Examination of the Coordinate Effects of Pseudomonas aeruginosa ExoS on Rac1
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感染与免疫杂志 2005年第9期
Department of Microbiology, Immunology and Cell Biology, West Virginia University Health Sciences Center, Morgantown, West Virginia 26505
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425
ABSTRACT
Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.
INTRODUCTION
Pseudomonas aeruginosa is a ubiquitous, environmentally beneficial bacterium that can adapt to become a highly virulent opportunistic pathogen in compromised individuals. The versatility and pathogenicity of P. aeruginosa are multifactorial and relate to its ability to respond to its environment by the regulated production of a variety of cell-associated and extracellular products. The establishment of infection begins with the adherence of P. aeruginosa to host cells through type IV pili or nonpilus adherence mechanisms (42, 50, 52). After colonization, the organism ensures its survival in the host through the secretion of virulence factors, including exotoxin A (24), hemolysins (40), elastases LasA and LasB (2, 20, 21), and pigments (53). In addition to secreted virulence factors, P. aeruginosa is able to directly affect eukaryotic cell function through the contact-dependent translocation of effector proteins by the type III secretion system (59). Four P. aeruginosa-type III secretory (TTS) effector proteins have been identified, ExoS, ExoT, ExoU, and ExoY, each affecting eukaryotic cell function differently. Notably, the TTS system is found in both clinical and environmental P. aeruginosa isolates, suggesting an essential role of TTS in P. aeruginosa overall growth and survival (8, 9, 49). The integral relationship between P. aeruginosa TTS effectors and eukaryotic cell function is also evident in the requirement of eukaryotic cofactors for the enzymatic function of ExoS, ExoT, ExoU, and ExoY (4, 14, 51, 60).
ExoS is the most extensively studied of the TTS effectors. Structure-function analyses identified ExoS as a bifunctional toxin, including separate domains with GTPase-activating protein (GAP) and carboxy-terminal ADP-ribosyltransferase (ADPRT) activities (18, 25, 26). In vitro studies and cell culture models of infection found the GAP activity of ExoS to target low-molecular-weight G (LMWG) proteins in the Rho family that regulate cytoskeletal structure (27, 41). Arginine at position 146 (R146) was identified as essential for ExoS GAP activity (18), and in three-dimensional structural analysis R146 was found to function as an arginine finger in stabilizing the transition state of the Rac-GTPase reaction (58). The ADPRT activity of ExoS also targets LMWG proteins but preferentially targets Ras superfamily proteins that are integral to cell signaling pathways (3, 5, 11, 12, 22, 33). In vitro kinetic analyses identified two residues involved in the ADPRT reaction of ExoS. Glutamic acid at position 379 (E379) contributes to the transfer of ADP-ribose to target substrates, and glutamic acid at position 381 (E381) functions as a catalytic residue (43).
Eukaryotic cell culture models of infection found TTS-translocated ExoS to have multiple and cell line-dependent effects on cell function. ExoS affects the cell growth, morphology, and adherence of epithelial and fibroblastic cell lines and also exerts antiphagocytic effects on macrophages (13, 38, 39). When ExoS GAP or ADPRT mutants were used to assess the role of each domain on HT-29 epithelial cell function, ADPRT activity was found to be required for effects of ExoS on cell growth and morphology, while minimal effects of ExoS GAP activity were detected (10). In studies of J774A.1 macrophages, ExoS GAP activity was found to exert an antiphagocytic function, whereas ExoS ADPRT activity was again linked to effects on cell morphology and adherence (45). The identification of effects of ExoS ADPRT activity in some cell lines and effects of ExoS GAP in other cell lines led us to explore how ExoS GAP and ADPRT functions were being coordinated and targeted within the host cell.
In examining the mechanism for the effects of ExoS ADPRT activity on cell morphology, a cell-line-dependent hierarchy of LMWG protein substrate modification was detected (12, 47). In human epithelial cells, early targets of ExoS ADPRT activity included plasma membrane-associated LMWG proteins, Ras and RalA, followed by membrane-associated Rab5, then Rab8, Rab11, and Rab7, and finally Rac1 and Cdc42 (12). Notably, the sequential targeting of ExoS ADPRT activity to substrates in human epithelial cells mimicked a vesicular retrograde trafficking process. In linking substrate modification to alterations in cell morphology, Ras and RalA are known to indirectly affect cytoskeletal structure, whereas Rho family proteins, such as Rac1, provide a more direct link for effects of ExoS on cytoskeletal structure. The recent identification of the non-G-protein substrates of ExoS ADPRT activity, the Ezrin/Radixin/Moesin (ERM) family of proteins, provides another mechanism by which ExoS can affect cytoskeleton dynamics (32). Notably, in studies of ExoS substrate specificity, it was recognized that ExoS GAP and ADPRT activity can both target Rac1, identifying Rac1 as a candidate protein for studying how the domain function of ExoS might be coordinated within the eukaryotic cell.
