Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1 mediated maturation and release of Interleukin-1β (IL-1β)*

Peter Schotte‡,†, Geertrui Denecker§,#,†, Aeke Van Den Broeke‡, Peter Vandenabeele§,

Guy Cornelis#,º, Rudi Beyaert‡,º,¶

From the Department of Molecular Biology, Unit for Molecular Signal Transduction in

Inflammation, Flanders Interuniversity Institute for Biotechnology and Ghent University,

B-9052 Zwijnaarde, Belgium and from the Biozentrum der Universität Basel, CH4056

Basel, Switzerland.

Running title: Rac1 in the maturation and release of IL-1β

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* Research was supported by the Swiss National Science Foundation, the Fund

for Scientific Research-Flanders (FWO-Vlaanderen), the Interuniversity Attraction Poles

(IUAP-V) and a EC-RTD grant. P.S. and G.D. are postdoctoral researchers with the

Flemish Institute For the Promotion of Scientific-Technological Research in the Industry

(IWT) and the FWO-Vlaanderen, respectively. The authors thank W. Burm and A.

Meeus for technical assistance. Dr. L. J. Mota, Dr. A. Boland, Dr. J Tschopp and Dr. P.

Caroni are acknowledged for kindly providing materials. The authors also thank Dr. L.J.

Mota and Dr. W. Declercq for fruitful discussion.

† These authors contributed equally to this work as first authors.

º These authors contributed equally to this work as senior authors.

¶ To whom correspondence should be addressed: Unit of Molecular Signal

Transduction in Inflammation, Department of Molecular Biomedical Research, VIB,

Ghent University, Technologiepark 927, B-9052 Ghent, Belgium. Tel. ++32-93313770,

Fax. ++32-93313609, E-mail: rudi@dmbr.ugent.be.

‡ Unit of Molecular Signal Transduction in Inflammation, and § Unit of Molecular

Signalling and Cell Death, Department of Molecular Biomedical Research, VIB, Ghent

University, Gent, Belgium.

# Division of Molecular Microbiology, Biozentrum, Universität Basel, Basel,

Switzerland. 1 The abbreviations used are: IL: interleukin, Asc: apoptosis-associated speck-like

protein containing a CARD, CARD: caspase recruitment domain, GAP: GTPase

activating protein, JNK: c-Jun N-terminal kinase, NF-κB: nuclear factor-κB, NOD:

nucleotide-binding oligomerization domain, MAP: mitogen activated protein, PYD: pyrin

domain, LIMK1: LIM-kinase 1, LDH: lactate dehydrogenase, WT: wild type, DN:

dominant-negative, CA: constitutive-active.

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Abstract

Yersinia bacteria can take control of the host cell by injecting so-called Yop effector

proteins into the cytosol of the cells to which they adhere. Using Yersinia enterocolitica

strains that are deficient for one or more Yops, we could show that YopE, and to a lesser

extend also YopT, interferes with the caspase-1 mediated maturation of pro-interleukin-

1β in macrophages. In addition, overexpression of YopE and YopT was shown to

prevent the auto-proteolytic activation of caspase-1 in a way that is dependent on their

inhibitory effect on Rho GTPases. Expression of constitutive-active or dominant-negative

Rho GTPase mutants, or treatment with Rho GTPase inhibitors, confirmed the role of

Rho GTPases, and in particular Rac1, in the auto-activation of caspase-1. Rac1 induced

caspase-1 activation was mediated by its effect on LIM kinase-1, which is targeting the

actin cytoskeleton. Rac-1 and LIM kinase-1 dominant-negative mutants were shown to

inhibit caspase-1 activation induced by overexpression of Asc, which is a caspase-1

activating adaptor protein. Moreover, Rac1 as well as YopE and YopT significantly

modulated caspase-1 oligomerization. These results highlight a previously unknown

function of Rho GTPases in the activation of caspase-1, and give new insight on the role

of YopE in immune-escape mechanisms of Yersinia.

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Introduction

A number of Gram-negative pathogens subvert the innate immune system of their host

by a virulence mechanism known as the type III secretion system. In the archetypal

Yersinia - Y. pestis, agent of bubonic plague, Y. pseudotuberculosis and Y.

enterocolitica - the type III secretion system is encoded on a 70-kb virulence plasmid (1).

By this mechanism, Yersinia bacteria adhering at the surface of eukaryotic cells inject

proteins - called Yops - across cellular membranes into the cytosol of these cells. Yop

proteins (YopE, T, H, M, O and P) are powerful effectors that take control of the host cell

by hijacking the intracellular machinery in order to interfere with the inflammatory

response and phagocytosis (2). The Yop effectors can be subdivided into three

functional groups. The first functional group, includes YopE, YopT, YopO/YpkA and

YopH which cooperatively lead to the destruction of the actin cytoskeleton and by doing

so prevent phagocytosis. YopE and YopT target Rho GTPase family members, which

are key players in cytoskeletal reorganization (3). YopE is a GTPase-activating protein

(GAP) for the Rho GTPase family (Rho, Rac and Cdc42) (4), switching off Rho GTPases

by accelerating GTP hydrolysis. YopT is a Ser/Thr protein kinase that becomes

autophosphorylated upon contact with actin, and modulates cytoskeleton dynamics. Its

real cellular target is still unknown but possible candidates are RhoA and Rac1, which

were shown to interact with the C-terminus of YopO (5). The second functional group,

consisting of YopP and, again, YopH, prevents the onset of the inflammatory response

(6). YopP prevents the activation of members of the MAP kinase kinase family and of

IκB kinase-β (6-9). In this way YopP efficiently shuts down NF-κB dependent signalling

pathways, preventing the production of pro-inflammatory cytokines such as TNF and IL-

8. In addition, because of the action of YopP, macrophages undergo cell death with the

rapid generation of pro-apoptotic tBid (10). YopH also belongs to this second functional

group because, besides its anti-phagocytic role, it also prevents the release of the

macrophage chemoattractant MCP-1 by blocking the phosphatidylinositol-3 kinase

pathway (11). Finally, the third group consists of YopM, whose exact function remains

unknown, though it is required for full virulence of Yersinia, as demonstrated in mouse

infection models (12), Recently, it was shown that YopM binds and promotes the kinase

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activity of PRK-2 (protein kinase C-like 2) and RSK1 (ribosomal S6protein kinase 1)

(13), which could explain the effect of YopM on the expression of genes involved in cell

cycle progression and cell growth (14).

