Pseudomonas aeruginosa Exoenzyme S ADP-ribosylates Ras at Multiple Sites*

Anand K. GanesanDagger , Dara W. FrankDagger , Ravi P. Misra§, Gudula Schmidt, and Joseph T. BarbieriDagger par

From the Departments of Dagger  Microbiology and § Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the  Institute for Pharmacology and Toxicology, University of Freiburg, Freiburg D-79104, Federal Republic of Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of ~2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue. SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras. Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues. Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type RasDelta CAAX and RasDelta CAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation. This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Small molecular weight GTP-binding proteins of the Ras superfamily play an integral role in eukaryotic signal transduction, controlling processes such as cell differentiation and proliferation (1). Several bacterial toxins covalently modify members of the Ras superfamily and alter transduction of intracellular signals. Clostridium botulinum C3 ADP-ribosylates Rho at Asn-41, inhibiting Rho-dependent differentiation (2). Clostridium sordelii lethal toxin glucosylates Ras at Thr-37, inhibiting the epidermal growth factor-stimulated p42/44 mitogen-activated protein kinase signaling pathway (3). Escherichia coli cytolethal necrotizing factor deaminates Gln-63 of Rho, inhibiting intrinsic GTPase activity (4, 5).

Conditions such as cystic fibrosis, leukemia, neutropenia, and burn wounds predispose individuals to infections by P. aeruginosa (6). Numerous factors, including the production of exoenzyme S (ExoS1; 453 amino acids), may contribute to the virulence of P. aeruginosa (7). ExoS, a member of the family of bacterial ADP-ribosyltransferases (8), requires the presence of a eukaryotic protein termed factor-activating exoenzyme S (FAS), a 14-3-3 protein, for expression of ADP-ribosyltransferase activity (9). Unique to most bacterial toxins, ExoS does not have rigid target protein specificity. In vitro, ExoS has been shown to ADP-ribosylate a number of targets, including IgG3 (10), apolipoprotein A-I (10), vimentin (11), and several members of the Ras superfamily (12). Analysis of chemical sensitivity indicated that ExoS ADP-ribosylates these proteins at arginine residues (10, 13).

Initial studies implicated a role for ExoS in the dissemination of P. aeruginosa from burn wounds (14) and in tissue destruction in chronic lung infections (15). However, recent studies have shown that the transposon mutant used in the earlier studies had a disruption in the gene encoding a component of its type III secretion system; thus, this mutant would have pleiotropic effects on the expression of other type III secreted factors in addition to ExoS (16). Two recent studies have implicated a role for the type III secretion apparatus in the delivery of ExoS into eukaryotic cells. Olson and co-workers (17) have shown that incubation of cultured cells with strains of P. aeruginosa expressing ExoS resulted in the ADP-ribosylation of Ras, whereas Forsberg and co-workers (18) have demonstrated that ExoS is cytotoxic to cultured cells when delivered by the type III secretion system of the heterologous host Yersinia.

In this study, we examined the biochemical aspects of the ADP-ribosylation of Ras by ExoS. We demonstrate that ExoS ADP-ribosylates c-Ha-Ras at more than one site, with Arg-41 being the preferred site of ADP-ribosylation. In addition, we show that infection of PC-12 cells by a strain of P. aeruginosa that produces ExoS inhibits nerve growth factor (NGF)-stimulated neurite outgrowth, which is the first demonstration that ADP-ribosylation by ExoS disrupts a Ras-mediated signal transduction pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The following reagents were purchased from the indicated manufacturers: [adenylate phosphate-32P]NAD and [35S]methionine, NEN Life Science Products; TnT7 quick coupled transcription/translation kit, Promega; Sculptor in vitro mutagenesis kit, Amersham Pharmacia Biotech; bovine serum albumin, Pierce; and DNA oligomers, Operon Technologies, Inc. Recombinant FAS and the pET16b c-Ha-Ras vector were gifts from H. Fu (Emory University). P. aeruginosa 388 exoenzyme S was purified as described previously (19). P. aeruginosa 388 and P. aeruginosa 388Delta ExoS were cultured as described previously (16).

Purification of His-tagged Ras Proteins-- Ras proteins were expressed in E. coli and purified as described previously (20). Briefly, protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin) were added to the concentrated cell suspensions, which were disrupted with a French press. The lysate was centrifuged (30,000 × g for 20 min) and filtered (0.45-µm filter), and His-tagged Ras proteins were purified by Ni2+ affinity chromatography (20). His-tagged Ras proteins were eluted with elution buffer containing 3 µM GTP and 10 mM MgCl2, diluted to 40% glycerol, and stored at -20 °C. These proteins were termed RasDelta CAAX.

