must be stabilized or properly oriented within the full-length IFNc
structure in order to be available for productive binding with
Inhibition of formation of the LcrV-IFNc complex
A set of synthetic glycoconjugates that mimic HS were
synthesized and tested for their ability to interact with IFNc
. These molecules were shown to inhibit the binding of IFNc
to heparin, the most active one displaying an IC50 value of 35
40 nM. Since the formation of the IFNc-HS and LcrV-IFNc
complexes share a common dependency on the basic residues
present in the C-terminus of IFNc, these data prompted us to
test whether these synthetic glycoconjugates would also inhibit
the IFNc-LcrV interaction in a Biacore study. We thus chose a
set of compounds, each composed of two tetrasaccharides (T),
hexasaccharides (H) or octasaccharides (O), which were linked to
poly(ethylene glycol)-based spacers of different lengths (Figure 4A).
Glycoconjugates were incubated with IFNc in a 0.5:1 molar ratio,
and the complexes were injected over immobilized LcrV. The
binding obtained at equilibrium was compared to that observed
for free IFNc, which was considered as 0% inhibition. Results
from three independent experiments indicated that all tested
compounds inhibited LcrV-IFNc assembly (Figure 4B), with the
octasaccharides being the best inhibitors (approx. 70% of
We further characterized the inhibition properties of 2O32,
chosen as the most potent inhibitor. Increasing ratios of
2O32:IFNc were injected over immobilized LcrV, and the binding
responses were analyzed as described above. The inhibition curve
of 2O32 is shown in Figure 4C. Fitting of the data leads to an IC50
of 75 nM, which is comparable to the IC50 value of 3540 nM
obtained for the inhibition of IFNc-heparin binding by 2O10 .
These data thus allowed the identification of efficient inhibitors of
the LcrV-IFNc interaction, which could constitute potential
candidates for preventing Yersinia dissemination over the course
The type III secretion system is a complex macromolecular
system employed by a number of human pathogens to inject toxins
directly into the cytoplasm of target eukaryotic cells and thus
initiate infection . LcrV is secreted by the T3SS and is a major
regulator and effector of virulence [12,34]. Passive protection
against Y. pestis was observed in mice using anti-V antibodies that
recognize one or more internal epitopes located between amino
acids 168 and 275, which underlines the highly protective
antigenic character of LcrV . LcrV interactions with the
immune system also include an anti-inflammatory role: LcrV is
able to trigger secretion of IL-10, a powerful anti-inflammatory
cytokine that prevents expression of different host inflammatory
factors [35,36]. This induction of secretion could be partly
explained by the binding of LcrV to receptor-bound IFNc .
The results presented here provide evidence that LcrV is able to
interact with free IFNc even in the absence of IFNcR. Addition of
IFNcR to IFNc did not improve binding, but in contrast partially
prevented optimal interaction of IFNc with immobilized LcrV.
IFNc residues involved in receptor binding (amino acids 1826
and 108124) are located in the globular structured part of the
molecule [23,24], and it has been shown that the C-terminal
domain of IFNc is partially buried within the IFNc-IFNcR
complex [24,25]. Our results demonstrate that LcrV recognizes
the unstructured flexible C-terminal domain of IFNc (residues
124143). These elements provide an explanation for the
decreased binding of IFNc to LcrV in the presence of IFNcR:
titration of IFNc by its receptor causes steric hindrance for LcrV
recognition, thus blocking the accessibility of the IFNc C-terminus
for interaction with LcrV. Notably, Abramov and co-workers ,
by using flow cytometry, reported that LcrV recognizes IFNc
when it is in complex with its receptor (IFNcR). Here, we employ
different biochemical and biophysical techniques to show that
IFNcR is not required for LcrV recognition of IFNc, thus
underlining its potential as a regulator of inflammation.
In addition, we show by surface plasmon resonance that the
interaction of IFNc with LcrV mostly relies on D1 and D2, the two
basic clusters present in the C-terminus of the protein. Basic residues
in both clusters were sequentially disrupted, which showed that D2
was slightly more important than D1 for the interaction. These
results are in accordance with previous observations made by
Abramov et al., who showed that a truncated form of IFNc lacking
cluster D2, and mouse IFNc, which includes only cluster D1, are
unable to interact with LcrV when added to U937 cells or alveolar
macrophages. Additionally, our results point out the prominent role
of cluster D1 in the strong affinity of IFNc for LcrV.