One of the most important inflammatory responses upon infection is the production of

interleukin-1β (IL-1β), which is a pleiotropic cytokine that is involved in the regulation of

both the innate and the acquired immune response (15). IL-1β expression in

macrophages is inducible in a NF-κB dependent way (16), and is synthesized as an

inactive proIL-1β precursor whose proteolytic maturation is controlled by the cysteine

protease caspase-1 (17). The latter is itself present in the cytosol as a 45-kDa precursor,

which can be induced to undergo a series of processing events necessary for its

activation. Although the mechanism of caspase-1 activation is still unclear, a multiprotein

complex (named the ‘inflammasome’) that is involved in the activation of procaspase-1

upon lipopolysaccharide stimulation was recently identified (18). The inflammasome

consists of caspase-recruitment domain (CARD) and pyrin domain (PYD) containing

proteins, including Asc (apoptosis-associated speck-like protein containing a CARD),

which mediate the oligomerization of procaspase-1 to facilitate its activation process

(19). The CARD of Asc binds the CARD of procaspase-1, whereas Asc’s PYD can

interact with the PYD of a subset of proteins belonging to the NOD protein family, which

have been implicated recently in innate recognition of bacteria and the induction of

inflammatory responses (20).

In the present work we addressed the question whether specific Yersinia Yop effector

proteins can interfere with the caspase-1 mediated maturation of proIL-1β. Therefore,

we investigated the effect of several Yop deficient Yersinia strains on the production of

mature IL-1β by infected macrophages. Our results point out a novel role for YopE in

modulating the inflammatory response of the macrophage during infection. In addition,

we demonstrate for the first time the involvement of Rho GTPases, and in particular

Rac1, in the regulation of caspase-1 activation.

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Materials and Methods Plasmids and antibodies. Wild type (WT) YopE, YopT and YopH were amplified by

polymerase chain reaction from the pYV40 plasmid from strain E40 (21) and cloned in

frame with an N-terminal peptide E-tag epitope (GAPVPYPDPLEPR) into pCAGGS-Etag

(22), which was cut with NotI and XmaI restriction enzymes. The inactive mutants (M)

YopER144A and YopTC139S were generated by overlapping polymerase chain reaction

using mutated primers. pYV40 plasmids for specific Yop effector knockouts have been

described previously (23,24). Expression plasmids for YopEWT and YopEM, which were

used for complementation of YopE knockout strains, were a gift from Dr. L.J. Mota. The

cDNA of human RhoA, Rac1, Cdc42 and their corresponding dominant-negative

(T17 N17 in Rac1 and Cdc42, T19 N19 in RhoA) and constitutive-active (Q61 L61 in

Cdc42, Q63 L63 in RhoA, G12 V12 in Rac1) mutants have been described previously

(25), and were a kind gift of Dr. J. Piette (Liège). The cDNA of Rho family members and

caspase-1 were amplified by polymerase chain reaction and cloned in frame with an N-

terminal E-tag or Flag-tag into pCAGGS, which was cut with NotI and XmaI restriction

enzymes. Overlapping polymerase chain reaction using constitutive-active Rac1 as a

template and mutated primers generated the constitutive-active mutants Rac1CA-F37L and

Rac1CA-Y40H, respectively. The mouse proIL-1β cDNA was cloned into pCAGGS-Etag

vector with an additional HA-tag at the 3'-end of the proIL-1β cDNA. All constructs were

confirmed by DNA sequence analysis. The expression plasmid for N-terminal Flag-

tagged human Asc (pCR3.V66-Met-Flag-Asc) and myc-tagged LIMK1 constructs were

kind gifts of Dr. Jurg Tschopp (Lausanne) and Dr. Pico Caroni (Basel, Switzerland),

respectively. The β-galactosidase-encoding plasmid pUT651 was purchased from Cayla

(Toulouse, France). A rabbit polyclonal antibody against recombinant murine caspase-1

was prepared by the Centre d'Economie Rurale (Laboratoire d'Hormonologie Animale,

Marloie, Belgium).

Bacterial strains and growth conditions. Escherichia coli Top10 or MC1061 were used

for standard manipulations; E. coli SM10 lambda pir+ was used to deliver mobile

plasmids into Y. enterocolitica (1). E. coli strains were routinely grown at 37oC in tryptic

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soy broth or on tryptic soy agar plates containing the appropriate antibiotics. Y.

enterocolitica bacteria were grown at 25oC in brain-heart infusion (BHI; Difco) or on

tryptic soy agar plates containing the appropriate antibiotics. Y. enterocolitica E40

strains and derivatives have been described before (23,24). For infections, bacteria were

diluted to an OD 0.1 in fresh BHI medium and incubated at 25oC for 120 min.

Subsequently, Yop secretion was induced by incubation for 30 min in a shaking water

bath (110 rpm) at 37°C. Prior to infection bacteria were washed with RPMI1640.