ADP-ribosylation of RasDelta CAAX by ExoS-- Reaction mixtures (25 µl) contained 0.2 M sodium acetate (pH 6.0), with the indicated amount of [adenylate phosphate-32P]NAD, and RasDelta CAAX in the presence and absence of FAS and/or ExoS or Delta N222 (a catalytic deletion peptide of ExoS) (20) at the indicated concentrations. Reactions were performed for 1 h at room temperature and stopped with 0.5 volume of gel loading buffer containing beta -mercaptoethanol and boiling. Samples were analyzed by SDS-PAGE followed by autoradiography.

Construction of M13mp18RasDelta CAAX and Site-directed Mutagenesis of RasDelta CAAX-- RasDelta CAAX was engineered by deletion polymerase chain reaction mutagenesis of pet16b c-Ha-Ras using primers containing EcoRI and BamHI restriction sites at the 5'- and 3'-ends of the gene, respectively. The resulting polymerase chain reaction product was digested with XbaI and BamHI and ligated into the pET15b vector. The EcoRI-BamHI fragment, encoding the RasDelta CAAX gene and the T7 promoter, was then cloned into M13mp18. M13mp18RasDelta CAAX was used as the template for site-directed mutagenesis. Mutagenesis was performed essentially following the instructions of Amersham Pharmacia Biotech. DNA primers were constructed to be 10 base pairs complementary to the DNA template flanking the mutation of interest and encoded a Arg-to-Ala substitution (GC to CG). The presence of the mutation was confirmed by sequence analysis. The mutants were designated RnA, where arginine at residue n of Ras was changed to alanine.

ADP-ribosylation of in Vitro Transcribed/Translated M13ras by ExoS-- M13mp18RasDelta CAAX (M13ras) or mutated M13mp18Ras Delta CAAX replicative form DNA was subjected to coupled in vitro transcription/translation (Promega). Transcription/translation reactions (12 µl) contained 0.5 µg of replicative form DNA and [35S]Met (specific activity of 98 Ci/mmol). An aliquot of the transcription/translation reaction (2 µl) was added to 4 volumes of gel loading buffer containing beta -mercaptoethanol and boiled for 5 min. This sample was subjected to SDS-PAGE followed by autoradiography to monitor the electrophoretic mobility of intrinsically labeled [35S]Met-RasDelta CAAX. The transcription/translation mixture was also subjected to ADP-ribosylation by ExoS in reaction mixtures (25 µl) containing 4 µl of the transcription/translation mixture and 0.6 M sodium acetate (pH 6.0) in the presence and absence of 0.1 µM FAS, 960 µM NAD, and either 12 or 1.2 nM ExoS as indicated. Reactions were stopped at the indicated times by adding 2 volumes of gel loading buffer containing beta -mercaptoethanol and boiling for 5 min. Samples were subjected to SDS-PAGE followed by autoradiography. The electrophoretic mobility of this sample was compared with the electrophoretic mobility of intrinsically labeled [35S]Met-RasDelta CAAX.

GTP Dissociation Kinetics of Wild-type Ras and RasDelta CAAXR41K-- The rate of GDP/GTP dissociation of wild-type RasDelta CAAX and RasDelta CAAXR41K was measured essentially as described previously (21). Briefly, 2 µM RasDelta CAAX was incubated in 4 mM EDTA, 50 mM Tris (pH 7.6), and 5 mM dithiothreitol for 10 min at 37 °C, and then 50 µM [alpha -32P]GTP (specific activity of 1.4 Ci/mmol) and 10 mM MgCl2 were added. After 30 min at 37 °C, a 40-fold excess of unlabeled GTP was added. At defined times, 25-µl aliquots were removed, filtered through nitrocellulose filters, and washed. Filter-bound radioactivity was determined by scintillation counting.

