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Infection and Immunity, January 2009, p. 348-359, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.01005-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity {triangledown}

Aimee M. deCathelineau{dagger} and Gary M. Bokoch*

Department of Immunology and Microbial Science and Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Received 11 August 2008/ Returned for modification 18 September 2008/ Accepted 10 October 2008


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ABSTRACT
 
Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.


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INTRODUCTION
 
Bacillus anthracis endospores enter the body through inhalation, ingestion, or cutaneous abrasions. Of the three routes of infection, inhalation results in the most severe form of the disease, which is typically asymptomatic prior to rapid onset of hypotension and shocklike symptoms, followed by death due to respiratory failure (20). The morbidity and mortality associated with anthrax infection is largely attributed to the actions of the anthrax exotoxins (20, 70). Similar to toxins produced by other successful bacterial pathogens, anthrax toxins target and inhibit signal transduction pathways that regulate the immune response, inflammation, and cell survival (3, 17, 28, 58, 67, 74), allowing for growth of the bacteria. Systemic anthrax is often fatal in the absence of early clinical intervention due to the combinatorial effects of the B. anthracis exotoxins and massive bacteremia. Therefore, understanding anthrax toxin biological activity is critical for successful treatment of the disease (73).

Anthrax toxins belong to the type A/B family of large clostridial cytotoxins, which share structural homology despite widely divergent biological activities (6, 15, 37). Type A/B toxins consist of an enzymatic "A" subunit and a "B" subunit which mediates the binding and translocation of the A subunit into the host cell. Anthrax toxin is unique in that it consists of two separate enzymatic toxin subunits: lethal factor (LF), a zinc-dependent metalloprotease (14, 21, 37, 66, 84), and edema factor (EF), a calcium- and calmodulin-dependent adenylate cyclase (48), which both utilize a common binding subunit, protective antigen (PA) (69). The combination of either LF or EF with PA results in the formation of lethal toxin (LT) and edema toxin (ET), respectively (for comprehensive reviews of anthrax toxin structure and function, see references 15 and 59).

Two cell surface receptors capable of binding PA, ATR1/TEM8, and CMG-2 have been identified (12, 76, 77). After binding to host cells, proteolytic cleavage of full-length PA (PA83) by furin-family proteases results in the production of a 63-kDa PA isoform (PA63) (7, 42). Oligomerization of PA63 results in competency for LF and EF binding, redistribution of the holoenzyme and receptor into cholesterol-enriched lipid raft domains, and internalization via clathrin-dependent endocytosis (2). Normal acidification of the endosomal compartment drives a pH-dependent conformational change in the PA heptamer, resulting in membrane insertion and pore formation, and allowing for translocation of EF and LF into the host cell cytoplasm (1, 27, 46).

The only known cytoplasmic targets of the LF metalloprotease are mitogen-activated protein kinase (MAPK) kinases, also known as the MAPK/ERK kinases (MKKs or MEKs, respectively) 1, 2, 3, 4, 6, and 7 (21, 68, 84, 85). MKKs in turn phosphorylate the MAPK family members p38, ERK1 and ERK2 (p42/44), and JNK/SAPK, which are essential for the regulation of the immune response and cell survival. LF cleaves MKKs at their NH2 termini, resulting in removal of the MAPK-binding "D" domain and preventing activation of appropriate downstream MAPK family members (82, 86). LT induces cytolysis and apoptosis in some specific cell types, such as primary human (sensitized) macrophages and endothelial cells, as well as in the RAW 264.7 and J774 murine macrophagelike cell lines (27, 40, 53, 67, 72). However, a clear causative connection between MKK cleavage and cell death has not been established.

The Rho family of low-molecular-weight GTPases regulate a variety of critical cellular functions, including cytoskeletal dynamics, cell adhesion, and migration, and the production of reactive oxygen species (9, 36, 75). Rho GTPases are also known to play important roles in the internalization of particles and fluids from the extracellular space through phagocytosis, macropinocytosis, and endocytosis (24, 47). We hypothesized that Rho GTPases might be involved in anthrax toxin internalization, trafficking, or activity. Statins, drugs which disrupt Rho GTPase posttranslational processing by inhibiting the synthesis of the isoprenoid precursor mevalonic acid (32, 52), are effective in inhibiting Rho GTPase-dependent biological responses (32, 52). Interestingly, statins have been shown to alter signal transduction by members of the MAPK family, the primary molecular target of LF activity (13, 60). We therefore examined the effect of statin treatment on cellular intoxication by LT and investigated the role(s) of Rho GTPase inhibition in this process.


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MATERIALS AND METHODS
 
Cell culture and reagents. RAW 264.7 macrophages and Cos7 cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco modified Eagle medium containing 10% heat-inactivated calf serum supplemented with L-glutamine, penicillin, and streptomycin at 37°C and 10% (RAW 264.7) or 5% (Cos7) CO2.