Culture, infection and transfection of cells. The murine macrophage cell line Mf4.4

(26), and the human embryonic kidney cell line HEK293T were cultured at 37°C in

RPMI1640 or DMEM, respectively, supplemented with 10% FBS, 2 mM L-glutamine,

penicillin (100 U/ml), streptomycin sulphate (100 µg/ml), sodiumpyruvate (1 mM) and β-

mercaptoethanol (2 x 10-5 M). Prior to infection, Mf4/4 cells were seeded at 106 cells/6-

well in medium without antibiotics. After 15 h, cells were infected at a multiplicity of

infection (m.o.i.) of 50 with the relevant Y. enterocolitica strains that were grown at 37°C

under conditions for moderate Yop induction (see above). Extracellular bacteria were

killed 2 h after infection by adding gentamicin (50 µg/ml). HEK293T cells were plated in

6-wells at 2x105 cells per well and transiently transfected by calcium phosphate-DNA

coprecipitation. 24 h after transfection, medium was removed and cells were lysed in

300 µl lysis buffer (50 mM Hepes, pH 7.6, 200 mM NaCl, 0.1% NP40, 5 mM EDTA).

Proteins were separated by SDS-PAGE and analysed by Western blotting with rabbit

polyclonal anti-caspase-1 and anti-IL-1β antibodies (R&D systems), respectively, with

mouse monoclonal anti-Flag-Horseradish peroxidase (Sigma-Aldrich) or anti-E-tag-

Horseradish peroxidase antibodies (Amersham Biosciences). Immunoreactivity was

revealed with the enhanced chemiluminescence method (NENTM Renaissance, NEN

Life Sciences Products). LDH release was assayed using Cytotox-one homogeneous

membrane integrity assay as described by the manufacturers protocol (Promega). β-

Galactosidase release was assayed using the Galactostar reporter gene assay system

(Applied Biosystems)

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Results YopE inhibits the release of IL-1β in Yersinia enterocolitica infected macrophages.

Yersinia has previously been shown to prevent NF-κB activation in infected cells in a

YopP-dependent manner (8). Therefore, it could be expected that the expression of NF-

κB dependent genes would be strongly increased in cells infected with a YopP-deficient

strain (YopP-) compared to cells infected with wild-type (WT) Yersinia. To verify this, we

compared the amount of IL-1β and IL-6 in the supernatant of Mf4/4 macrophages that

were infected with either YopP- or WT Y. enterocolitica. To our surprise, only the levels

of IL-6 but not those of IL-1β were increased in the supernatant of YopP- infected cells

(Fig. 1A). Nevertheless, intracellular proIL-1β levels in the corresponding cell lysates

were considerably higher in cells infected with the YopP- strain (Fig. 1A, inset). It should

be mentioned that only the precursor form of IL-1β could be detected in these cell

extracts. The above observations indicated a role for another Yop effector protein in the

regulation of IL-1β maturation and its release in the cell supernatant. Therefore, we

infected macrophages with a Y. enterocolitica strain that is deficient for all six Yop

effectors (∆HOPEMT, Fig. 1B), which indeed resulted in a large increase of IL-1β levels

in the supernatant (Fig. 1B). All together, the above results demonstrate that, besides

the YopP-mediated down regulation of proIL-1β production at the transcriptional level,

other Yop effectors could specifically inhibit the maturation and release of IL-1β. To

analyse which Yop effector controls the release of IL-1β, we constructed five double

knockout Y. enterocolitica strains, which are negative for YopP and for any one of the

five other Yops: YopPE-, YopPT-, YopPO-, YopPH- and YopPM- strains. After infection of

macrophages with each of these double deficient bacteria, we analysed again the

secretion of IL-1β in the supernatant. IL-1β release was still strongly inhibited in

macrophages infected with the double mutant bacteria YopPT-, YopPO-, YopPH- and

YopPM-, although a small but reproducible increase in IL-1β could be detected in

YopPT- infected cells (Fig. 1B). However, cells infected with the YopPE- strain released

significant higher levels of IL-1β, suggesting that YopE is at least partially responsible for

the inhibition of IL-1β release as seen upon infection with WT or YopP-deficient Yersinia.

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Since IL-1β levels released by YopPE- and YopPT- infected cells were still lower than

those released from cells infected with the ∆HOPEMT mutant, one could expect that

cells infected with a triple YopPET- mutant would release comparable amounts of IL-1β

as those infected with the ∆HOPEMT mutant. However, we were unable to see a

reproducible and significant difference between cells infected with YopPET- or YopPE-

mutants (data not shown). YopE is a GAP for Rho GTPases, in particular Rac1 (27),

switching them off by accelerating GTP hydrolysis. To analyse if the GAP activity of

YopE is required for inhibition of IL-1β release, we complemented the YopPE- strain with

wild-type YopE (YopEWT) or with a mutant of YopE (YopEM) that lacks the GAP activity

(Fig. 1C). Complementation of the YopPE- strain with YopEWT, but not with YopEM,

restored the potential of Yersinia to prevent the release of IL-1β in the medium of

infected macrophages. In contrast, intracellular expression levels of proIL-1β were

independent of YopE (Fig. 1C). A previous report showed that installation of the Yersinia

secretion apparatus into the cell membrane of the macrophage results in pore formation

and the release of cytosolic proteins such as lactate dehydrogenase (LDH), which could

be prevented by YopE. Moreover, a role for Rho GTPases and rearrangements of the

cytoskeleton in the regulation of this pore formation was illustrated (28). In agreement

with the latter observation, YopE deficiency also resulted in a significant increase of LDH

and proIL-1β release in our experiments (Fig. 1C and 1D, respectively), which coincided

with an apoptotic morphology of the cells (data not shown). However, also a clear band

corresponding to mature active IL-1β could be seen in the supernatant of YopPE-

infected cells, implicating that YopE affects the release as well as the proteolytic

maturation of IL-1β. The latter was further confirmed by the fact that identical results

could be obtained when the IL-1 levels in the supernatant were measured with a CTLL

cell proliferation assay that detects specifically mature bio-active IL-1β (data not shown)

(29). In conclusion, our results indicate that the GAP activity of YopE is responsible for

the inhibition of the maturation and secretion of IL-1β in Yersinia infected macrophages.