PC-12 Cell Culture-- PC-12 cells were passaged in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. Twenty-four hours prior to an experiment, ~4 × 103 cells were seeded per well in a 12-well culture dish coated with poly-L-lysine. For differentiation, the culture medium was changed to Dulbecco's modified Eagle's medium containing 1% horse serum and 100 ng/ml NGF (Boehringer Mannheim).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

ADP-ribosylation of RasDelta CAAX by ExoS-- ExoS ADP-ribosylates several eukaryotic proteins in vitro, including vimentin, IgG, and several members of the Ras superfamily (10, 13). In this study, the ADP-ribosylation of c-Ha-Ras by ExoS was examined. To facilitate expression and purification, a deletion peptide of Ras (termed RasDelta CAAX) was expressed as a His-tagged protein in E. coli. RasDelta CAAX possesses the complete GTP-binding domain and effector functions of Ras, but lacks the four carboxyl-terminal residues, which comprises the farnesylation sequence necessary for membrane targeting. ADP-ribosylation of RasDelta CAAX by ExoS showed an absolute requirement for FAS and NAD (Fig. 1). ADP-ribosylated RasDelta CAAX possessed a slower electrophoretic mobility as determined by SDS-PAGE relative to non-ADP-ribosylated RasDelta CAAX. Other experiments showed that at saturation, ExoS had ADP-ribosylated RasDelta CAAX at a stoichiometry of ~2 mol of ADP-ribose bound per mol of RasDelta CAAX (Table I). This suggested that ExoS ADP-ribosylated RasDelta CAAX at potentially two sites. Similar results were obtained when ExoS ADP-ribosylated full-length c-Ha-Ras (data not shown).


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Fig. 1.   ADP-ribosylation by ExoS alters the electrophoretic mobility of RasDelta CAAX. RasDelta CAAX was ADP-ribosylated by ExoS in the presence (+) or absence (-) of the indicated reagents. The band with the fast electrophoretic mobility corresponds to RasDelta CAAX, whereas the band with slow electrophoretic mobility corresponds to ADP-ribosylated RasDelta CAAX. Reaction mixtures (25 µl) contained 0.2 M sodium acetate (pH 6.0), 300 µM NAD (specific activity of 1.6 Ci/mmol), and 30 µM histidine-Ras fusion protein in the presence and absence of 1 µM FAS and/or 0.1 µM ExoS. Reactions were performed for 1 h at room temperature and analyzed by SDS-PAGE (upper panel) followed by autoradiography (lower panel).

                              
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Table I
Biochemical analysis of recombinant wild-type Ras and RasDelta CAAXR41K

ADP-ribosylation of in Vitro Transcribed/Translated RasDelta CAAX by ExoS-- Biochemical characterization of eukaryotic proteins that had been ADP-ribosylated by ExoS indicated that arginine was the site of ADP-ribosylation (10). Since the presence of two potential sites for ADP-ribosylation within Ras could complicate a biochemical determination of the preferred site for ADP-ribosylation, a molecular approach was used to determine if ExoS ADP-ribosylated Ras at a preferred arginine residue. Briefly, the gene encoding RasDelta CAAX was subcloned downstream of a T7 promoter, which was then subcloned into M13mp18 (M13ras). The replicative form of M13ras was isolated and subjected to in vitro transcription/translation using [35S]Met as an intrinsic label. The in vitro transcription/translation mixture, containing [35S]Met-RasDelta CAAX, was subjected to ADP-ribosylation by ExoS. ADP-ribosylated [35S]Met-RasDelta CAAX was measured as a shift in the electrophoretic mobility of [35S]Met-RasDelta CAAX. The requirements for the ADP-ribosylation of in vitro translated [35S]Met-RasDelta CAAX by ExoS are shown in Fig. 2. In vitro translated [35S]Met-RasDelta CAAX migrated as a distinct 28-kDa band on SDS-PAGE. Incubation of in vitro translated [35S]Met-RasDelta CAAX with exogenous NAD, FAS, and ExoS resulted in the detection of two electrophoretic mobility shifted forms of RasDelta CAAX. This was consistent with ExoS ADP-ribosylating Ras at two distinct sites. RasDelta CAAX showed little electrophoretic mobility shifting in reactions lacking NAD, whereas two electrophoretic mobility shifted forms of RasDelta CAAX were observed in reactions lacking FAS. This indicated that the reticulocyte lysates used for in vitro transcription/translation of RasDelta CAAX contained a small amount of NAD and saturating amounts of FAS relative to the concentrations used in the ADP-ribosyltransferase assay.


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Fig. 2.   ADP-ribosylation of in vitro transcribed/translated RasDelta CAAX requires the addition of exogenous NAD and ExoS. In vitro transcribed/translated [35S]Met-RasDelta CAAX was subjected to ADP-ribosylation by ExoS in the presence (+) or absence (-) of the indicated reagents for 10 or 120 min at room temperature. ExoS (12 nM) was added to reaction mixtures as indicated. Reaction mixtures were analyzed by SDS-PAGE followed by autoradiography. Electrophoretic migrations are indicated as RasDelta CAAX (arrow) and ADP-ribosylated RasDelta CAAX (*, single-shifted RasDelta CAAX; **, double-shifted RasDelta CAAX).