Rac1 is a member of the Rho family of LMWG proteins involved in the reorganization of actin at the plasma membrane, leading to membrane ruffling and the formation of lamellipodia and focal complexes (36, 44). Like all LMWG proteins, the activation state of Rac1 depends upon guanine nucleotide binding. Guanine nucleotide exchange factors (GEFs) and GAPs modulate the nucleotide-bound state of LMWG proteins via binding to the Switch I and Switch II regions (57). GEFs catalyze the exchange of GDP for GTP, and GAPs activate the intrinsic GTPase activity of LMWG proteins. The Switch domains form a maneuverable pocket cleft that undergoes a conformational change in response to GTP binding and GTP hydrolysis. This structural movement alters the ability of LMWG proteins to interact with and signal through downstream cellular effectors. GTP bound Rac1 associates with the membrane through an isoprenylated C terminus. Upon the inactivation of Rac1 by GAP, RhoGDI, a guanine dissociation inhibitor, dissociates GDP-bound Rac1 from the membrane and sequesters it in a GDP-bound state in the cytosol (30, 35, 37).
The present study examines the effects of ExoS ADPRT and GAP activities on Rac1, using the differential effects of the two domains on Rac1 function, and cell line differences in Rac1 ADP-ribosylation to examine how ExoS activity is coordinated within eukaryotic cells. The ADP-ribosylation of Rac1 in human epithelial cell lines was found to be associated with Rac1 activation and membrane localization. Conversely, in cell lines where Rac1 was not ADP-ribosylated, ExoS GAP functioned to maintain Rac1 in an inactive form. The studies highlight the potential of host cell properties to dictate and coordinate the function of the ExoS GAP and ADPRT domains.
MATERIALS AND METHODS
Bacterial strains and culture conditions. P. aeruginosa strains 388 (25), 388exoS (an ExoS isogenic mutant of strain 388 [28]), and strain PA103exoUexoT::Tc (PA103UT; a derivative of strain PA103 that lacks production of known TTS effectors [56]) were provided by Dara Frank (Medical College of Wisconsin, Milwaukee). Strain PA103UT was used to express pUCP plasmid-encoded ExoS, an R146A ExoS-GAP mutant, an E379A/E381A ExoS-ADPRT mutant, or a GAP-ADPRT inactive R146A/E379A/E381A ExoS mutant, which have been previously described (10). P. aeruginosa strains were grown in ExoS induction medium (TSBD-N) (25) for 16 h prior to coculture with eukaryotic cells, as previously described (39).
Eukaryotic cell culture. All cell lines, with the exception of RHEC and WI-38, were obtained from the American Type Cell Culture Collection (Manassas, VA). RHEC cells were provided by Carwile LeRoy, and WI-38 cells were provided by Daohong Zhou; both are investigators from the Medical University of South Carolina (Charleston). RHEC and WI-38 cells were cultured in Dulbecco modified Eagle medium. Other cell lines were cultured in media specified by the American Type Culture Collection. All media were supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml (Gibco-BRL, Gaithersburg, MD). Cells were cultured at 37°C in 5% CO2 to 95% air.
Coculture of eukaryotic cells with bacteria. For bacterial infection, eukaryotic cells were seeded at a density of 105 cells/ml and grown for 24, 48, or 72 h, depending on the cell line, to 60 to 90% confluence. Medium was removed, and bacteria were diluted based on an optical density at 590 nm of ca. 107 CFU/ml in cell line-specific medium containing 0.6% bovine serum albumin (BSA). Bacteria were added to eukaryotic cells at a multiplicity of infection of 30 to 100, and cells were cocultured for 2 to 6 h as indicated. All experiments included eukaryotic cells cultured in medium containing 0.6% BSA, but without bacteria, as a control.
Analysis of ADP-ribosylation of LMWG proteins. Ex vivo ADP-ribosylation of endogenous Rac1 by TTS-ExoS was assessed based on altered protein mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses after the coculture of eukaryotic cells with bacteria, as previously described (12). To examine Rac1 ADP-ribosylation, cells were lysed in Laemmli electrophoresis buffer (29), resolved by SDS-12% PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) (55), and probed with a mouse monoclonal antibody to Rac1 (BD-Transduction Laboratories, Lexington, KY), followed by the addition of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (Fc specific; Sigma). Rac1 was visualized by enhanced chemiluminescence (Amersham Pharmacia, Arlington Heights, IL).