Moreover, this also implies a role for Rho GTPases in the process of proIL-1β

maturation and IL-1β release.

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Specific Yop effector proteins and Rho GTPases regulate procaspase-1 activation.

Because proIL-1β maturation is mediated by caspase-1, we next analysed the effect of

YopE on caspase-1 mediated proIL-1β maturation in HEK293T cells that were

transiently transfected with procaspase-1 and proIL-1β. In these conditions, procaspase-

1 is partially converted to an active p20/p10 form due to its autocatalytic processing

upon overexpression, resulting in the maturation of the 33 kDa proIL-1β to the bio-active

17 kDa form (Fig. 2A). Moreover, caspase-1 auto-activation also results in the induction

of cell death, which can be assayed by the release of cotransfected β-galactosidase into

the medium (30,31). Additional co-expression of YopEWT, but not of the catalytically

inactive mutant YopEM, inhibited the autocatalytic processing of procaspase-1 as well as

the proteolytic maturation of proIL-1β. Furthermore, also β-galactosidase release into the

medium was partially inhibited. This demonstrates that YopE can interfere with the

autocatalytic processing of caspase-1 through its GAP activity, leading to a decrease in

proIL-1β maturation and caspase-1 mediated cell death. Interestingly, co-expression of

YopT had a similar inhibitory effect as YopE on the proteolytic activation of procaspase-

1 and proIL-1β (Fig. 2A). YopT functions as a cysteine protease that cleaves off the

prenylated C-terminus of Rho, Rac and Cdc42, leading to their release from the plasma

membrane and their irreversible inactivation (32). A mutant of YopT (YopTM) that has

lost its proteolytic activity on Rho GTPases, was no longer able to prevent the

processing of procaspase-1 and proIL-1β. In contrast to YopE and YopT, co-expression

of the effector protein YopH, which is a tyrosine phosphatase that has been shown to

dephosphorylate proteins from focal adhesions and other signalling complexes (33-35),

had no effect on caspase-1 activity. Although YopH can lead to a rearrangement of the

actin cytoskeleton (36), an effect on Rho GTPases has not been reported. These

experiments show that YopE and YopT can prevent the autocatalytic processing and the

activation of caspase-1 by interfering with the function of Rho GTPases. The clear

inhibitory effect of YopT expression on proIL-1β maturation is somewhat contradictory to

the small increase in IL-1β release in cells infected with a YopPT- double knockout as

described in our previous experiments (Fig. 1B). Most likely this can be explained by a

difference in the Yop concentration in cells upon overexpression or infection,

respectively. In this context, it should be mentioned that whereas YopE and YopT

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inactivate RhoA, Rac1, and Cdc42 in vitro or upon overexpression (27,32), when they

are injected into cells by Yersinia, they are remarkably specific, inactivating selectively

Rac1 and RhoA, respectively (27,37). Therefore, the more potent effect of YopE deletion

on proIL-1β maturation in infected cells suggests a major role for Rac1. In order to

confirm the role of Rho GTPases, and in particular Rac1, in the proteolytic activation of

caspase-1, HEK293T cells were cotransfected with procaspase-1, proIL-1β, β-

galactosidase, and constitutive-active (CA) mutants of RhoA, Rac1 and Cdc42. We

hypothesized from our previous experiments that the constitutive-active Rho GTPases

should promote the autocatalytic processing of procaspase-1 and the corresponding

maturation and secretion of IL-1β. Indeed, IL-1β levels were significantly increased in the

supernatant of cells overexpressing RhoACA, Cdc42CA or Rac1CA. Serial dilution of the

transfected plasmid DNA concentration clearly showed that Rac1CA is much more

efficient than RhoA or Cdc42 (Fig. 2B), which is in agreement with the more pronounced

release of IL-1β in YopPE- versus YopPT- infected cells (Fig. 1B) and the described

preferential effect of YopE on Rac1 (27). As expected, Rac1CA also enhanced the

autoproteolytic activation of procaspase-1 and the release of β-galactosidase (Fig. 2C),

whereas RhoACA and Cdc42CA did not (data not shown). Reversely, transfection of a

dominant-negative mutant of Rac1 (Rac1DN) resulted in a decrease of caspase-1 auto-

activation and, consequently, in a decrease of IL-1β and β-galactosidase release (Fig.

2D and E). It should be mentioned that the ectopic expression levels of Rac1 that are

needed to affect caspase-1 activation are extremely low, being under the detection limit

in the case of Rac1DN (Fig. 2E and data not shown). All together, the above

observations imply an important role for Rho GTPases, especially Rac1, in the

regulation of caspase-1 activity.

Pharmacological modulation of Rho GTPase activity affects caspase-1 activation and IL-1β

maturation.