ADP-ribosylation of RasDelta CAAX Containing Single Arginine Mutations by ExoS-- M13ras single-stranded DNA was subjected to site-directed mutagenesis in which 12 individual Arg-to-Ala mutations (each mutant was designated RnA (Arg at residue n of Ras mutated to an Ala) were introduced into the ras open reading frame. M13ras replicative form DNA, encoding the wild type or an individual Arg-to-Ala mutation, was subjected to in vitro transcription/translation. In vitro translated [35S]Met-RasDelta CAAX was ADP-ribosylated by ExoS at a concentration of ExoS such that a single preferred site for ADP-ribosylation would be detected. Wild-type [35S]Met-RasDelta CAAX and 11 of the individual Arg-to-Ala mutants of RasDelta CAAX, with the exception of RasDelta CAAXR41A, showed an electrophoretic mobility shift upon ADP-ribosylation by ExoS. In contrast, RasDelta CAAXR41A did not show an electrophoretic mobility shift upon incubation with ExoS. These data suggested that Arg-41 was the preferred site of ADP-ribosylation by ExoS (Fig. 3).


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Fig. 3.   Arg-41 is the preferred site of ADP-ribosylation of Ras by ExoS. Twelve site-directed Arg-to-Ala mutants of RasDelta CAAX were produced (R41A, R169A, R68A, R73A, R97A, R102A, R123A, R128A, R164A, R135A, R149A, and R161A). In vitro transcribed/translated [35S]Met-RasDelta CAAX was ADP-ribosylated by ExoS. Reaction mixtures were subjected to SDS-PAGE followed by autoradiography. The - lanes represent the electrophoretic mobility of [35S]Met-RasDelta CAAX proteins, whereas the + lanes represent the electrophoretic mobility of the [35S]Met-RasDelta CAAX proteins following incubation with 1.2 nM ExoS and 960 µM NAD for 1 h at room temperature. WT, wild type.

ADP-ribosylation of in vitro translated [35S]Met-RasDelta CAAX (the wild type and RnA mutants) was also assayed at higher concentrations of ExoS such that multiple sites within Ras would be ADP-ribosylated (Fig. 4). With the exception of RasDelta CAAXR41A, ExoS ADP-ribosylated wild-type RasDelta CAAX and the RnA mutants, with the appearance of two sequential electrophoretic mobility shifts, suggestive of the presence of double ADP-ribosylated Ras. In contrast, ExoS ADP-ribosylated RasDelta CAAXR41A to only a single electrophoretic mobility shift, which was consistent with the presence of only a single site for ADP-ribosylation. These data also indicated that the second site of ADP-ribosylation was not at a specific arginine residue and that more than one arginine could be ADP-ribosylated at the second site. Since a third ADP-ribosylation event was not observed, it appeared that ADP-ribosylation at the second site excluded a third ADP-ribosylation event.


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Fig. 4.   Exoenzyme S does not ADP-ribosylate Ras at a distinct secondary site. Twelve site-directed Arg-to-Ala mutants of RasDelta CAAX were produced (R41A, R169A, R68A, R73A, R97A, R102A, R123A, R128A, R164A, R135A, R149A, and R161A). In vitro transcribed/translated [35S]Met-RasDelta CAAX was ADP-ribosylated by ExoS. Reaction mixtures were subjected to SDS-PAGE followed by autoradiography. The - lanes represent the electrophoretic mobility of [35S]Met-RasDelta CAAX proteins, whereas the + lanes represent the electrophoretic mobility of [35S]Met-RasDelta CAAX proteins following incubation with 12 nM ExoS and 960 µM NAD for 1 h at room temperature. WT, wild type.

ADP-ribosylation of Several Arg-41 Mutants of Ras by ExoS-- To support the identification of Arg-41 as the preferred site for ADP-ribosylation by ExoS, several more conservative site-directed mutations were engineered at residue 41, lysine (RasDelta CAAXR41K) and glutamine (RasDelta CAAXR41Q). As observed for RasDelta CAAXR41A, low concentrations of ExoS did not ADP-ribosylate either RasDelta CAAXR41K or RasDelta CAAXR41Q (Fig. 5). A second positive control, RasDelta CAAXG12V, was ADP-ribosylated by ExoS as measured by the generation of electrophoretic mobility shifted Ras.


