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Targeting Rac1 by the Yersinia effector protein YopE inhibits caspase-1-mediated maturation and release of interleukin-1beta.

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 extent, YopT interfere with the caspase-1-mediated maturation of prointerleukin-1beta in macrophages. In addition, overexpression of YopE and YopT was shown to prevent the autoproteolytic 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 autoactivation 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.

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 (Yersinia pestis, an agent of bubonic plague, Yersinia pseudotuberculosis, and Yersinia 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 mem-branes into the cytosol of these cells. Yop proteins (YopE, YopT, YopH, YopM, YopO, and YopP) 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) 1 for the Rho GTPase family (Rho, Rac, and Cdc42) (4), switching off Rho GTPases by accelerating GTP hydrolysis. YopT is a cysteine protease that cleaves off the C-terminal prenyl membrane anchor from Rho GTPases and releases them into the cytosol where they are inactive (5). YopO 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 (6). The second functional group consisting of YopP and again YopH prevents the onset of the inflammatory response (7). YopP prevents the activation of members of the mitogen-activated protein kinase kinase family and of IB kinase-␤ (7-10). As a result, YopP efficiently shuts down NF-B-dependent signaling pathways, preventing the production of proinflammatory cytokines such as tumor necrosis factor-␣ and interleukin-8 (IL-8).
In addition, because of the action of YopP, macrophages undergo cell death with the rapid generation of proapoptotic tBid (11). YopH also belongs to this second functional group because, besides its anti-phagocytic role, it also prevents the release of the macrophage monocyte chemoattractant protein-1 by blocking the phosphatidylinositol 3-kinase pathway (12). Finally, the third group consists of YopM whose exact function remains unknown, although YopM is required for full virulence of Yersinia as demonstrated in mouse infection models (13), Recently, it was shown that YopM binds and promotes the kinase activity of protein kinase C-like 2 and ribosomal S6 protein kinase 1 (14), which could explain the effect of YopM on the expression of genes involved in cell cycle progression and cell growth (15).
One of the most important inflammatory responses upon infection is the production of IL-1␤, which is a pleiotropic cytokine that is involved in the regulation of both the innate and the acquired immune response (16). IL-1␤ expression in macrophages is inducible in a NF-B-dependent way (17) and is synthesized as an inactive proIL-1␤ precursor whose proteolytic maturation is controlled by the cysteine protease caspase-1 (18). The latter itself is 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 identified recently (19). The inflammasome consists of caspase-recruitment domain (CARD) and pyrin domain containing proteins including Asc (apoptosis-associated speck-like protein containing a CARD), which mediate the oligomerization of procaspase-1 to facilitate its activation process (20). The CARD of Asc binds the CARD of procaspase-1, whereas the pyrin domain of Asc can interact with the pyrin domain of a subset of proteins belonging to the nucleotide-binding oligomerization domain protein family, which has been implicated recently in innate recognition of bacteria and the induction of inflammatory responses (21).
In this work, we addressed the question of whether specific Yersinia Yop effector proteins can interfere with the caspase-1-mediated maturation of proIL-1␤. Therefore, we investigated the effect of several Yop-deficient Yersinia strains on the production of mature IL-1␤ 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.

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 (22) and cloned in-frame with an N-terminal peptide E-tag epitope (GAPVPYPDPLEPR) into pCAGGS-E-tag (23), which was cut with NotI and XmaI restriction enzymes. The inactive mutants (M) YopE R144A and YopT C139S were generated by overlapping polymerase chain reaction using mutated primers. pYV40 plasmids for specific Yop effector knock-outs have been described previously (24,25). Expression plasmids for YopE WT and YopE M , which were used for complementation of YopE knock-out strains, were a gift from Dr. L. J. Mota. The cDNA of human RhoA, Rac1, Cdc42, and their corresponding dominant-negative (Thr 17 3 Asn 17 in Rac1 and Cdc42 and Thr 19 3 Asn 19 in RhoA) and constitutive-active (Gln 61 3 Leu 61 in Cdc42, Gln 63 3 Leu 63 in RhoA, and Gly 12 3 Val 12 in Rac1) mutants have been described previously (26) and were a kind gift of Dr. J. Piette (Liège, Belgium). 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 Rac1 CA-F37L and Rac1 CA-Y40H , respectively. The mouse proIL-1␤ cDNA was cloned into pCAGGS-E-tag vector with an additional HA tag at the 3Ј-end of the proIL-1␤ cDNA. All of the 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 LIM kinase-1 (LIMK1) constructs were kind gifts of Dr. Jurg Tschopp (Lausanne, Switzerland) and Dr. Pico Caroni (Basel, Switzerland), respectively. The ␤-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 ϩ was used to deliver mobile plasmids into Y. enterocolitica (1). E. coli strains were routinely grown at 37°C in tryptic soy broth or on tryptic soy agar plates containing the appropriate antibiotics. Y. enterocolitica bacteria were grown at 25°C in brain-heart infusion (Difco) or on tryptic soy agar plates containing the appropriate antibiotics. Y. enterocolitica E40 strains and derivatives have been described previously (24,25). For infections, bacteria were diluted to an optical density of 0.1 in fresh brain-heart infusion medium and incubated at 25°C 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 RPMI 1640 medium.