Anthrax toxins were a generous gift from S. H. Leppla (National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) or were purchased from BEI Resources (Manassas, VA). Catalytically inactive LF (E687C) was also a generous gift from N. S. Duesbery (Van Andel Research Institute, Grand Rapids, MI). Bafilomycin A1 (Sigma) was added to cells 30 min prior to anthrax toxin treatment and remained present during assays. Clostridium difficile toxin B (50 ng/ml; List Biologicals) was added to cells for 30 min and was removed prior to anthrax toxin treatment. To assess toxin B activity, cell lysates were analyzed by Western blotting with monoclonal antibody 102 (BD Pharmaceuticals), which only detects nonglucosylated Rac1. Total Rac1 was detected with monoclonal antibody 24E8 (Upstate Biologicals). C3 transferase was purchased from Cytoskeleton, Inc., and was incubated with cells for 4 h at 1.0 µg/ml prior to LT treatment. Lovastatin and mevalonolactone were purchased from Sigma. Mevastatin, simvastatin, fluvastatin, GGTI-286, and FTI-277 were purchased from Calbiochem. Statins, prenyl-transferase inhibitors, and mevalonolactone were added to culture media for 16 to 20 h prior to and were removed during LT treatment (statins remained present during the extended time course experiment of Fig. 1B).


Figure 1
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  		FIG. 1. Statins attenuate LT action. (A) Schematic diagram of the cholesterol biosynthetic pathway. Statins inhibit cholesterol and isoprenoid synthesis by blocking the activity of HMG-CoA reductase and production of mevalonolactone. (B) Statins protect RAW cells from LT-mediated death up to 9 h. RAW cells were pretreated with mevastatin, simvastatin, or fluvastatin prior to treatment with anthrax LT (500 ng of PA/ml, 100 ng of LF/ml), and viability was measured after 3, 6, or 9 h by using an MTT assay. Survival of RAW cells, even at 9 h, is significantly increased compared to untreated cells. The data are mean percent cell viabilities compared to untreated controls (± the standard deviation [SD]) from four independent experiments (**, P < 0.01; *, P < 0.05). (C) RAW cells were pretreated with mevastatin (Mev) in the presence or absence of mevalonolactone (Mvl) supplementation for 18 h prior to LT treatment (500 ng of PA/ml, 100 ng of LF/ml). Neither mevalonolactone supplementation nor ethanol vehicle (Veh) alone had an effect on cell viability in response to LT. However, mevastatin decreased LT-mediated cell death, an effect which was reversed by the addition of mevalonolactone. The data are the mean percent cell viabilities compared to untreated controls (± the SD) from three independent experiments (*, P < 0.05 versus untreated control). (D) Mevastatin treatment also delayed MEK2 cleavage in LT-treated RAW cells (500 ng of PA/ml, 100 ng of LF/ml) at 2 h, and mevastatin-mediated attenuation of MEK2 cleavage was also reversed by the addition of mevalonolactone. Levels of the cytosolic protein RhoGDI are shown as a loading control. The data are representative of three independent experiments. (E) Treatment of RAW cells with the geranylgeranyltransferase inhibitor GGTI-286 (GGTI), but not the farnesyltransferase inhibitor FTI-277 (FTI), mimics the effect of statins on LT-mediated death. The data are mean percent cell viabilities compared to untreated controls (± the SD) from three independent experiments (*, P < 0.05 versus untreated control). (F) GGTI-286 (GGTI), like the mevastatin (Mev), also prevented MEK2 cleavage in response to LT treatment (500 ng of PA/ml, 100 ng of LF/ml) at 2 h, whereas FTI-277 (FTI) did not, as with the untreated (Unt) or vehicle (not shown) controls. The data are representative of two independent experiments. (G) The effect of mevastatin on Rho GTPase activity was assessed by using GST-RBD pulldown assays for GTP-loaded RhoA. Although untreated cells had low levels of active RhoA, mevastatin treatment (Mev) significantly increased GTP loading of RhoA, an effect that was reversed with supplementation of mevalonolactone (Mvl) to the culture medium during mevastatin treatment. The data shown are representative of two independent experiments.

GTPase activity assays. The levels of GTP-bound (active) Rac1 and RhoA were assessed by their ability to bind to their respective effector domains in affinity assays—either the p21 binding domain of PAK1 (PBD) or the RhoA binding domain of Rhotekin (RBD), as described previously (8, 79). A 1/20 proportion of input lysate was immunoblotted as a measure of total Rac1 or RhoA levels. Parallel gels were immunoblotted with antibodies specific for the NH2-terminal of MEK2 to assess LT activity; Western blots for RhoGDI were used to assess equal loading. The levels of active GTPase, determined by densitometry, were normalized to the total GTPase levels, and the fold activation was expressed as a ratio to similarly normalized samples from unstimulated controls.

MTT viability assays. RAW 264.7 macrophages plated in 96-well microtiter dishes were assayed for viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 2 h of LT incubation, the tetrazolium salt MTT (Sigma) was added to a final concentration of 0.2 mg/ml, and the cells were cultured for an additional 1 h and then washed with phosphate-buffered saline (PBS) and dissolved in dimethyl sulfoxide. The absorbance of the dissolved formazan precipitate was measured on a microplate reader at 560 nm. The viability was calculated based on averages of triplicate measurements for each sample and is presented as a percentage of the untreated controls.

Detection of SDS-resistant oligomeric PA. RAW 264.7 cells were cultured in six-well plates and were either untreated or pretreated with 30 nM bafilomycin A1 or toxin B at 100 ng/ml for 30 min. Cells were subsequently incubated with LT (1,000 of PA ng/ml, 250 ng of LF/ml) for the indicated time periods. Cells were washed three times with ice-cold PBS, and cell lysates were prepared in PBS-1% NP-40 supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM aprotinin, and 10 mM Na3VO4. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride for Western blot analysis. Rabbit anti-PA polyclonal antibody (7349.3) was made in our laboratory by immunization with purified PA.