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Fig. 5.   ADP-ribosylation of wild-type RasDelta CAAX and several Ras mutants by ExoS. In vitro transcribed/translated [35S]Met-RasDelta CAAX and [35S]Met-RasDelta CAAX mutants (R41A, R41K, R41Q, and G12V) were incubated alone (-) or with (+) 1.2 nM ExoS and 960 µM NAD for 1 h at room temperature. Reaction mixtures were subjected to SDS-PAGE followed by autoradiography. The asterisk denotes single-shifted [35S]Met-RasDelta CAAX proteins.

ADP-ribosylation of RasDelta CAAX and RasDelta CAAXR41K by ExoS-- To address ExoS-mediated ADP-ribosylation of Ras at Arg-41 in more detail, RasDelta CAAX and RasDelta CAAXR41K were analyzed as targets for ADP-ribosylation. Bacterially produced RasDelta CAAX and RasDelta CAAXR41K were expressed and purified to similar levels and reacted with polyclonal antisera to Ras by Western blotting (Fig. 6). To determine whether RasDelta CAAX and RasDelta CAAXR41K were functional, the GTP dissociation kinetics were measured. Both RasDelta CAAX and RasDelta CAAXR41K exhibited similar GTP dissociation kinetics (Table I), which indicated that the R41K mutation did not alter the global functional properties of Ras. Others have reported that mutations at Arg-41 did not alter the GTP dissociation kinetics (21).


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Fig. 6.   Western blotting of wild-type RasDelta CAAX and His-tagged RasDelta CAAXR41K. Wild-type RasDelta CAAX (WT), RasDelta CAAXR41K (K41), and soybean trypsin inhibitor (SBTI; 2 µg) were subjected to SDS-PAGE. One gel was stained for protein (A), and proteins in the second gel were transferred to nitrocellulose filters and incubated with a Ras polyclonal antibody (Upstate Biotechnology, Inc.) followed by incubation with 125I-protein A. The filter was subjected to autoradiography (B).

Next, the ADP-ribosylation of RasDelta CAAX and RasDelta CAAXR41K by ExoS was studied. At saturation, ExoS ADP-ribosylated wild-type RasDelta CAAX to a stoichiometry of ~2 mol of ADP-ribose incorporated per mol of Ras (Table I), whereas ExoS ADP-ribosylated RasDelta CAAXR41K to a stoichiometry of 0.7 mol of ADP-ribose incorporated per mol of Ras. Examination of the Coomassie Blue-stained gel and autoradiogram showed that, relative to non-ADP-ribosylated Ras, ADP-ribosylated RasDelta CAAX migrated with an altered electrophoretic mobility on SDS-PAGE, whereas ADP-ribosylated RasDelta CAAXR41K did not have an altered electrophoretic mobility on SDS-PAGE (Fig. 7). The fact that ADP-ribosylated RasDelta CAAXR41K did not have an altered electrophoretic mobility in this experiment is due to fact that this experiment was performed using a 12% polyacrylamide gel, whereas the second ADP-ribosylation event was observed upon separation on a 15% polyacrylamide gel, as seen in Fig. 4. Under linear velocity conditions, ExoS ADP-ribosylated wild-type RasDelta CAAX at a faster velocity than RasDelta CAAXR41K. A representative graph depicting the velocity of the ADP-ribosylation of wild-type RasDelta CAAX and RasDelta CAAXR41K by ExoS is shown in Fig. 8. The average of three independent experiments indicated that ExoS ADP-ribosylated wild-type RasDelta CAAX at 5-fold greater velocity than RasDelta CAAXR41K (Table I).


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Fig. 7.   ADP-ribosylation of wild-type Ras and RasDelta CAAXR41K by ExoS. Wild-type Ras and RasDelta CAAXR41K were ADP-ribosylated by Delta N222. Reaction mixtures (25 µl) contained 0.2 M sodium acetate (pH 6.0), 100 µM NAD (specific activity of 1.6 Ci/mmol), and 5 µM histidine-Ras fusion protein in the presence of 100 nM FAS and 50 nM Delta N222. Reactions were performed at room temperature and stopped at the indicated times, and samples were analyzed by SDS-PAGE. The gel was stained for protein (A) and subjected to autoradiography (B).