To further confirm the role of Rho GTPases in caspase-1 activation and IL-1β

production, we analysed the effect of Clostridium difficile Toxin B. The latter is a

glucosyltransferase that covalently links a glucose moiety on a critical threonine residue

of Rho, Rac and Cdc42 (38), thus impairing the docking of the GTPases on their

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effectors. Similarly, we also tested the effect of the geranylgeranyl transferase inhibitor

GGTI-2147, which prevents the prenylation and membrane localization of Rho GTPases

(39). Western blot analysis revealed that treatment of procaspase-1 and proIL-1β

expressing cells with Toxin B or GGTI-2147 significantly prevents the proteolytic auto-

activation of caspase-1 and maturation of proIL-1β (Fig. 2F). These results further

demonstrate that Rho GTPases play an important role in the regulation of caspase-1

activation, and the corresponding caspase-1 mediated maturation and release of IL-1β.

The effect of Rac1 on caspase-1 activation is independent of its effect on the JNK

pathway, but is mediated by LIM kinase-1

The functions of Rho GTPases, first assigned to the regulation of the organisation of the

actin cytoskeleton, have been extended to many other cellular processes, including

activation of the c-Jun N-terminal kinase (JNK) (3,40). Moreover, Rac-induced

cytoskeleton reorganisation and JNK activation are the result of independent Rac-

induced signalling pathways (41,42). To dissect which signalling pathway is important in

the Rac1-induced-activation of caspase-1, we used specific point mutants of Rac1CA that

are defective in either JNK activation (Rac1CA-Y40H) or actin reorganisation (Rac1CA-F37L)

(41,42). Transfection of cells with Rac1CA or Rac1CA-Y40H promoted the proteolytic

activation of cotransfected procaspase-1, as well as the corresponding release of

mature IL-1 into the medium, to a similar extent (Fig. 3A). In contrast, cotransfection with

Rac1CA-F37L was unable to promote the activation of caspase-1 and the release of IL-1.

Therefore, we can conclude that Rac1-mediated signalling to JNK activation is not

involved in caspase-1 activation. In contrast, caspase-1 activation seems to be limited to

the Rac1-mediated control of the actin cytoskeleton. LIM kinase-1 (LIMK1) is known to

participate in Rac1 mediated actin cytoskeletal reorganization by phosphorylating cofilin

(43). Interesting, overexpression of constitutive-active LIMK1 did promote the activation

of caspase-1, whereas a dominant-negative form of LIMK1 could abrogate the Rac1-

induced activation of caspase-1 (Fig. 3B). These results demonstrate that the Rac1-

induced activation of caspase-1 is mediated by its effect on LIMK1 and the actin

cytoskeleton.

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Rac1 controls the Asc-mediated activation and oligomerization of caspase-1.

The molecular mechanism of caspase-1 activation is still largely unknown. The caspase

recruitment domain (CARD) containing protein Asc has been shown to function as a

caspase-1 activating adaptor protein by mediating the assembly of a caspase-1

signalling complex that promotes the activation of caspase-1 and the proteolytic

maturation of proIL-1β (19,44). To analyse if Rac1 and LIMK1 can modulate the Asc-

mediated activation of caspase-1, we cotransfected cells with expression vectors for

procaspase-1, Asc, proIL-1β and either YopE, YopT, LIMK1DN or Rac1DN. Western blot

analysis of caspase-1, as well as analysis of the production of bio-active IL-1β shows

that Asc mediated caspase-1 activation can be inhibited by Rac1DN and LIMK1DN, as

well as by the Yop effector proteins YopE and YopT (Fig. 4A). To analyse if constitutive-

active Rac1CA could still further promote the Asc-mediated caspase-1 activation, we co-

expressed either Asc or Rac1CA with procaspase-1 to a level that does not lead to a

significant activation of caspase-1. However, co-expression of Rac1CA and Asc resulted

in a strong activation of caspase-1 (Fig. 4B). These data suggest that Rac1 and LIMK1

can regulate the Asc-mediated activation of caspase-1. We were unable to study the

effect of Rac1 on Asc-induced caspase-1 oligomerization because overexpression of

Asc led to the redistribution of caspase-1 oligomers to the insoluble cell fraction (data

not shown), which is consistent with the previously described formation of filament-like

aggregates upon Asc overexpression (45). To analyse further whether Rac1 modulates

caspase-1 activation by affecting its oligomerization, we made use of the fact that

caspase-1 oligomerization can be forced by overexpression in HEK293T cells, and

analyzed in a co-immunoprecipitation experiment whether Rac1, YopE and YopT could

affect oligomerization of two enzymatically inactive (C284A) procaspase-1 molecules

that were fused with a different epitope-tag. As illustrated in Figure 5, a dominant-

negative Rac mutant (Rac1DN), as well as YopE and YopT, could prevent the

oligomerization of procaspase-1. In contrast, coexpression of constitutive-active Rac1

(Rac1CA) could promote the formation of procaspase-1 oligomers. These experiments

demonstrate that Rac1 modulates caspase-1 activation by affecting its oligomerization.

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Discussion

In order to survive and proliferate, pathogens acquired sophisticated mechanisms to

interfere with the host response to infection. Yersinia spp. and many other pathogens

such as Salmonella spp. and Pseudomonas spp., developed a ‘molecular syringe’ called

type III secretion system to inject their effectors into the host cell and as such interfere

with several signalling cascades involved in inflammation and phagocytosis. The

Yersinia effector YopP, which controls the activity of NF-κB by blocking I-κB kinase B

and MAP kinase kinase activity, is so far the best-characterized anti-inflammatory Yop.

Until recently, four of the six injected effectors of Yersinia were believed to target

elements of the cytoskeleton in order to block phagocytosis. However, recent reports

illustrated that some of the anti-phagocytic effectors, such as YopH, can also interfere

with signalling pathways of the immune defence system, leading for example to a down

regulation of the Fc-mediated oxidative burst in macrophages and neutrophils (11,46,47).