MKK cleavage assays. At the designated time point after the addition of LT, cells were placed on ice and washed twice with 2 ml of ice-cold (4°C) PBS/well and then lysed in PBS-1% NP-40 supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM aprotinin, and 10 mM Na3VO4. Next, 15 µg of lysate was resolved by SDS-PAGE and immunoblotted for the NH2 terminus of MEK2 (sc-524; Santa Cruz), MKK3 (sc-960; Santa Cruz), MKK4 (sc-964; Santa Cruz), or RhoGDI (as a loading control). Several exposures were acquired, and densitometric analysis was done within an appropriate linear range by using a Bio-Rad GS-800 calibrated densitometer and Quantity One 1-D analysis software (Bio-Rad).

Cos7 cells were transfected with Lipofectamine Plus (Invitrogen) for 12 h according to the manufacturer's instructions, and LT treatment (250 ng of LF/ml, 1,000 ng of PA/ml) was carried out for 2 h with dominant-negative Rho GTPase mutants, for 90 min (data not shown) and 2 h with constitutively active Rho GTPase mutants, or for 2.5 h and 5 h with MEK1 wild-type or mutant constructs.

For in vitro MKK cleavage assay, RAW cells were treated with toxin B as described and then lysed in PBS-1% NP-40 in the presence of protease inhibitors, and the lysates were cleared by centrifugation. LF was added to lysate supernatants at 1.0 µg/ml for 15 min 37°C, and the reaction was stopped by boiling in SDS sample buffer. LF activity was assessed by Western blotting for the NH2 terminus of MEK2 as previously described.


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RESULTS
 
Statins attenuate LF action. Certain murine-derived macrophagelike cell lines, such as RAW 264.7 and J774.1, undergo rapid cell death in response to LT (27, 34, 53, 72). We used this observation to examine the effect of statins on LT activity. A class of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, commonly referred to as statins, are widely used for therapeutic treatment of hypercholesterolemia. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonic acid, a rate-limiting intermediate in the cholesterol biosynthetic pathway and the immediate precursor of the isoprenoids (32, 52) (Fig. 1A).

RAW macrophages were treated with either mevastatin or the synthetic statin derivatives simvastatin or fluvastatin for 12 h prior to the addition of LT, and cell viability was determined after 3, 6, or 9 h of LT treatment (Fig. 1B). Statins did not adversely affect cell viability in untreated cells when present for less than 24 h (data not shown). However, statin treatment maintained RAW cell viability above 60% of untreated control cells even after 9 h of LT treatment, suggesting that statins may be effective inhibitors of LT action. The observed effects of mevastatin treatment could be ameliorated by supplementation of the media with mevalonolactone, the rate-limiting biosynthetic pathway intermediate downstream of HMG-CoA reductase (Fig. 1C). Neither mevalonolactone alone nor its vehicle control (ethanol) had an effect on LT-mediated cell death.

Cytolysis of RAW cells in response to LT is dependent on LF activity, since metalloproteinase inhibitors prevent death (42), as do mutations in the LF zinc binding domain (H686A, H690A) or in the catalytic domain (E687C) (21, 35, 41). However, no direct molecular link between LF metalloprotease activity and cell death has been demonstrated. In order to determine whether statins also affected MKK cleavage by LF, RAW macrophages were pretreated with mevastatin and subsequently treated with LT, and samples were analyzed for MEK2 cleavage. Similar to the attenuation of LT-induced cell death, mevastatin also decreased LF-mediated MEK2 cleavage, and addition of mevalonolactone to mevastatin-treated cells ablated mevastatin-mediated rescue of MEK2 cleavage (Fig. 1D).

Supplementation of mevastatin-treated cells with mevalonolactone could potentially restore synthesis of both prenylation intermediates and cholesterol (see Fig. 1A). However, statin treatment under the conditions of our experiments, i.e., short durations and in the presence of cholesterol in the medium (due to serum), does not alter cellular cholesterol levels (43). Statins have been additionally shown to have potent anti-inflammatory effects, attributed to the inhibition of geranylgeranyl pyrophosphate (GGpp) synthesis and the resulting inhibition of Rho GTPase function (51, 52). In order to determine whether statin-mediated inhibition of LT activity could take place through specific effects on the isoprenylation of small GTPases, we examined the effects of CAAX-peptidomimetic direct inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277) on LT activity. Pretreatment of RAW macrophages with GGTI-286, which blocks geranylgeranylation of Rho GTPases (50), protected RAW cells from LT-induced death and decreased MEK2 cleavage (Fig. 1E and F). However, FTI-277, which does not inhibit Rho GTPase geranylgeranylation but does inhibit farnesylation of Ras subfamily GTPases (49, 80) did not attenuate LF activity.

The posttranslational addition of isoprenyl groups, either Fpp (Ras subfamily) or GGpp (Rho/Rab subfamilies), to their C termini mediates the ability of cytosolic GTPases to interact with membranes and effector proteins (81). Isoprenylation is also important for GTPase binding to GDP dissociation inhibitors (GDIs), regulatory proteins that sequester inactive (GDP bound) GTPases in the cytosol and prevent activation by exchange factors (19). Inhibition of GTPase isoprenylation by statin treatment does not inhibit Rho GTPase activation per se and in fact often results in an increase in GTP-loading, apparently due to the release from GDI inhibition (16). However, functional GTPase inhibition is conferred by inappropriate subcellular localization due to the lack of isoprenylation (10, 16, 87). In order to determine whether statin treatment was effectively modifying Rho GTPase activity under our experimental conditions, lysates from RAW macrophages pretreated for 18 to 24 h with mevastatin were subjected to affinity-based GTPase activity assays. An increase in GTP-bound (active) Rac1 (not shown) and RhoA was evident in mevastatin-treated cells (Fig. 1G). The addition of mevalonolactone had no effect on GTP loading on its own but reversed GTP loading of RhoA in the presence of mevastatin. We also observed that membrane localization of GTPases was perturbed after statin treatment (data not shown). We conclude that mevastatin was effective in altering normal regulation of RhoA GTPase activation and that this was likely to be the direct result of statin-mediated inhibition of isoprenylation.