In vitro ADP-ribosylation of Rac1 was performed with 10 μg of purified Rac1 (12) loaded with 10 μM GTPS, according to the procedure of Antonny et al. (1). For ADP-ribosylation reactions, 1 μg of Rac1-GTPS was incubated for 30 to 60 min at 25°C in 200 mM Tris acetate (pH 6.0) containing the indicated amount of purified His-tagged ExoS, 10 mM NAD, 1 mM MgCl2, and a source of the eukaryotic ExoS ADPRT cofactor—either 10 μl of wheat germ extract or 1 μM 14-3-3 protein (Upstate Biotechnology, Lake Placid, NY)—in a final reaction volume of 50 μl.
To monitor the incorporation of ADP-ribose into Rac1 and Rac1 mutants (see below), 2 μCi of nicotinamide adenine [adenylate-32P]dinucleotide (32P-NAD; 200 mCi/mmol; Amersham Biosciences) was added to 15-μl ADPRT reaction mixtures containing 200 mM Tris acetate, 100 μM NAD, 150 ng of His-Rac1 or His-Rac1 mutant, 250 nM 14-3-3, and either 0 or 5 nM purified His-ExoS. The reaction was terminated with 4x Laemmli sample buffer and heating for 3 min at 95°C. Reactions were resolved by SDS-PAGE, analyzed by autoradiography, and relative differences in the incorporation of radioactivity into Rac1 proteins were quantified by densitometry.
Analysis of the site of ADP-ribosylation by ExoS on Rac1. To examine the preferred site of ExoS ADP-ribosylation on Rac1, each of the nine arginine residues of Rac1 (R66, R68, R94, R102, R120, R163, R174, R185, and R187), potential targets of ExoS ADP-ribosylation, was mutated to a lysine. Mutations were introduced into Rac1 cloned into pET15b (12) by using the QuikChange PCR-based site-directed mutagenesis system (Strategene, La Jolla, CA). The primers designed to introduce specific mutations along with diagnostic restriction sites, are listed in Table 1. Rac1 proteins were expressed and purified as His-tagged proteins, by using previously described methods (7, 12).
PAK1 coprecipitation (pull-down) of active Rac1 in vitro and ex vivo. GST-PAK1:GSH beads for pull-down reactions were constructed by amplifying the cDNA corresponding to the Rac/Cdc42 binding domain (amino acids 50 to 134 of PAK1) (PBD) by using the forward primer 5'-CCCGGATCCAAAAAGAAAGAGAAAGAGCGGCC-3', containing a BamHI restriction site, and the reverse primer 5'-CCCGAATTCAGCTGACTTATCTGTAAAGCTCATG-3', containing an EcoRI restriction site (restriction sites are underlined). The PCR product was digested with BamHI and EcoRI and ligated into the pGEX4T vector (Amersham Pharmacia) for expression as a glutathione S-transferase (GST) fusion protein. The construct was verified by DNA sequence analysis and transformed into E. coli BL21(DE3) for protein expression. After induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside), bacteria were lysed by using a French pressure cell (Thermo IEC, Needham Heights, MA), and cleared lysates were bound to glutathione (GSH)-agarose beads. PAK1-GST bound to GSH-agarose beads was quantified by SDS-PAGE and gel densitometry analysis with BSA as a standard.
In vitro Rac1-PAK1 pull-down (PD) reactions were performed by adding 100 ng of either Rac1-GDP, Rac1-GTPS or ADP-ribosylated Rac1-GTPS to 5 μg of PAK1-GST:GSH-agarose beads in coimmunoprecipitation (Co-IP) lysis buffer (30 mM HEPES [pH 7.5], 1% Triton X-100, 10 mM NaCl, 10% glycerol, 1 mM EGTA, 25 mM NaF, 1 mM Na3VO4, 10 mM -glycerophosphate, and protease inhibitors). The mixture was rotated for 1 h at 4°C, the beads were washed three or four times with Co-IP wash buffer (50 mM HEPES [pH 7.5], 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, 20 mM NaF), resuspended in Laemmli electrophoresis sample buffer, resolved by SDS-PAGE, and immunoblotted for Rac1 as described above.