The caspase-1 inhibitory effect of YopE described in the present study reveals a novel

function of Yop effector proteins in regulating the inflammatory response against

Yersinia infection. By this, Yersinia not only interferes with the production of proIL-1β at

the transcriptional level (by means of YopP), but also prevents the caspase-1 mediated

maturation and secretion of already preformed IL-1β. Because caspase-1 is also

responsible for the maturation of proIL-18, a similar effect of YopE can be expected on

the production and secretion of mature IL-18 during infection, which is indeed supported

by our own experiments (data not shown). It should be mentioned that, although the

effect of YopE and YopT on caspase-1 activation was comparable upon overexpression

in HEK293T cells and mediated by their effect on Rho GTPases (as shown by the use of

specific YopE and YopT mutants), the effect of YopE was much more pronounced in the

case of Yersinia infected macrophages. Although the underlying mechanism of Rho

GTPase inhibition by YopE and YopT is different, they both have been shown to inhibit

Rac1, RhoA and Cdc42 in vitro or upon overexpression (4,27,32,37). However, in

Yersinia infected macrophages, YopE and YopT have been shown to specifically inhibit

Rac1 and RhoA, respectively (27,37). The latter observation, together with our own data

showing that IL-1β release from YopPE- infected macrophages is much higher

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compared to the release from YopPT- infected cells, point to a major role for Rac1 in

caspase-1 activation and the proteolytic maturation of proIL-1β in Yersinia infected

macrophages. Interestingly, IL-1β levels in the supernatant of cells infected with the

∆HOPEMT- strain were reproducibly higher compared to those found upon infection with

a YopPE- strain (Fig. 1B), suggesting a role for another Yop effector protein as well.

Because IL-1 levels were also slightly increased in the case of YopPT- infection, one

could expect also a contribution of YopT. We were unable to see a reproducible

difference between cells infected with YopPET- or YopPE- mutants (data not shown).

Most likely, efficient inhibition of IL-1β production can only be achieved by a co-operative

effect of YopE, YopT, YopH and YopO on the cytoskeleton, as it is for the anti-

phagocytic function of these Yop effector proteins. More direct evidence for the

involvement of Rho GTPases, and in particular Rac1, in the activation process of

caspase-1 was obtained by comparing the effect of ectopic expression of Rac1, RhoA

and Cdc42 dominant-negative or constitutive-active mutants on caspase-1 auto-

activation, showing that Rac1 was by far the most potent GTPase to promote the

secretion of IL-1β as a result of enhanced caspase-1 activation. It should be mentioned

that in contrast to the clear inhibitory effect of a dominant-negative Rac1 mutant on

caspase-1 activation, ectopic expression of dominant-negative mutants of Cdc42 and

RhoA rather enhanced the activation of caspase-1 (data not shown). Several groups

have described analogous effects in which overexpression of dominant-negative forms

of RhoA and Cdc42, led to a phenotype consistent with activation of Rac1 (48). It is

presumed that distinct Rho family members transduce competing signals such that a

balance of their interplay determines the ultimate signalling pathway. Therefore,

overexpression of dominant-negative RhoA and Cdc42 could lead to the activation of

endogenous Rac1, which is then responsible for the observed increase in IL-1β

production.

The mechanism of caspase activation has been an intriguing topic of research in the

past 10 years. Surprisingly, just recently some progress has been made in

understanding the specific activation mechanism of caspase-1. Several models have

been suggested, all of which are based on activation by ‘induced proximity’ of

procaspase-1. Specific NOD (nucleotide-binding oligomerization domain) proteins, a

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growing family of cytosolic proteins that have been implicated recently in innate

recognition of bacteria and the induction of inflammatory responses, organize a

molecular platform (also known as ‘the inflammasome’) linking specific bacterial signals

to the downstream activation of caspase-1 (18,20,49). Asc (also known as TMS1 or

PYCARD) is an important adaptor between procaspase-1 and several NOD family

members, mediating caspase-1 oligomerization (18). Our observation that Rac1 could

modulate the Asc-induced proteolytic activation of caspase-1 and corresponding proIL-

1β maturation suggests that Rac1 directly affects the oligomerization of caspase-1,

which could indeed be confirmed in a coimmunoprecipitation experiment. Interestingly,

Asc has been described to be associated with filamentous or speck-like elements in the

cell (50), retaining Asc into an insoluble cell fraction (45). Therefore, it is not unlikely that

Rac1-induced changes in the actin cytoskeleton influence the intracellular localisation of

Asc and the associated activation of caspase-1. Although Rac1 also couples to

signalling pathways that are not directly linked to cytoskeletal changes, we were able to

exclude a role for Rac1-induced JNK activation. Indeed, a Rac1 mutant that is defective

in p65PAK binding, and thus also in JNK activation, but still results in cytoskeletal

reorganisation (41,42,51), could still enhance the auto-activation of caspase-1. In

addition, we could show an involvement of LIMK1 in Rac1-induced caspase-1 activation.

LIMK1 is known to phosphorylate and inactivate cofilin, leading to the accumulation of

actin filaments (43,52). These results further suggest a crucial role for Rac1-induced

cytoskeletal changes in the formation or functioning of a molecular platform for caspase-

1 oligomerization and activation.

Several pathogens produce toxins and virulence factors that target Rho proteins (53).

Some of these toxins inhibit Rho functioning by ADP-ribosylation (e.g. Clostridium

botulinium C3 transferase) or glucosylation (Clostridium difficile toxin B), and others

activate them by deamidation (e.g. Escherichia coli CNF1) and transglutamination (e.g.