Inactivation of Rho GTPases with toxin B inhibits LT activity. In order to examine whether Rho GTPases contribute to the observed inhibitory effects on anthrax LT, we used C. difficile toxin B. Toxin B specifically inactivates Rho GTPases, but not other members of the low-molecular-weight GTPase superfamily such as Rab, Ras, or Rap family members, by monoglucosylation of a threonine residue in the nucleotide binding domain (Thr35 for Cdc42 and Rac1, Thr37 for RhoA) (37, 38).

RAW 264.7 cells were treated with toxin B prior to the addition of anthrax LT, and cell viability was again assessed by using an MTT assay (Fig. 2A). Toxin B did not adversely affect cell viability at the concentrations or duration of exposure used in these experiments (data not shown). However, toxin B pretreatment significantly inhibited LT-mediated cell death compared to untreated controls, supporting the idea that the inhibition of Rho GTPase function contributes to statin-mediated attenuation of LT uptake or function. Toxin B also consistently decreased LF-mediated MEK2 cleavage (Fig. 2B). The effects of toxin B were not limited to MEK2, since the cleavage of MKK3 and MKK4 was also consistently reduced in toxin B-treated cells. A monoclonal antibody that only recognizes the nonglucosylated form of Rac1 (30) indicated that the toxin B dose (50 ng/ml) and incubation time (30 min) used in these experiments was sufficient to fully glucosylate and inactivate Rac1 (Fig. 2C). The biological efficacy of toxin B was also verified by its ability to prevent cell spreading and ruffling in PMA-treated cells (data not shown).


Figure 2
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  		FIG. 2. Inhibition of Rho GTPases with C. difficile Toxin B mimics statin activity and inhibits anthrax LT activity. (A) RAW cells were pretreated with toxin B prior to treatment with LT (500 ng of PA/ml, 100 ng of LF/ml), and cell viability was assessed by MTT assay after 3 h of treatment. Rho GTPase inactivation by toxin B increased the viability of LT-treated cells compared to cells treated with LT alone. The data are mean percent cell viabilities compared to untreated controls (± the SD) from four independent experiments. (*, P < 0.05; **, P < 0.01.) (B) RAW macrophages were pretreated with toxin B prior to incubation with LT (500 ng of PA/ml, 100 ng of LF/ml) for various amounts of time. Samples were analyzed for MKK cleavage by immunoblot for the NH2 terminus of MEK2, MKK3, or MKK4. Toxin B pretreatment attenuated LT-mediated MEK2 cleavage. Similar results were also observed for cleavage of MKK3 and MKK4. Levels of RhoGDI are shown as a loading control. The data shown are representative of two (MKK3 and MKK4) or three (MEK2) independent experiments. Densitometry was used to quantify levels of intact MEK2. MEK2 levels were normalized to levels of RhoGDI and are expressed as a percentage of untreated controls. There was a significant difference in MEK2 cleavage at intermediate (e.g., 90 min) time points. (C) Activity of toxin B under conditions used for these experiments was confirmed by using Western blot analysis. Total Rac1 levels are equal in both untreated and toxin B-treated cells (top panel). However, nonglucosylated Rac1 is not detectable in toxin B-treated (50 ng/ml; 30 min) cells (bottom panel). The data shown is representative of three independent experiments. (D) Inhibition of RhoA alone did not affect LT-mediated death. RAW cells were pretreated with a cell-permeable C3T (1.0 µg/ml) for 4 h prior to treatment with LT (500 ng of PA/ml, 100 ng of LF/ml), and cell viability was measured by MTT assay at 3 h after LT treatment. The data are mean percent cell viabilities compared to untreated controls (± the SD) from three independent experiments. (E) RAW cells were treated with C3T, and MEK2 cleavage was assayed by Western blotting after 2 h treatment with LT (500 ng of PA/ml; 100 ng of LF/ml). RhoGDI levels are shown as a loading control. Inhibition of RhoA alone with C3T did not inhibit LT-mediated MEK2 cleavage. The data shown are representative of two independent experiments.

In order to further distinguish which Rho GTPase family member may be required for LT activity, we tested the effect of a cell-permeable version of the Clostridium botulinum C3 transferase (C3T) in RAW macrophages. C3T specifically inactivates only RhoA-C through ADP-ribosylation of an asparagine residue (N41) within the active site (4). In contrast to toxin B, C3T had no effect on cell viability or MEK2 cleavage in the presence of LT (Fig. 2D and E, respectively). To confirm that C3T was active under the conditions used in these experiments, we used the glutathione S-transferase-rhotekin (Rho) binding domain (GST-RBD) pulldown assay for RhoA activation to show that treatment of RAW cells with C3T for as little as 2 h was effective at inhibiting RhoA activation (data not shown).