Culture, Infection, and Transfection of Cells-The murine macrophage cell line Mf4/4 (27) and the human embryonic kidney cell line HEK293T were cultured at 37°C in RPMI 1640 or Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin sulfate, 1 mM sodium pyruvate, and 2 ϫ 10 Ϫ5 M ␤-mercaptoethanol. Prior to infection, Mf4/4 cells were seeded at 10 6 cells/6-well plates in medium without antibiotics. After 15 h, cells were infected at a multiplicity of infection 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 g/ml). HEK293T cells were plated in 6-well plates at 2 ϫ 10 5 cells/well and transiently transfected by calcium phosphate-DNA coprecipitation. 24 h after transfection, medium was removed and cells were lysed in 300 l of lysis buffer (50 mM Hepes, pH 7.6, 200 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA). Proteins were separated by SDS-PAGE and analyzed by Western blotting with rabbit polyclonal anti-caspase-1 and anti-IL-1␤ antibodies (R&D systems), respectively, with mouse monoclonal anti-FLAG-horseradish peroxidase (Sigma) or anti-E-tag-horseradish peroxidase antibodies (Amersham Biosciences). Immunoreactivity was revealed with the enhanced chemiluminescence method (NEN TM Renaissance, PerkinElmer Life Sciences Products). Lactate dehydrogenase release was assayed using Cytotox-one homogeneous membrane integrity assay as described by the manufacturer's protocol (Promega). ␤-Galactosidase release was assayed using the Galactostar reporter gene assay system (Applied Biosystems).

RESULTS
YopE Inhibits the Release of IL-1␤ in Y. enterocolitica infected macrophages-Yersinia has previously been shown to prevent NF-B activation in infected cells in a YopP-dependent manner (9). Therefore, it could be expected that the expression of NF-B-dependent genes would be increased strongly in cells infected with a YopP-deficient strain (YopP Ϫ ) compared with cells infected with WT Yersinia. To verify this theory, we compared the amount of IL-1␤ and IL-6 in the supernatant of Mf4/4 macrophages that were infected with either YopP Ϫ or WT Y. enterocolitica. To our surprise, only the levels of IL-6 but not those of IL-1␤ were increased in the supernatant of YopP Ϫinfected cells (Fig. 1A). Nevertheless, intracellular proIL-1␤ levels in the corresponding cell lysates were considerably higher in cells infected with the YopP Ϫ strain (Fig. 1A, inset). It should be mentioned that only the precursor form of IL-1␤ could be detected in these cell extracts. The above observations indicated a role for another Yop effector protein in the regulation of IL-1␤ 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 (Fig. 1B, ⌬HOPEMT), which indeed resulted in a large increase of IL-1␤ levels in the supernatant (Fig. 1B). All together, the above results demonstrate that, besides the YopP-mediated downregulation of proIL-1␤ production at the transcriptional level, other Yop effectors could specifically inhibit the maturation and release of IL-1␤. To analyze which Yop effector controls the release of IL-1␤, we constructed five double knock-out Y. enterocolitica strains, which are negative for YopP and for any one of the five other Yops: YopPE Ϫ , YopPT Ϫ , YopPO Ϫ , YopPH Ϫ , or YopPM Ϫ strains. After infection of macrophages with each of these double-deficient bacteria, we analyzed again the secretion of IL-1␤ in the supernatant. IL-1␤ release was still strongly inhibited in macrophages infected with the double mutant bacteria YopPT Ϫ , YopPO Ϫ , YopPH Ϫ , and YopPM Ϫ , although a small but reproducible increase in IL-1␤ could be detected in YopPT Ϫ -infected cells (Fig. 1B). However, cells infected with the YopPE Ϫ strain released significant