Bordetella dermonecrotic toxin). Other type III injected virulence factors manipulate the

regulatory GTPase cycle of Rho GTPase family members by acting as GAPs (e.g.

Pseudomonas aeruginosa ExoS and ExoT) or guanine nucleotide exchange factors

(GEFs, e.g. Salmonella SopE). In view of our present observations, it can therefore be

expected that at least some of these pathogens may also interfere with the inflammatory

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response by targeting Rac1-mediated activation of caspase-1. Intriguingly, Salmonella is

known to promote an inflammatory response by activating caspase-1, using an unknown

mechanism that involves the association of caspase-1 with the Salmonella effector SipB

(54). Because, Salmonella also injects SopE, a GEF for Rho GTPases (55), our

observations suggest that apart from SipB, also SopE may play a role in the activation of

caspase-1 during Salmonella infection.

Once again, the study of host-pathogen interactions revealed eukaryotic cell processes

not understood before. In the present study, we have found a new role for the Yersinia

effector proteins YopE and YopT in down regulating the inflammatory response, and we

have highlighted a previously unknown function of Rho GTPases in the activation of

caspase-1 and the release of IL-1β. As for its anti-phagocytic defence, Yersinia seems

to inhibit the production of pro-inflammatory cytokines by a complex interplay between

several Yop effectors that act at multiple levels. Intriguingly, modulation of caspase-1

mediated inflammation might also occur during infection with several other pathogens

such as Clostridium spp., Salmonella spp. and Pseudomonas spp., which encode

proteins that are also known to target specific Rho GTPases. Our findings are therefore

not only relevant for the development of tools that target caspase-1 in several

inflammatory diseases, but may also give new insights in drug design for treating

infectious diseases.

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Legends

Fig. 1. YopE inhibits the release of IL-1β in Y. enterocolitica infected macrophages. (A)

Mf4/4 macrophages were infected with wild type (WT) or YopP-deficient (YopP-)

derivatives of Y. enterocolitica E40. Non-infected (NI) Mf4/4 cells were used as a control.

2 h after the beginning of infection, gentamicin (50 µg/ml) was added to kill extracellular

bacteria. 4 h later, cell supernatants were collected and cytosolic cell lysates were

prepared. IL-1β or IL-6 release in the supernatant was analysed by ELISA specific for IL-

1β (Quantikine, R&D Systems) or IL-6 (Biotrak, Amersham Biosciences). (A, inset)

Cytosolic proteins were subjected to SDS-PAGE and transferred to Hybond nitrocellulose

membranes (Amersham Biosciences). The blot was probed with polyclonal antibodies

against IL-1β. (B) Mf4/4 cells were infected with WT or Yersinia enterocolitica strains

deficient for one or multiple Yop effector proteins, as indicated, and analysed for IL-1β

release as described in (A). (C) Mf4/4 cells were infected with YopPE-, or YopPE- strains

that were complemented with wild type (YopEWT) or mutant YopE (YopEM) as indicated,

and analysed for IL-1β release as described in (A). The lower part of panel C shows the

intracellular expression levels of proIL-1β as revealed by Western blot analysis of the

corresponding cell lysates, and the relative LDH activity released into the medium of

infected cells compared to non-infected cells, as revealed by the Cytotox-one

homogeneous membrane integrity assay (Promega). (D) Mf4/4 cells were infected as

described under (C) and TCA precipitated proteins in the medium were analysed by

Western blot analysis for the presence of proIL-1β and mature IL-1β (* indicates a non-

specific band).

Fig. 2. Specific Yop effector proteins and Rho GTPases regulate procaspase-1

activation. (A) HEK293T cells were transiently transfected with expression plasmids

encoding procaspase-1 (100 ng), proIL-1β (200 ng), β-galactosidase (100ng) and 4 ng

of either empty vector (EV) or an expression vector encoding wild-type (WT) YopE,

YopT or YopH or the catalytically inactive mutants (M) YopER144A or YopTC139S, all fused

N-terminally with an E-tag. Cell lysates were subjected to SDS-PAGE and transferred to

Hybond nitrocellulose membranes. The blot was probed with polyclonal antibodies against

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caspase-1 and reprobed with anti-IL-1β. p20 and p22 represent specific processing

products of caspase-1. The expression of YopE, -T and -H was confirmed using the

monoclonal anti-E-tag-HRP antibody. Cell death was measured 24h after transfection by

the release of β-galactosidase into the medium using the Galactostar reporter gene

assay system and values are expressed as % of the amount that was released into the

medium of cells that were only transfected with procaspase-1 and proIL-1β. (B-E)

HEK293T cells were transiently transfected with the indicated amounts of expression

vectors for the constitutive-active (CA) mutants RhoAQ63L (RhoACA), Rac1G12V (Rac1CA)

or Cdc42Q61L (Cdc42CA), or an expression vector for the dominant-negative (DN) mutant

Rac1T17N (Rac1DN), together with expression plasmids encoding procaspase-1 (100 ng),

proIL-1β (200 ng), and β-galactosidase (100ng). Secretion of mature bio-active IL-1β (B-

D) was assayed 24 h after transfection in a CTLL cell proliferation assay (29). Values are

expressed in pg/ml and corrected for transfection efficiency as reflected by β-galactosidase

activity measured in the corresponding cell lysates. Standard deviation (n=3) was <10%.

Cell extracts were subjected to SDS-PAGE and transferred to Hybond nitrocellulose

membranes. Expression of transfected Rho GTPases was confirmed by Western blotting

using the monoclonal anti-E-tag-HRP antibody. To detect caspase-1 processing (C, E), the

blot was probed with polyclonal antibodies against caspase-1. (F) HEK293T cells were

transfected with expression plasmids for procaspase-1 (100 ng) and proIL-1β (200 ng).