Finally, we investigated whether LT activity could be attenuated using the dominant-negative Rho GTPase mutants, Rac1(T17N), Cdc42(T17N), and RhoA(T17N). This experiment was performed using a Cos7 cell system where the high transfection efficiencies required for these GTPase mutants to be effective could be readily attained. Somewhat surprisingly, we observed that each dominant-negative Rho GTPase mutant attenuated MEK2 cleavage by LT in Cos7 cells (Fig. 3A and B). The observed inhibition by the dominant-negative RhoA contrasted with the lack of effect of C3T we observed (see above). It is possible that the dominant-negative RhoA is competitively inhibiting an exchange factor that is also active on Rac1 and/or Cdc42 along with RhoA. In contrast, the C3 exoenzyme would be completely specific for Rho inhibition. Expression of constitutively activated Rac1(Q61L), Cdc42(Q61L), or RhoA(Q63L) did not significantly alter MEK2 cleavage in response to LT under these conditions (Fig. 3C and D).


Figure 3
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  		FIG. 3. Expression of dominant-negative mutants of Rho family GTPases inhibits LT-mediated MEK cleavage. (A) Cos7 cells were either untransfected (Unt) or transfected in the absence of DNA (mock), with an empty vector control (EVC) or with dominant-negative constructs of Rac (N17), RhoA (N19), or Cdc42 (N17). Cells were treated with LT (250 ng of LF/ml and 1,000 ng of PA/ml) for 2 h, and MEK2 cleavage was assessed by Western blotting (top panel). RhoGDI levels are shown for comparison of sample loading (center panel), and expression of the EGFP-epitope tagged constructs is demonstrated (bottom panel). A representative example of four independent experiments is shown. (B) Intact MEK2 levels from four independent experiments were quantified by densitometry, normalized to RhoGDI levels, and are expressed as a percentage of untreated controls, ± the SD. While transfection alone or with an empty vector control did not affect MEK2 cleavage, expression of N17Rac1, N19RhoA, or N17Cdc42 resulted in a significant increase (P < 0.005) in MEK2 levels in LT-treated cells. (C) Cells transfected with constitutively active mutant constructs of Rac (Q63L), RhoA (Q61L), or Cdc42 (Q63L) were treated with LT (250 ng of LF/ml and 1,000 ng of PA/ml) for 2 h. MEK2 cleavage was assessed by Western blotting. Equal loading of total MEK2 is demonstrated by immunoblotting with an antibody specific for the COOH-terminal of MEK2 and expression of the EGFP-epitope tagged constructs is demonstrated (bottom panel). A representative of three independent experiments is shown. (D) Intact MEK2 levels from three independent experiments were quantified by densitometry, normalized to RhoGDI levels (data not shown), and are expressed as a percentage of untreated controls ± the SD.

LT activates Rho GTPases. Based on the preceding results, we next investigated whether Rho GTPases were activated in response to cellular intoxication with LT. Moderately increased RhoA activity was observed in RAW 264.7 cells within 5 min of LT addition and decreased to basal levels over the next 2 h (Fig. 4A). In contrast, we were not able to consistently detect activated Rac1 in the RAW cells, perhaps due to relatively high levels of endogenous RhoA activity. We therefore examined the effects of LT on Rac1 activity in the Cos7 cell system instead. A low, but detectable level of active Rac1 was present in unstimulated, serum-starved Cos7 cells that increased substantially within 15 min of toxin addition and remained elevated up to 90 min (Fig. 4B). Rac1 activity returned to basal levels by the 2-h time point, coincident with the time at which MEK2 cleavage becomes evident in this cell type. Rac1 activation, normalized to total Rac1, ranged from 1.5- to 3.8-fold at the 30-min time point in three independent experiments.


Figure 4
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  		FIG. 4. LT activates Rac and RhoA. (A) RAW cells were treated with LT (500 ng of PA/ml; 100 ng of LF/ml) and analyzed for GTP-bound (active) RhoA by using a GST-RBD pulldown assay. Lower panels show parallel immunoblots for total RhoA (1/20 of input lysates), the NH2 terminus of MEK2 as a measure of LT activity (as in Fig. 1D), and RhoGDI as a loading control for the MEK2 blots. The data shown are representative of three independent experiments. RhoA pulldown assays were quantified by densitometry and normalized using total RhoA from matched samples as loading controls. Activity is expressed as a fold increase relative to unstimulated controls. RhoA activity moderately increased up to 1.5-fold is detectable as early as 5 min after LT stimulation and remained increased up to the 90-min time point. Activation of PA alone or LTE687C was assayed at the 30-min time point. The data represent an average of three independent experiments ± the SD. (B) Activation of Rac in Cos7 cells treated with LT (1,000 ng of PA/ml, 250 ng of LF/ml) and assessed for GTP-bound Rac1 by using a GST-PBD pulldown assay. Lower panels show parallel immunoblots for total Rac1 (1/20 of input lysates), the NH2 terminus of MEK2 to demonstrate LT activity (loaded for equal protein concentration), and RhoGDI as a loading control for the MEK2 immunoblots. The data shown are representative of three independent experiments. Rac1 pulldown assays were quantified by densitometry and normalized using total Rac1 from matched samples as loading controls. Activity is expressed as a fold increase relative to unstimulated controls. Increased (twofold) Rac activity is detectable by 15 min of LT stimulation and persists to the 90-min time point. The data represent an average of three independent experiments ± the SD. (C) Rac activation in response to PA alone or LTE687C was assayed at 30 min after stimulation in Cos7 cells. The data shown is representative of three independent experiments ± the SD.