Ex vivo Rac1-PAK1 PD reactions were performed after a 4- to 6-h coculture of eukaryotic cells with bacteria, as indicated. Eukaryotic cells were lysed in Co-IP lysis buffer for 30 min on ice, and lysates were cleared by centrifugation for 5 min at 16,000 x g at 4°C. An aliquot of cleared lysate was removed for determination of total cellular Rac1. PAK1-GST:GSH beads (10 μg) were added to the remaining lysate and the PD reaction mixture was processed and analyzed for Rac1 as described above. Cells cultured without bacteria, cells cultured with non-ExoS-producing P. aeruginosa strains (388S or PA103UT pUCP), and cells cultured with PA103UT expressing GAP-ADPRT inactive ExoS (ExoS-R146A/E379A/E381A) served as negative controls in these studies.
Cellular localization of Rac1. Eukaryotic cells were seeded in 60- or 100-mm dishes and cocultured with the indicated bacterial strain for 2 to 6 h. Bacteria were removed, and eukaryotic cells were washed in phosphate-buffered saline (PBS) prior to fractionation by using either a homogenization or a digitonin extraction procedure. In the homogenization procedure, cells were harvested by using trypsin-EDTA, the cell suspension was then centrifuged at 400 g for 5 min and washed with PBS and then homogenization medium (250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl [pH 7.4]). The pellet was resuspended in homogenization medium and lysed with 16 strokes of a Potter-Elvehjem homogenizer. The sample was centrifuged at 1,000 x g to remove whole cells and nuclear debris and then centrifuged at 116,000 x g. The resulting pellet (membrane fraction) was resuspended in homogenization medium in an amount equivalent to the supernatant (cytosolic fraction). In the digitonin extraction procedure, cells were lysed in ice-cold digitonin extraction buffer (0.01% digitonin, 10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, and P8340 protease inhibitor cocktail [Sigma]). Lysed cells were separated into cytosolic and membrane fractions by centrifugation at 16,000 x g for 15 min at 4°C. Cytosolic and membrane fractions were equilibrated relative to volume in Laemmli sample buffer. All samples were resolved by SDS-PAGE and immunoblotted for Rac1, as described above. Similar results were obtained from both fractionation procedures.
Immunofluorescence staining. Cellular localization of endogenous Rac1 in relation to bacterial contact was examined by seeding HT-29 and J774A.1 cells on chamber slides (Nalge Nunc International, Rochester, NY) and coculturing for 2.5 or 5 h with strain PA103UT expressing ExoS or ExoS mutants. Cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, and then blocked with 1% BSA-5% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS for 30 min at room temperature. Prior to permeabilizing the cells, extracellular Pseudomonas was stained with a polyclonal guinea pig antibody to P. aeruginosa (Biogenesis, Kensington, NH), followed by incubation with anti-guinea pig Alexa Fluor 546 conjugate (Molecular Probes, Eugene, OR). Cells were then washed three times with PBS and refixed with 4% paraformaldehyde for 30 min. HT-29 cells were permeabilized and blocked for 30 min with 0.2% Triton X-100-1% BSA in PBS. J774A.1 cells were permeabilized with chilled 100% methanol for 10 min at –20°C and then blocked for 30 min at room temperature with 1% BSA-5% normal goat serum in PBS. Endogenous Rac1 was stained with an anti-Rac1 monoclonal antibody (BD Transduction Laboratories, Lexington, KY). Cells were washed with PBS and incubated with anti-mouse Alexa Fluor 488 conjugate (Molecular Probes). Slides were mounted in ProLong antifade reagent (Molecular Probes) and examined by confocal microscopy (Carl Zeiss, Inc., Jena, Germany).
RESULTS
Cell line dependence of ExoS ADP-ribosylation of Rac1. Rac1 ADP-ribosylation by ExoS was found to be cell line dependent. In coculture studies with the ExoS-producing strain 388 or the isogenic 388exoS mutant, Rac1 ADP-ribosylation was detected in human and simian epithelial and fibroblastic cells, after a 4- to 6-h coculture period (represented in Fig. 1A). In comparison, Rac1 ADP-ribosylation by TTS-ExoS was not detected in rodent cell lines or in macrophage or lymphocytic cell lines of either human or rodent origin (Fig. 1B), even though other LMWG proteins were ADP-ribosylated by ExoS in these cell lines (45, 47). ADP-ribosylation was assessed based on a decrease in protein mobility in SDS-PAGE analysis, which was previously determined to reflect the addition of an ADP-ribose moiety (12). Notably, Rac1 from cell lines that do not allow TTS-ExoS ADP-ribosylation can be ADP-ribosylated by ExoS in vitro, supporting the idea that in situ cellular properties, rather than intrinsic protein properties, were determining Rac1 ADP-ribosylation (12, 47).