Medium was replaced 4 h after transfection with medium containing GGTI-2147 (10 µM,

Calbiochem) or Toxin B (10 pM, Calbiochem). Cells were lysed 24 h after transfection

and proteins were subjected to SDS-PAGE and transferred to Hybond nitrocellulose

membranes. The blot was probed with polyclonal antibodies against caspase-1 (upper

panel) and reprobed with anti-IL-1β (lower panel).

Fig. 3. Rac1 signalling towards LIMK1 but not JNK is required for Rac1-mediated

activation of caspase-1. (A) HEK293T cells were transiently transfected with empty

vector (EV) or an expression vector (50ng) encoding the constitutive-active (CA)

mutants Rac1CA, Rac1CA-F37L or Rac1CA-Y40H, together with expression plasmids

encoding procaspase-1 (100 ng), proIL-1β (200 ng) and a β-galactosidase (100ng)

reporter plasmid. (B) HEK293T cells were transiently transfected with an expression

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Schotte et al., 20

vector encoding procaspase-1 (50ng) together with empty vector (EV; 500ng), or

constitutive-active LIMK1 (200ng), constitutive-active Rac1CA (50ng), dominant-negative

mutant of LIMK1. Cell extracts were subjected to SDS-PAGE and transferred to Hybond

nitrocellulose membranes. The blot was probed with polyclonal antibodies against

caspase-1. Expression of Rac1 and LIMK1 was confirmed using the monoclonal anti-E-

tag-HRP or an anti-myc-tag antibody, respectively. Supernatant was harvested 24 h after

transfection of HEK293T cells, and secretion of mature bio-active IL-1β was assayed in a

CTLL cell proliferation assay. Values are expressed in pg/ml and corrected for transfection

efficiency as reflected by β-galactosidase activity measured in the corresponding cell

lysates. Standard deviation (n=3) was <10%.

Fig. 4. Asc-mediated activation of caspase-1 is modulated by Rac1. (A) HEK293T cells

were transiently transfected with an expression vector (50ng) encoding procaspase-1

(50ng) and Asc (50ng) as indicated, together with empty vector (EV; 600ng) or an

expression plasmid encoding YopT, YopE or the dominant-negative mutants of LIMK1DN

or Rac1DN. (B) HEK293T cells were transiently transfected with an expression vector

encoding procaspase-1 (50ng) and Asc (20ng) as indicated, together with 20ng empty

vector (EV) or an expression plasmid encoding Rac1CA. Cell extracts were subjected to

SDS-PAGE and transferred to Hybond nitrocellulose membranes. The blot was probed

with polyclonal antibodies against caspase-1. Expression of Rac1, YopE and YopT was

confirmed using the monoclonal anti-E-tag-HRP antibody. The expression of LIMK1 and

Asc was confirmed using a monoclonal anti-myc or an anti-Flag-HRP antibody,

respectively. Supernatant was harvested 24 h after transfection and secretion of mature

bio-active IL-1β was assayed in a CTLL cell proliferation assay. Values are expressed in

pg/ml and corrected for transfection efficiency as reflected by β-galactosidase activity

measured in cell lysates. Standard deviation (n=3) was <10%.

Fig. 5. Rac1, as well as YopE and YopT, regulate the oligomerization of procaspase-1.

Co-immunoprecipitation assays were performed using lysates from HEK293T cells that

were transiently transfected with plasmids (100ng) encoding enzymatically inactive E-

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tagged procaspase-1 (E) and Flag-tagged procaspase-1 (F), and 20 ng of either empty

vector (EV) or an expression vector encoding YopE, YopT, Rac1DN or Rac1CA.

Immunoprecipitates were prepared using anti-Flag antibody adsorbed to protein G-

sepharose and analyzed by SDS-PAGE/immunoblotting using anti-E-tag-HRP antibody.

Expression of Rac1, YopE and YopT was confirmed using the monoclonal anti-E-tag-HRP

antibody.

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IL-1

b (

pg

/ml)

Ra

c1

CA

EV

12500

10000

Rac1

CA

-Y40H

Ra

c1

CA

-F37L

A

Caspase-1

LIM

K1

CA

EV

EV

+ LIMK1DN

EV

20

0n

g

50

0n

g

+ Rac1CA

Procaspase-1

p22

p20

B

Rac1

LIMK1

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Procaspase-1

p22

Ra

c1

DN

LIM

K1

DN

Yo

pT

Yo

pE

EV

Caspase-1 + Asc

Figure 4

Asc

YopE/YopTLIMK1/Rac1

BA

Caspase-1 Asc + Caspase-1

Rac1CA

AscR

ac

1C

A

EV

Ra

c1

CA

EV

15000

10000

5000

0

IL-1

b (

pg

/ml)

25000

20000

15000

10000

5000

0

IL-1

b (

pg

/ml)

25000

20000

30000

Procaspase-1

p22

Ca

sp

as

e-1

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E-Procaspase-1

F+

E +

Ra

c1

DN

FE

IP : anti-FW : anti-E

F+

E

F+

E +

Ra

c1

CA

F+

E +

Yo

pE

F+

E +

Yo

pT

E-Procaspase-1

Rac1

F-Procaspase-1

Figure 5

Yop

Total Expression

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Cornelis and Rudi BeyaertPeter Schotte, Geertrui Denecker, Aeke Van Den Broeke, Peter Vandenabeele, Guy R.

)β (IL-1βmaturation and release of interleukin-1Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1 mediated

published online April 1, 2004J. Biol. Chem. 

  10.1074/jbc.M401245200Access the most updated version of this article at doi:

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