higher levels of IL-1␤, suggesting that YopE is at least partially responsible for the inhibition of IL-1␤ release as seen upon infection with WT or YopP-deficient Yersinia. Because IL-1␤ levels released by YopPE Ϫ -and YopPT Ϫ -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 Ϫ mutant would release comparable amounts of IL-1␤ as those infected with the ⌬HOPEMT mutant. However, we were unable to see a reproducible and significant difference between cells infected with YopPET Ϫ or YopPE Ϫ mutants (data not shown). YopE is a GAP for Rho GTPases, particularly Rac1 (28), switching them off by accelerating GTP hydrolysis. To analyze whether the GAP activity of YopE is required for inhibition of IL-1␤ release, we complemented the YopPE Ϫ strain with wild-type YopE (YopE WT ) or with a mutant of YopE (YopE M ) that lacks the GAP activity (Fig. 1C). Complementation of the YopPE Ϫ strain with YopE WT , but not with YopE M , restored the potential of Yersinia to prevent the release of IL-1␤ in the medium of infected macrophages. In contrast, intracellular expression levels of proIL-1␤ were independent of YopE (Fig. 1C). A previous report (29) shows 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, 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. In agreement with the latter observation, YopE deficiency also resulted in a significant increase of lactate dehydrogenase and proIL-1␤ release in our experiments (Fig. 1, C and D, respectively), which coincided with an apoptotic morphology of the cells (data not shown). However, also a clear band corresponding to mature active IL-1␤ could be seen in the supernatant of YopPE Ϫ -infected cells, implicating that YopE affects the release as well as the proteolytic maturation of IL-1␤. This role of YopE 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 bioactive IL-1␤ (data not shown) (30). In conclusion, our results indicate that the GAP activity of YopE is responsible for the inhibition of the maturation and secretion of IL-1␤ in Yersinia infected macrophages. Moreover, this also implies a role for Rho GTPases in the process of proIL-1␤ maturation and IL-1␤ release.

FIG. 1. YopE inhibits the release of IL-1␤ in Y. enterocolitica infected macrophages.
A, Mf4/4 macrophages were infected with WT or YopP-deficient (YopP Ϫ ) 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 g/ml) was added to kill extracellular bacteria. 4 h later, cell supernatants were collected and cytosolic cell lysates were prepared. IL-1␤ or IL-6 release in the supernatant was analyzed by enzyme-linked immunosorbent assay specific for IL-1␤ (Quantikine, R&D Systems) or IL-6 (Biotrak, Amersham Biosciences). 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-1␤. B, Mf4/4 cells were infected with WT or Y. enterocolitica strains deficient for one or more Yop effector proteins as indicated and analyzed for IL-1␤ release as described in A. C, Mf4/4 cells were infected with YopPE Ϫ or YopPE Ϫ strains that were complemented with wild type (YopE WT ) or mutant YopE (YopE M ) as indicated and analyzed for IL-1␤ release as described in A. The lower part of panel C shows the intracellular expression levels of proIL-1␤ as revealed by Western blot analysis of the corresponding cell lysates and the relative lactate dehydrogenase activity released into the medium of infected cells compared with non-infected cells as revealed by the Cytotox-one homogeneous membrane integrity assay (Promega). D, Mf4/4 cells were infected as described in C, and trichloroacetic acid-precipitated proteins in the medium were analyzed by Western blot analysis for the presence of proIL-1␤ and mature IL-1␤. Asterisk indicates a nonspecific band.