PcrV is the homologue of LcrV in the well-studied Pseudomonas
aeruginosa TTS system . Both proteins share 41% sequence
identity and their role in translocon assembly and toxin
translocation have been proposed to be similar [16,29]. However,
when PcrV was tested for direct binding to IFNc by surface
plasmon resonance, no interaction was observed. LcrV contains a
number of acidic residues including LEEL3235 and DEEI203206
motifs that have been proposed as being involved in IFNc binding
through electrostatic interactions with the C-terminal positive
charges of IFNc . The crystal structure of LcrV, which shows
that the molecule is shaped like a dumbbell with both N- and
Ctermini on the same side (Figure 3D) , indicates that both
motifs are exposed on the protein surface, thus being potentially
available for interactions with other protein partners (Figure 3D).
The surface of LcrV, however, is highly acidic, as indicated by the
elevated number of exposed negative charges (shown in red in
Figure 3D), and thus the participation of additional acidic patches
in the interaction with IFNc cannot be ruled out. Interestingly, the
sequence of PcrV displays a region with sequence similarity to the
LEEL3235 motif of LcrV (residues QEEL2932), but lacks an acidic
region which could correspond to LcrVs DEEI203206 motif. The
absence of this specific surface acidic patch could explain why
PcrV does not interact with IFNc in spite of a high degree of
homology with LcrV. Of note, it has been shown that recombinant
LcrV and PcrV also differ in their ability to induce IL-10 or
suppress TNF-a production by stimulated macrophages :
while LcrV elicits an extended immunosuppressive response,
PcrVs interactions with the immune system may be limited to its
immunogenic character [44,45]. These elements are particularly
interesting when considered in light of the respective degree of
virulence of both pathogens: plague bacilli must kill their host to
survive in nature , which may require a rapid and global
neutralization of the host immune system, whereas P. aeruginosa is
considered as an opportunistic microorganism, able to develop a
chronic infection that will survive the immune response.
The relevance of the IFNc-LcrV interaction in vivo remains to be
investigated. It has been shown that LcrV displays differential
targeting profile in the host: it is located on the extracellular surface
of the bacterium [46,47], where it may act as an assembly platform
for the translocation pore [15,29,48], but it is also released from
Yersinia into the extracellular medium [49,50]. The latter authors
have also reported that a pool of LcrV may be injected into the host
cell cytoplasm, where it could interfere with cellular processes
required to eliminate Yersinia during infection. A global model of
LcrV targeting was proposed, which suggests its dynamic
association at the bacterial surface, allowing partial release in the
surrounding medium . This model is consistent with an
immunomodulatory role for LcrV: released LcrV would be free to
diffuse away and have a broadly local or even a systemic effect
during an infection process. We propose that this pool of secreted
LcrV is involved in the interaction with IFNc, acting as a
longdistance weapon to prevent the multiple effects of the cytokine.
In light of this model, it appears crucial to develop new molecules
that could interfere with the extended anti-immune properties of
LcrV. The oligosaccharides used in this study constitute particularly
interesting candidates as they inhibit the interaction between LcrV
and IFNc with a high efficiency. Further ex vivo and in vivo studies will
be needed to test their anti-Yersinia potential, but several factors are
encouraging: i) their small size allows an efficient synthesis of
homogenous preparations, ii) they are heparin-like, and thus
presumably non-immunogenic compounds, and as such they should
be well tolerated by the organism, iii) they were specifically designed
to interact with IFNc , and they represent interesting scaffold
structures for further developments.
Cytokine network perturbation is one of the strategies exhibited by
pathogenic bacteria to counteract the host immune system .
The proteins responsible for these effects have been named
bacteriokines . LcrV can be classified into this group since
i) a protein A-LcrV fusion induces suppression of TNF-a and IFNc in
spleen homogenates of mice, which promotes in vivo survival of the
bacteria [14,19], ii) a polyhistidine fusion of LcrV causes an increase
of IL-10 expression at the spleen level , and iii) in the present
study, we show that LcrV also directly interacts with IFNc in vitro.
This additive anti-inflammatory mechanism probably enables the
invading bacteria to achieve a lethal cellular burden before an
effective specific immune response can be initiated. Therefore, the
identification of specific inhibitors of this interaction, provided in this
study, is of major importance and provides interesting potential
candidates for the development of new antibiotics against Yersinia spp.
The authors wish to thank Ina Atree (CEA-Grenoble) for the kind gift of
the LcrV-expressing plasmid, Izabel Berard and Eric Forest (LSMP, IBS)
for mass spectrometry analyses, Andres Palencia (EMBL Grenoble) for
help with isothermal calorimetry trials and fluorescence studies, and Els
Saesen (IBS) for purification of IFNc.
Conceived and designed the experiments: CG HLJ JMJ AD. Performed
the experiments: CG DB SS. Analyzed the data: CG HLJ JMJ AD.
Contributed reagents/materials/analysis tools: HLJ JMJ AD. Wrote the
paper: CG HLJ AD.
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