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Fig. 8.   Velocity of the ADP-ribosylation of RasDelta CAAX and RasDelta CAAXR41K by ExoS. Wild-type Ras and RasDelta CAAXR41K were ADP-ribosylated by Delta N222. Reaction mixtures (25 µl) contained 0.2 M sodium acetate (pH 6.0), 30 µM NAD (specific activity of 12.8 Ci/mmol), and 2.5 µM histidine-Ras fusion protein in the presence of 2.5 nM FAS and 2.5 nM Delta N222. Reactions were performed at room temperature and stopped after 7.5, 15, 30, and 60 min. Samples were analyzed by SDS-PAGE, and radioactivity was quantitated by scintillation counting.

Infection of PC-12 Cells with P. aeruginosa Expressing ExoS Inhibits NGF-stimulated Neurite Formation-- Arg-41 is located next to the switch 1 region of Ras (residues 30-39), a domain that interacts with downstream effectors (Fig. 9) (22). Thus, ADP-ribosylation of Ras at Arg-41 could potentially disrupt eukaryotic signal transduction by inhibiting the interaction of Ras with downstream effectors. The ability of ExoS to disrupt eukaryotic signal transduction was determined by analysis of NGF-stimulated neurite outgrowth in PC-12 cells. NGF-stimulated neurite outgrowth in PC-12 cells follows a Ras-mediated signal transduction pathway, although there are also Ras-independent components to the pathway (23). Since ExoS is secreted from P. aeruginosa via a type III secretion pathway (16), ExoS was delivered into PC-12 cells by infection with P. aeruginosa. PC-12 cells were incubated alone, with a strain of P. aeruginosa that produced ExoS (388), or with a strain of P. aeruginosa that was genetically deleted for exoS (388Delta ExoS). After 2 h, PC-12 cells were washed to remove unbound bacteria and incubated alone or with NGF. At 24 h post-infection, PC-12 cells were examined by phase microscopy. PC-12 cells that were incubated in the absence of bacteria and NGF showed a rounded morphology, typical of unstimulated cells (Fig. 10). In contrast, PC-12 cells that had been incubated alone or with a strain of P. aeruginosa that lacks exoS (388Delta ExoS) and then incubated with NGF had undergone a morphological change, where the cells had spread and exhibited neurite outgrowth. PC-12 cells that had been incubated with the ExoS-expressing strain of P. aeruginosa followed by incubation with NGF showed a rounded morphology, similar to the morphology of non-infected, non-NGF-stimulated cells. These data indicated that infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited NGF-stimulated signal transduction leading to neurite outgrowth. In other experiments, we observed that transient expression of ExoS from a cytomegalovirus promoter also inhibited NGF-mediated neurite outgrowth in PC-12 cells.2 Together, these data indicated that inhibition of NGF-mediated neurite outgrowth was due to the ADP-ribosyltransferase activity of ExoS and not a general cytotoxicity of ExoS or P. aeruginosa for PC-12 cells.


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Fig. 9.   Alignment of members of the family of small molecular weight GTP-binding proteins with respect to Arg-41 of Ras. Switch 1 and switch 2 are two domains of Ras that change conformation with respect to the presence of GDP/GTP. Switch 1 also represents a site of effector interaction within Ras.


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Fig. 10.   Uncoupling of NGF stimulation of neurite formation by P. aeruginosa expression of ExoS. PC-12 cells were incubated alone or were infected with P. aeruginosa 388 or 388Delta ExoS at a multiplicity of infection of 10:1 (bacteria to PC-12 cells) for 3 h at 37 °C. Cells were washed to remove unbound bacteria and incubated in differentiation medium containing penicillin/streptomycin alone or containing 0.1 µg/ml NGF. After 24 h, the morphology of the PC-12 cells was examined by phase microscopy.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Earlier studies predicted that ExoS ADP-ribosylates eukaryotic proteins at arginine residues (10, 12). We initially pursued a biochemical analysis of the site of ADP-ribosylated Ras. However, we found that, during proteolysis, a considerable amount of radiolabel was released as ADP-ribose and that the radiolabel that remained peptide-associated was present in several peptides.2 This, in addition to the observation that ExoS ADP-ribosylated Ras to a stoichiometry of ~2 (Table I), prompted a molecular approach for the characterization of the site of ADP-ribosylation. The subsequent determination that ExoS ADP-ribosylates Ras at multiple sites is consistent with our inability to identify a specific arginine as the preferred site of ADP-ribosylation by biochemical approaches. Characterization of in vitro transcribed/translated forms of Ras that each possessed one of 12 individual Arg-to Ala substitutions identified Arg-41 as the preferred site for ADP-ribosylation since the Arg-41 mutants of Ras were not ADP-ribosylated by low concentrations of ExoS. Also, examination of the velocities of ADP-ribosylation of Ras by ExoS showed that Arg-41 was essentially completely ADP-ribosylated prior to the appearance of the second ADP-ribosylation event, which was consistent with Arg-41 being the high affinity site of ADP-ribosylation within Ras.2