Serum-starved Cos7 cells typically have a flattened morphology, with few actin stress fibers and a low level of phalloidin staining. In support of the observed Rac1 activation, when Cos7 cells were stimulated with LT they exhibited substantial membrane ruffling. Membrane ruffling was quantified by scoring individual cells from randomly acquired digitized images (obtained in three independent experiments) for the presence or absence of membrane ruffles. Approximately 44% of LT-treated cells (n = 250) compared to ~22% of LTE687C-treated (n = 267) or <18% of PA-treated (n = 277) cells or untreated cells (n = 277) were positive for membrane ruffling.

The observation that Rho GTPase activity is measurable as early as 5 to 15 min after LT stimulation in two cell types suggests that activation occurs in response to receptor ligation, rather than through catalytic action of the toxin. To test this hypothesis, cells were stimulated with LT, the catalytically inactive LTE687C, or PA alone for 30 min, and the GTPase activity was determined. Both RhoA and Rac1 (Fig. 4A and C, respectively) were activated in response to LT, with either the WT LF or catalytically inactive LFE687C subunit. While Rac1 activation was stimulated by PA alone in the Cos7 cells, RhoA was not activated in the RAW 264.7 cells in the absence of the LF catalytic subunit.

Rho GTPase inhibition does not disrupt endocytosis of LT. Since Rho GTPases have previously been shown to be involved in the regulation of clathrin-dependent endocytosis (47), we hypothesized that their inhibition might affect LT internalization and processing. LT binding was assessed in initial studies using biotinylated PA and flow cytometry. The biotinylated PA used in these experiments was found to be comparably functional to unlabeled PA for LT-induced cell death (data not shown). In addition, binding of biotinylated PA could be inhibited by preincubation of the cells with unlabeled PA (data not shown). Both untreated and cells in which Rho GTPases had been inactivated with toxin B exhibited similar levels of PA binding at 4°C (Fig. 5A). LT internalization, measured as a decrease in surface labeled PA when cells were warmed to 37°C, also appeared to be similar in toxin B-treated and untreated cells.


Figure 5
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  		FIG. 5. Toxin B does not affect PA binding or pore formation. (A) Binding and uptake of biotinylated PA by RAW macrophages in the absence (solid histograms) or presence (outlined histograms) of toxin B was assessed using phycoerythrin-streptavidin staining and flow cytometry. Toxin B pretreatment did not significantly alter the binding (4°C) or uptake (37°C, 1 h) of PA. The data are representative of three independent experiments. (B) RAW macrophages were untreated (lanes 0), treated with bafilomycin A1 (lanes B), or treated with toxin B (lanes T) prior to the addition of LT (100 ng of LF/ml, 1,000 ng of PA/ml) for 1 h. MEK2 cleavage occurs in untreated cells, but not in bafilomycin A1- or toxin B-treated cells (bottom panels; immunoblots for RhoGDI are shown as a loading control). Cell lysates were examined for the presence of the SDS-resistant form of PA (indicated as "Oligomeric PA" in the top panel), which is induced upon exposure to acidic pH. While bafilomycin A1 treatment prevented pH-induced conformational change in the PA heptameric pore as expected, the SDS-resistant form of the PA heptamer is clearly present in toxin B-treated cells, suggesting that inactivation of Rho GTPases does not alter normal endocytic trafficking or acidification, which are required for release of the anthrax toxin catalytic subunits into the cytoplasm. Levels of the full-length (PA83) and furin-cleaved (PA63) PA isoforms are shown for loading comparisons. The data shown are representative of four independent experiments. (C) MEK2 cleavage was assessed in an in vitro assay by adding LF (1,000 ng/ml) directly to cleared lysates from untreated (0), toxin B-treated (TxB), or mevastatin (Mev)-treated RAW cells, and samples were incubated at 4 or 37°C for 15 min. Inactivation of Rho GTPases did not directly inhibit MEK2 cleavage by LF. The data shown are representative of three independent experiments.

Once internalized, the heptameric PA pre-pore undergoes an acid pH-dependent conformational change which results in an alteration of its migration during SDS-PAGE (54, 57). The formation of the mobility-resistant, or SDS-resistant, oligomerized PA was analyzed to determine whether Toxin B effects anthrax toxin processing (Fig. 5B). As a positive control, we used the macrolide antibiotic bafilomycin A1, which prevents endosomal acidification through selective inhibition of the vacuolar H+-ATPase (11). SDS-resistant PA could be detected in untreated cells and was not present in bafilomycin A1-treated cells at 1 h after the addition of LT. SDS-resistant oligomeric PA levels in toxin B-treated cells were observed to be similar to the levels in untreated controls, whereas MEK2 cleavage was inhibited, suggesting that internalization and endocytic processing of the PA heptamer occurs normally even though MKK cleavage does not.

Finally, we examined the effect of toxin B or mevastatin on LF-mediated MEK2 cleavage by using an in vitro assay. RAW macrophages were either left untreated or were treated with Toxin B or mevastatin as described for previous experiments. LF was then added directly to cleared cytosolic extracts, and MEK2 cleavage was assessed either at 4°C or after a brief incubation period at 37°C. MEK2 was cleaved in under 15 min in all three samples (Fig. 5C). Similarly, no differences in cleavage were noted at different (limiting) concentrations of LT (data not shown). These data suggest that the action of Rho GTPases occurs after toxin internalization but is likely not due to direct inhibitory effects on substrate cleavage. It is still possible that Rho GTPase activity may be important either for proper trafficking of anthrax lethal toxin and/or its exit from the endocytic pathway.