Specific Yop Effector Proteins and Rho GTPases Regulate
Procaspase-1 Activation-Because proIL-1␤ maturation is mediated by caspase-1, we next analyzed the effect of YopE on caspase-1-mediated proIL-1␤ maturation in HEK293T cells that were transiently transfected with procaspase-1 and proIL-1␤. In these conditions, procaspase-1 is partially converted to an active p20/p10 form because of its autocatalytic processing upon overexpression, resulting in the maturation of the 33-kDa proIL-1␤ to the bioactive 17-kDa form ( Fig. 2A). Moreover, caspase-1 autoactivation also results in the induction of cell death, which can be assayed by the release of cotransfected ␤-galactosidase into the medium (31,32). Additional coexpression of YopE WT , but not of the catalytically inactive mutant YopE M , inhibited the autocatalytic processing of procaspase-1 as well as the proteolytic maturation of proIL-1␤. Furthermore, ␤-galactosidase release into the medium also 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-1␤ maturation and caspase-1-mediated cell death. Interestingly, coexpression of YopT had a similar inhibitory effect as YopE on the proteolytic activation of procaspase-1 and proIL-1␤ ( Fig. 2A). YopT functions as a cysteine protease that cleaves off of the prenylated C termini of Rho, Rac, and Cdc42, leading to their release from the plasma membrane and their irreversible inactivation (5). A mutant of YopT (YopT M ) that had lost its proteolytic activity on Rho GTPases was no longer able to prevent the processing of procaspase-1 and proIL-1␤. In contrast to YopE and YopT, coexpression of the effector protein YopH, which is a tyrosine phosphatase that has been shown to dephosphorylate proteins from focal adhesions and other signaling complexes (33)(34)(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-1␤ maturation is somewhat contradictory to the small Cell lysates were subjected to SDS-PAGE and transferred to Hybond nitrocellulose membranes. The blot was probed with polyclonal antibodies against caspase-1 and reprobed with anti-IL-1␤. p20 and p22 represent specific processing products of caspase-1. The expression of YopE, YopT, and YopH was confirmed using the monoclonal anti-E-tag-HRP antibody. Cell death was measured 24 h after transfection by the release of ␤-galactosidase into the medium using the Galactostar reporter gene assay system, and values were expressed as the percentage of the amount that was released into the medium of cells that were only transfected with procaspase-1 and proIL-1␤. B-E, HEK293T cells were transiently transfected with the indicated amounts of expression vectors for the CA mutants RhoA Q63L (RhoA CA ), Rac1 G12V (Rac1 CA ), or Cdc42 Q61L (Cdc42 CA ) or an expression vector for the DN mutant Rac1 T17N (Rac1 DN ) together with expression plasmids encoding procaspase-1 (100 ng), proIL-1␤ (200 ng), and ␤ -galactosidase (100 ng). Secretion of mature bioactive IL-1␤ (B-D) was assayed 24 h after transfection in a CTLL cell proliferation assay (30). Values are expressed in pg/ml and corrected for transfection efficiency as reflected by ␤-galactosidase activity measured in the corresponding cell lysates. Mean Ϯ S.D. (n ϭ 3) was Ͻ10%. 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 and 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-1␤ (200 ng). Medium was replaced 4 h after transfection with medium containing geranylgeranyl transferase inhibitor 2147 (10 M, 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-1␤ (lower panel).
increase in IL-1␤ release in cells infected with a YopPT Ϫ double knock-out as described in our previous experiments (Fig. 1B). Most probably, 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 inactivate RhoA, Rac1, and Cdc42 in vitro or upon overexpression (5, 28), when they are injected into cells by Yersinia, they are remarkably specific, inactivating selectively Rac1 and RhoA, respectively (28,37). Therefore, the more potent effect of YopE deletion on proIL-1␤ maturation in infected cells suggests a major role for Rac1. 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-1␤, ␤-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-1␤. Indeed, IL-1␤ levels were significantly increased in the supernatant of cells overexpressing RhoA CA , Cdc42 CA , or Rac1 CA . Serial dilution of the transfected plasmid DNA concentration clearly showed that Rac1 CA is much more efficient than RhoA or Cdc42 (Fig. 2B), which is in agreement with the more pronounced release of IL-1␤ in YopPE Ϫ -versus YopPT Ϫ -infected cells (Fig. 1B) and the described preferential effect of YopE on Rac1 (28). As expected, Rac1 CA also enhanced the autoproteolytic activation of procaspase-1 and the release of ␤-galactosidase (Fig. 2C), whereas RhoA CA and Cdc42 CA did not (data not shown). Reversibly, transfection of a dominantnegative mutant of Rac1 (Rac1 DN ) resulted in a decrease of caspase-1 autoactivation and, consequently, in a decrease of IL-1␤ and ␤-galactosidase release (Fig. 2, D and E). It should be mentioned that the ectopic expression levels of Rac1 that are needed to affect caspase-1 activation are extremely low under the detection limit in the case of Rac1 DN (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-1␤ Maturation-To further confirm the role of Rho GTPases in caspase-1 activation and IL-1␤ production, we analyzed 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 effectors. Similarly, we also tested the effect of the geranylgeranyl transferase inhibitor 2147, which prevents the prenylation and membrane localization of Rho GTPases (39). Western blot analysis revealed that treatment of procaspase-1-and proIL-1␤-expressing cells with Toxin B or geranylgeranyl transferase inhibitor 2147 significantly prevents the proteolytic autoactivation of caspase-1 and maturation of proIL-1␤ (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-1␤.