Analysis of the single Arg mutants of Ras did not identify a specific Arg as the second site for ADP-ribosylation since all of the single Arg mutants of Ras, with the exception of Ras R41A, could be double ADP-ribosylated. These data indicated that one of several arginine residues could constitute the second site of ADP-ribosylation within Ras. Since a third ADP-ribosylation event was not observed, it appeared that the ADP-ribosylation at one arginine residue excluded a third ADP-ribosylation event. This suggests that the arginines that are targeted for the second ADP-ribosylation event are located in close proximity in the three-dimensional structure of Ras. These data also showed that the ADP-ribosylation at Arg-41 and that at the second arginine were independent events and not the result of sequential, ordered ADP-ribosylation reactions since the Arg-41 mutants could be ADP-ribosylated at the second arginine residue. There is precedence for the ADP-ribosylation of proteins at two sites. Aktories and co-workers (24) recently showed that an endogenous eukaryotic ADP-ribosyltransferase modifies actin at Arg-95 and Arg-372.

The ability of ExoS to ADP-ribosylate Ras at multiple arginine residues assists in addressing an earlier report by Coburn et al. (12) that described the ADP-ribosylation of several members of the Ras superfamily by ExoS and the subsequent prediction that ExoS ADP-ribosylated Ras at Arg-123 (13). Our data could be consistent with Arg-123 being one of the secondary sites of ADP-ribosylation within Ras.

Examination of the x-ray crystallographic structure of Ras (25) predicts that Arg-41 lies adjacent to the switch 1 domain, which has been shown to function in the interactions with downstream effectors, particularly Raf (22). Alignment of the primary amino acid sequences of members of the Ras superfamily of small molecular weight GTP-binding proteins showed that Arg-41 is not a highly conserved residue. Thus, it appears that ExoS can potentially ADP-ribosylate the Arg-41 homologue of Ras, Ral, and Rap, but that members of the Rab or Rho family will not be targeted for ADP-ribosylation by ExoS at the Arg-41 homologue. However, the observation that ExoS may ADP-ribosylate Ras at a second arginine residue poses the possibility that other members of the Ras superfamily that lack the Arg-41 homologue, including Rho and Rab, may be targeted for ADP-ribosylation by ExoS at an arginine residue that corresponds to the second site of ADP-ribosylation within Ras. The ADP-ribosylation of other Ras superfamily members by ExoS is currently under investigation.

Neurite outgrowth in PC-12 cells is a multistep process that is initiated by NGF binding to the Trk membrane tyrosine kinase. NGF binding stimulates Trk kinase activity, which results in the phosphorylation of several effector molecules that activate Ras. This pathway has both Ras-dependent and -independent components. Several studies have suggested that Ras is a dominant element in the transmission of NGF signals from Trk, including microinjection of antibodies to Ras, which interferes with the signaling pathway (26), or introduction of dominant inhibitory Ras alleles into PC-12 cells, which inhibits NGF-induced neurite outgrowth. In addition, transfection of constitutively active Ras, in the absence of NGF stimulation, is sufficient to induce neurite outgrowth (26). The Ras switch 1 domain is required for this signal transduction process since point mutations within the switch 1 regions of constitutively active RasV12 interfere with neurite outgrowth (21). The fact that ExoS preferentially modifies Ras at a region adjacent to the switch 1 domain and that infection of PC-12 cells with strains of P. aeruginosa expressing ExoS results in an inhibition of neurite outgrowth in PC-12 cells is consistent with the model that ExoS inhibits Ras-mediated signal transduction in vivo. However, since ExoS has the potential to modify a wide variety of targets in vitro, it is conceivable that ExoS can disrupt PC-12 cell signal transduction by the ADP-ribosylation of components of this signal transduction pathway. Future studies will focus on whether ExoS disrupts PC-12 cell signal transduction via modification of other components involved in PC-12 cell signal transduction.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants RO1-AI-30162 and K04-AI-01087 (to J. T. B.), Grants RO1-AI-31665 and K04-AI-01289 (to D. W. F.), and Grants R55GM/OD51856 and R29NS36256 (to R. P. M.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

par To whom correspondence should be addressed: Dept. of Microbiology, Medical College of Wisconsin, 8701 Watertown Plk. Rd., Milwaukee, WI 53226. Tel.: 414-456-8412; Fax: 414-456-6535; E-mail: toxin{at}mcw.edu.