Inhibition of Rho GTPases induces activation of the MAPK pathway. Cell survival in RAW cells is regulated by MAPK signal transduction, and LT-mediated death in other murine macrophage-like cell lines is mimicked by pharmacologic inhibition of p38 MAPK (67). Therefore, we examined the phosphorylation states of p38 MAPK, JNK, and p42/44 (ERK1 and ERK2) after Rho GTPase inhibition. Although no phosphorylation was observed in untreated controls, all three MAPK family members were phosphorylated in toxin B-treated cells (Fig. 6A). Since MAPK phosphorylation is the result of MKK activity, we also examined whether MEK1/2, as a representative MKK, was phosphorylated in the presence of toxin B and other Rho GTPase inhibitors used in these studies. RAW macrophage MEK1/2 was indeed phosphorylated on serine residues 218 and 222 in the presence of toxin B but not in untreated cells (Fig. 6B). Similar results for MAPK and MEK1/2 phosphorylation states were observed following statin treatment (data not shown). While MEK1/2 was also found to be phosphorylated in mevastatin-treated cells, it was not phosphorylated in the presence of mevalonolactone alone or when mevastatin-treated cells were supplemented with mevalonolactone (Fig. 6C).


Figure 6
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  		FIG. 6. Inactivation of Rho GTPases with toxin B or statins activates MAPK signaling pathways. (A) Toxin B-treated RAW macrophages were treated with LT (100 ng of LF/ml; 500 ng of PA/ml), and lysates were analyzed for phospho-p38, phospho-ERK1/2, or phospho-JNK/SAPK by Western blotting (left panel). Total MAPK levels are shown to demonstrate equal loading (right panel). Toxin B treatment resulted in activation of all three archetypal MAPK family members. However, the levels of phospho-MAPKs clearly decrease with increased duration of LT treatment. The data shown are representative of three independent experiments. (B) RAW macrophages were treated with toxin B prior to treatment with LT (100 ng of LF/ml; 500 ng of PA/ml). Lysates were analyzed for S218/S222 phosphorylated MEK1/2 by Western blotting. Toxin B treatment increased the levels of phospho-MEK1/2 compared to untreated cells. The data shown are representative of four independent experiments. (C) RAW cells were untreated (Unt), treated with mevastatin (Mev), treated with mevalonolactone (Mvl), or treated with mevastatin and mevalonolactone supplementation combined (Mev+Mvl) for 24 h. Mevastatin-induced phosphorylation of MEK1/2 is reversed with mevalonolactone supplementation. The data shown are representative of three independent experiments.


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DISCUSSION
 
The pathogenesis of B. anthracis has come under increased scrutiny over the past several years due to the use of anthrax spores as a bioweapon in the United States in 2001 and the potential for anthrax to be used in this capacity in the future. The mortality rate associated with inhalation anthrax, which approaches 100%, is attributed to the actions of the anthrax toxins. Systemic toxin levels rapidly increase as bacterial titers rise unabated, suppressing the immune response, disrupting normal tissue homeostasis, and rendering antibiotic intervention ineffective at late stages of the infection. Therefore, the development of novel mechanistic approaches to inhibit the immunosuppressive and deleterious effects of the anthrax toxins is critical.

The studies described here focus on the regulation of anthrax LT, a zinc-dependent metalloprotease which cleaves MKKs, thereby modulating MAPK signal transduction. Although the major intracellular target(s) of LT has been identified, the consequences of LT activity are not well understood. LT exhibits pleiotropic effects on leukocytes, resulting in disarmament of host immune defense, while preventing the release of proinflammatory cytokines in macrophages and dendritic cells through the disruption of normal signal transduction pathways and the induction of apoptosis (3, 67, 68, 71). Both LT and ET inhibit the production of reactive oxygen species (17) and chemotaxis (22) by neutrophils. Further, LT inhibits lymphocyte proliferation and the initiation of acquired immunity (25, 63, 64). Notably, the Rho family GTPases have been implicated in the regulation of many of these processes, as well as in the endocytic and trafficking pathways which govern LT internalization.

We demonstrate that inhibition of Rho GTPase signaling using drugs (statins) which inhibit Rho GTPase posttranslational processing, as well as toxin-mediated protein modification and transient transfection of dominant inhibitory mutants, decreases LT-mediated death in the RAW 264.7 murine macrophage-like cell line and attenuates MKK cleavage in both RAW 264.7 and Cos7 cells. Both the statin family of HMG-CoA reductase inhibitors and C. difficile toxin B, which inhibit Rho GTPase activity by distinct mechanisms, had similar antagonistic effects on LT activity. Consistent with the participation of Rho GTPases in the action of LT, we observed a marked activation of Rac1 and RhoA as early as 5 min after the addition of LT, which persisted during the time period preceding the overt cleavage of MKK substrates.

The ability of the dominant-negative mutants of Rac1, RhoA, and Cdc42 used in these studies to inhibit MEK2 cleavage when expressed in Cos7 cells suggests that either the activity of all three Rho GTPases serendipitously affect LT activity or that the observed inhibition of MEK2 cleavage reflects a more generalized response to Rho GTPase inhibition. One possibility would be disruption of cytoskeletal structures or dynamics. Indeed, when we treated RAW macrophages with latrunculin A, which sequesters actin monomers, or cytochalasin B, which caps the barbed end of actin filaments, prior to the addition of anthrax LT, we observed decreases in both LT-induced cell death and in cleavage of MKK substrates, even though the viability of non-toxin-treated cells was maintained (data not shown). However, interpretation of this result is complicated by the prior report that cytochalasin D blocks PA oligomerization (56), suggesting that cytoskeletal disruption would have an early effect on toxin uptake.