The Effect of Rac1 on Caspase-1 Activation Is Independent of Its Effect on the c-Jun N-terminal Kinase (JNK) Pathway but Is
Mediated by LIM Kinase-1-The functions of Rho GTPases first assigned to the regulation of the organization of the actin cytoskeleton have been extended to many other cellular processes including activation of the JNK (3,40). Moreover, Racinduced cytoskeleton reorganization and JNK activation are the result of independent Rac-induced signaling pathways (41,42). To dissect which signaling pathway is important in the Rac1-induced activation of caspase-1, we used specific point mutants of Rac1 CA that are defective in either JNK activation (Rac1 CA-Y40H ) or actin reorganization (Rac1 CA-F37L ) (41,42). Transfection of cells with Rac1 CA or Rac1 CA-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 Rac1 CA-F37L was unable to promote the activation of caspase-1 and the release of IL-1. Therefore, we can conclude that Rac1mediated signaling 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. LIMK1 is known to participate in Rac1-mediated actin cytoskeletal reorganization by phosphorylating cofilin (43). Interestingly, 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.
Rac1 Controls the Asc-mediated Activation and Oligomerization of Caspase-1-The molecular mechanism of caspase-1 activation is still largely unknown. The CARD-containing protein Asc has been shown to function as a caspase-1-activating adaptor protein by mediating the assembly of a caspase-1 signaling complex that promotes the activation of caspase-1 and the proteolytic maturation of proIL-1␤ (20,44). To analyze whether Rac1 and LIMK1 can modulate the Asc-mediated activation of caspase-1, we cotransfected cells with expression vectors for procaspase-1, Asc, proIL-1␤, and YopE, YopT, LIMK1 DN , or Rac1 DN . Western blot analysis of caspase-1 as well as analysis of the production of bioactive IL-1␤ shows that Asc-mediated caspase-1 activation can be inhibited by Rac1 DN and LIMK1 DN as well as by the Yop effector proteins YopE and YopT (Fig. 4A). To analyze whether constitutive-active Rac1 CA could still further promote the Asc-mediated caspase-1 activation, we coexpressed either Asc or Rac1 CA with procaspase-1 to a level that does not lead to a significant activation of caspase-1. However, coexpression of Rac1 CA 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 analyze 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 coimmunoprecipitation experiment whether or not 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 Fig. 5, a dominantnegative Rac mutant (Rac1 DN ) as well as YopE and YopT could prevent the oligomerization of procaspase-1. In contrast, coexpression of constitutive-active Rac1 (Rac1 CA ) could promote the formation of procaspase-1 oligomers. These experiments demonstrate that Rac1 modulates caspase-1 activation by affecting its oligomerization. DISCUSSION To survive and proliferate, pathogens acquired sophisticated mechanisms to interfere with the host response to infection. Yersinia sp. and many other pathogens such as Salmonella sp. and Pseudomonas sp. developed a "molecular syringe" called type III secretion system to inject their effectors into the host cell and as such interfere with several signaling cascades in-volved in inflammation and phagocytosis. The Yersinia effector YopP, which controls the activity of NF-B by blocking IB kinase B and mitogen-activated protein 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 to block phagocytosis. However, recent reports illustrated that some of the anti-phagocytic effectors such as YopH can also interfere with signaling pathways of the immune defense system, leading to a down-regulation of the F c -mediated oxidative burst in macrophages and neutrophils, for example (12,46,47). The caspase-1 inhibitory effect of YopE described in this study revealed a novel function of Yop effector proteins in regulating the inflammatory response against Yersinia infection. Thus, Yersinia not only interferes with the production of proIL-1␤ at the transcriptional level (by means of YopP) but also prevents the caspase-1-mediated maturation and secretion of already preformed IL-1␤. 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 (4,5,28,37). However, in Yersinia infected macrophages, YopE and YopT have been shown to specifically inhibit Rac1 and RhoA, respectively (28,37). The latter observation together with our own data showing that IL-1␤ release from YopPE Ϫ -infected macrophages is much higher compared with the release from YopPT Ϫ -infected cells point to a major role for Rac1 in caspase-1 activation and the proteolytic maturation of proIL-1␤ in Yersinia infected macrophages. Interestingly, IL-1␤ levels in the supernatant of cells infected with the ⌬HOPEMT Ϫ strain were reproducibly higher compared with those found upon infection with a YopPE Ϫ 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 Ϫ infection, one could expect also a contribution of YopT. We were unable to see a reproducible difference between cells infected with YopPET Ϫ or YopPE Ϫ mutants (data not shown). Most probably, efficient inhibition of IL-1␤ production can only be achieved by a cooperative 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 autoactivation, showing that Rac1 was by far the most potent GTPase to promote the secretion of IL-1␤ 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 signaling 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-1␤ production.