1 The abbreviations used are: ExoS, exoenzyme S; FAS, factor-activating exoenzyme S; NGF, nerve growth factor; PAGE, polyacrylamide gel electrophoresis.

2 A. K. Ganesan and J. T. Barbieri, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Wiesmuller, L., and Wittinghofer, F. (1994) Cell. Signalling 6, 247-267[Medline] [Order article via Infotrieve]
  2. Aktories, K., Mohr, C., and Koch, G. (1992) Curr. Top. Microbiol. Immunol. 175, 115-131[Medline] [Order article via Infotrieve]
  3. Just, I., Selzer, J., Hofmann, F., Green, G. A., Aktories, K. (1996) J. Biol. Chem. 271, 10149-10153[Abstract/Free Full Text]
  4. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729[CrossRef][Medline] [Order article via Infotrieve]
  5. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Florentini, C., and Boquet, P. (1997) Nature 387, 729-733[CrossRef][Medline] [Order article via Infotrieve]
  6. Bodey, G., Bolivar, R., Fainstein, V., and Jadeja, L. (1983) Rev. Infect. Dis. 5, 279-313[Medline] [Order article via Infotrieve]
  7. Nicas, T. I., and Iglewski, B. H. (1985) Antibiot. Chemother. 36, 40-48[Medline] [Order article via Infotrieve]
  8. Krueger, K. M., and Barbieri, J. T. (1995) Clin. Microbiol. Rev. 8, 34-47[Abstract]
  9. Fu, H., Coburn, J., and Collier, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2320-2324[Abstract]
  10. Knight, D. A., and Barbieri, J. T. (1997) Infect. Immun. 65, 3304-3309[Abstract]
  11. Coburn, J., Dillon, S. T., Iglewski, B. H., Gill, D. M. (1989) Infect. Immun. 57, 996-998[Medline] [Order article via Infotrieve]
  12. Coburn, J., Wyatt, R. T., Iglewski, B. H., Gill, D. M. (1989) J. Biol. Chem. 264, 9004-9008[Abstract/Free Full Text]
  13. Coburn, J. (1992) Curr. Top. Microbiol. Immunol. 175, 133-143[Medline] [Order article via Infotrieve]
  14. Nicas, T. I., Bradley, J., Lochner, J. E., Iglewski, B. H. (1985) J. Infect. Dis. 152, 716-721[Medline] [Order article via Infotrieve]
  15. Nicas, T. I., Frank, D. W., Stenzel, P., Lile, J. D., Iglewski, B. H. (1985) Eur. J. Clin. Microbiol. 4, 175-179[Medline] [Order article via Infotrieve]
  16. Yahr, T. L., Goranson, J., and Frank, D. W. (1996) Mol. Microbiol. 22, 991-1003[Medline] [Order article via Infotrieve]
  17. McGuffie, E. M., Frank, D. W., Vincent, T. S., Olson, J. C. (1997) General Meeting of the American Society for Microbiology, May 4-8, 1997, Miami Beach, FL, p. 56
  18. Frithz-Lindsten, E., Du, Y. D., Rosqvist, R., and Forsberg, A. (1997) Mol. Microbiol. 25, 1125-1139[Medline] [Order article via Infotrieve]
  19. Kulich, S. M., Frank, D. W., and Barbieri, J. T. (1993) Infect. Immun. 61, 307-313[Abstract]
  20. Knight, D. A., Finck-Barbancon, V., Kulich, S. M., Barbieri, J. T. (1995) Infect. Immun. 63, 3182-3186[Abstract]
  21. Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., Yokoyama, S. (1994) Oncogene 9, 2153-2157[Medline] [Order article via Infotrieve]
  22. Marshall, M. S. (1993) Trends Biochem. Sci. 18, 250-254[CrossRef][Medline] [Order article via Infotrieve]
  23. Kaplan, D. R., and Stephens, R. M. (1994) J. Neurobiol. 25, 1404-1417[Medline] [Order article via Infotrieve]
  24. Just, I., Sehr, P., Jung, M., van Damme, J., Puype, M., Vandekerckhove, J., Moss, J., Aktories, K. (1995) Biochemistry 34, 326-333[Medline] [Order article via Infotrieve]
  25. Milburn, M. V., Tong, L., deVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., Kim, S. H. (1990) Science 245, 939-945
  26. Szeberenyi, J. (1996) Neurobiology 4, 1-11[Medline] [Order article via Infotrieve]


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