Previous studies have suggested that factors in addition to the direct LF-MKK interaction may influence MKK cleavage in cells. Kau et al. showed that inhibition of a calyculin A-sensitive phosphatase prevents LT mediated cell death and MKK cleavage (39). Our data would also be consistent with Rho GTPases regulating the phosphorylation state of MKKs through a phosphatase activity, thereby modulating their susceptibility to LT action. Mutation of residues within the LF-interacting region of MEK1 that are conserved among all MKKs abolishes cleavage by LF (83). These residues map to the hydrophobic interior of MEK1 (61), suggesting that a conformational change, perhaps regulated by posttranslational modifications including phosphorylation, may be necessary for efficient interaction with LF. However, we were unable to obtain definitive evidence that phosphorylation of S218 or S222, residues which control MEK1 activity, are directly responsible for the observed inhibition of its cleavage. It remains possible that the phosphorylation of other, or additional, sites is required for protection of MKKs from LF-mediated cleavage.

There also remain possible alternative explanations for GTPase-mediated interference with LT activity. In the context of MAP kinase kinase kinase 4 (MEKK4)-mediated signaling, dominant-negative mutations of these GTPases block activation of downstream MAPK family members (31), suggesting that Rho GTPase activity is required for proper assembly of MAPK signaling modules. Indeed, the regulation of MKK activation is influenced not only by posttranslational modification by kinases and phosphatases but also by protein-protein and lipid interactions which influence subcellular localization (5, 29, 44). MEK1 is localized to peripheral membrane adhesion structures dependent on phosphorylation by p21 (Rac and Cdc42)-activated kinase (PAK1) (23, 78). Association with p14 and MP1 at the late endosome, a locale proximal to the site of LF release into the cytoplasm, could also affect the rate at which this MKK is cleaved (55). Such associations and redistributions for other MKKs may be affected by GTPase activation states and could sequester MKKs from LF action.

Although the PA oligomerization appears to occur normally in the presence of toxin B, we cannot rule out that endosomal trafficking and cytoplasmic release of the LF catalytic toxin subunit is affected by statins or toxin B. Specific Rho GTPase family members, such as RhoB, are involved in the maturation of endosomal multivesicular bodies (26), which are thought to be important for the translocation of LF from the endosomal compartment into the cytosol (1).

Statins as a potential therapeutic approach to delay the onset of LT action. Statins prevent lipid modification (prenylation) of low-molecular-weight GTPases by blocking mevalonic acid production, a precursor to the formation of Fpp and GGpp. Prenylation of Rho GTPases is important for their regulation and function (87). In particular, C-terminal geranylgeranylation of Rho GTPases directs correct localization of GTPases to the plasma membrane, where they become activated to interact with and stimulate effectors. In the absence of prenylation, membrane translocation is blocked, rendering the GTPases functionally inactive.

It is of substantial interest that the statins, as well as toxin B, antagonize LT action. Under our experimental conditions, cholesterol availability has been shown to not be rate limiting (43). In addition, our data show that the effects of statins on LT activity can be mimicked by use of a specific geranylgeranylpyrophosphyl transferase inhibitor, indicating that the observed antagonistic effects of the statins are likely attributable to disruption of Rho GTPase isoprenylation. The data from multiple labs indicates that many of the beneficial effects of statins come from inhibition of isoprenylation of Rho GTPases, as opposed to their effects on cholesterol synthesis (10, 16, 45, 51, 52). Since the Rho GTPases are critical regulators of the inflammatory response (9), it is not surprising that statins are effective therapeutic agents in the treatment of chronic inflammatory diseases (51). The pleiotropic effects of statins may have other benefits for the counteraction of pathogen-mediated intoxication: statins are known to inhibit the production of proinflammatory cytokines, including interleukin-1β and tumor necrosis factor alpha (33), as well as activation of NF-{kappa}B (62, 65) and NADPH oxidase (10, 18), all of which are important mediators that contribute to anthrax pathogenesis. Since statins are safe and effective pharmacologic agents that are routinely used therapeutically for the reduction of plasma cholesterol, it will be of great interest to examine the prophylactic effects of statin treatment during the course of B. anthracis infection of live animals.


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ACKNOWLEDGMENTS
 
We acknowledge the expert technical assistance of Bruce Fowler and Ben Bohl throughout these experiments, and we thank Hui Zhang for assistance with ongoing experiments.

This study was supported by grant CI000095 from the Centers for Disease Control (to G.M.B.) and by a postdoctoral fellowship from the National Arthritis Foundation (to A.M.D.).


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FOOTNOTES
 
* Corresponding author. Mailing address: The Scripps Research Institute, 10550 N. Torrey Pines Road, IMM14/R211, La Jolla, CA 92037. Phone: (858) 784-8217. Fax: (858) 784-8218. E-mail: bokoch{at}scripps.edu Back

{triangledown} Published ahead of print on 20 October 2008. Back

Editor: B. A. McCormick

{dagger} Present address: Rockefeller University Press, 1114 First Ave., 3rd Fl., New York, NY 10065. Back


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Infection and Immunity, January 2009, p. 348-359, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.01005-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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