The mechanism of caspase activation has been an intriguing topic of research for the past 10 years. Surprisingly, just re-cently 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 nucleotide-binding oligomerization domain proteins, a growing family of cytosolic proteins that have been implicated recently in innate FIG. 4. Asc-mediated activation of caspase-1 is modulated by Rac1. A, HEK293T cells were transiently transfected with expression vectors (50 ng) encoding procaspase-1 and Asc as indicated together with empty vector (EV) (600 ng) or an expression plasmid encoding YopT or YopE or the dominant-negative mutants of LIMK1 DN or Rac1 DN . B, HEK293T cells were transiently transfected with an expression vector encoding procaspase-1 (50 ng) and Asc (20 ng) as indicated together with 20 ng of EV or an expression plasmid encoding Rac1 CA . 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 bioactive IL-1␤ was assayed in a CTLL cell proliferation assay. Values are expressed in pg/ml and corrected for transfection efficiency as reflected by ␤-galactosidase activity measured in cell lysates. Mean Ϯ S.D. (n ϭ 3) was Ͻ10%.

FIG. 5. Rac1 as well as YopE and
YopT regulate the oligomerization of procaspase-1. Coimmunoprecipitation assays were performed using lysates from HEK293T cells that were transiently transfected with plasmids (100 ng) encoding enzymatically inactive E-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, Rac1 DN , or Rac1 CA . 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. 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 (19,21,49). Asc (also known as TMS1 or PYCARD) is an important adaptor between procaspase-1 and several nucleotide-binding oligomerization domain family members, mediating caspase-1 oligomerization (19). Our observation that Rac1 could modulate the Asc-induced proteolytic activation of caspase-1 and corresponding proIL-1␤ 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 as being associated with filamentous or specklike 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 localization of Asc and the associated activation of caspase-1. Although Rac1 also couples to signaling 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 p65 PAK binding and thus also in JNK activation but still results in cytoskeletal reorganization (41,42,51) could still enhance the autoactivation of caspase-1. In addition, we could show an involvement of LIMK1 in Rac1induced 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 C 3 transferase) or glucosylation (C. difficile toxin B), and others activate them by deamidation (e.g. E. coli cytotoxic necrotizing factor 1) 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 (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 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 guanine nucleotide exchange factor for Rho GTPases (55), our observations suggest that apart from SipB, SopE also may play a role in the activation of caspase-1 during Salmonella infection.
Once again, the study of host-pathogen interactions revealed the eukaryotic cell processes not understood before. In this 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-1␤. As for its anti-phagocytic defense, Yersinia seems to inhibit the production of proinflammatory cytokines by a complex interplay among several Yop effectors that act at multiple levels. Intriguingly, modulation of caspase-1mediated inflammation might also occur during infection with several other pathogens such as Clostridium sp., Salmonella sp., and Pseudomonas sp., which encode proteins that are also known to target specific Rho GTPases. Therefore, our findings are not only relevant for the development of tools that target caspase-1 in several inflammatory diseases but may also give new insights into drug design for treating infectious diseases.