Preferred site of ExoS ADP-ribosylation of Rac1. To further understand the effects of ExoS ADP-ribosylation on Rac1 function, residues targeted by ExoS ADPRT activity in Rac1 were explored. This analysis was approached by individually mutating the nine arginine residues in Rac1 (potential sites of ExoS ADP-ribosylation) to lysine and then examining the mutated proteins for their ability to be ADP-ribosylated by ExoS in vitro. Analyses of the efficiency of ExoS ADP-ribosylation of Rac1 (determined based on a shift in Rac1 mobility by SDS-PAGE) found the R66K and R68K Rac1 mutants to be less efficiently ADP-ribosylated by ExoS than wild-type (WT) Rac1 or the other Rac1 mutants (Fig. 2A). Using 32P-NAD to further assess ExoS ADP-ribosylation of Rac1 and Rac1 mutants, the R66K and R68K mutants were again found to incorporate radiolabeled ADP-ribose less efficiently than the other forms of Rac1. Based on densitometry measurements, the incorporation of radiolabeled ADP-ribose into R66K and R68K was calculated to be 194 ± 159 and 611 ± 230, respectively, which compared to densitometry measurements of 1,779 ± 122 for WT Rac1 and a mean densitometry measurement for the other Rac1 mutants of 2,615 ± 1,707 (range, 1,167 to 5,841). In dose-response studies, both the R66K and the R68K mutants exhibited a shift in mobility upon exposure to higher concentrations of ExoS (Fig. 2B). The results are consistent with R66 and R68 functioning as preferred but not absolute sites of ExoS ADP-ribosylation, with R66 appearing to be the most efficiently modified site.
Effect of ExoS ADP-ribosylation of Rac1 on function. (i) Interaction of ADP-ribosylated Rac1 with PAK1. R66 and R68 reside within the switch II domain of Rac1, a region that can affect GAP-mediated Rac1 inactivation. Activation of Rac1 results in structural changes in the switch I and switch II regions that allow GTP-bound Rac1 to interact with PAK1. Experimentally, Rac1 interaction with PAK1 can be used to assess Rac1 activation in pull-down reactions with PAK1-GST:GSH coupled agarose beads, as represented in Fig. 3A. Rac1 can be ADP-ribosylated by ExoS in either its GDP or GTP-bound form (Fig. 3B). In examining the effects of ExoS ADP-ribosylation of Rac1 on its interaction with PAK1, GTP (but not GDP)-bound ADP-ribosylated Rac1 was found to interact with PAK1 (Fig. 3C). The results indicate that ADP-ribosylation of Rac1 by ExoS (evident by the shift in Rac1 mobility) does not sterically interfere with Rac1 binding to PAK1.
To assess whether Rac1 ADP-ribosylation affected Rac1 activation or its ability to interact with PAK1 within eukaryotic cells, HT-29 human epithelial cells were cocultured with the ExoS-producing strain 388 or strain 388exoS, and Rac1 in cell lysates was examined for its ability to interact with PAK1-GST:GSH beads. As shown in Fig. 3D, Rac1 ADP-ribosylated by strain 388 interacted more efficiently with PAK1 than did Rac1 from cells treated with strain 388exoS or with no bacteria. The data support that ADP-ribosylation of Rac1 within eukaryotic cells by TTS-ExoS does not interfere with Rac1 interaction with PAK1 and appears to contribute to Rac1 activation. The results are consistent with ExoS ADP-ribosylating Rac1 at residue R66 or R68 interfering with GAP-mediated Rac1 inactivation, resulting in increased levels of active Rac1.
(ii) Cellular localization of ADP-ribosylated Rac1. Rac1, like other Rho-family proteins, cycles between a cytosolic, inactive GDP-bound form and a membrane-associated, active GTP-bound form (54). When cells are in steady state, Rac1 is primarily cytosolic and bound to RhoGDI. To assess the effects of TTS-ExoS on endogenous Rac1 localization, HT-29 cells were cocultured with strain 388 or 388exoS for 5 h, and cytosolic and membrane fractions were obtained by Potter-Elvehjem homogenization and centrifugation. As shown in Fig. 3E, Rac1 from cells exposed to non-ExoS-producing strain 388exoS localized primarily to the cytosol. However, upon exposure to ExoS-producing strain 388, Rac1 localized to the membrane fraction in conjunction with its ADP-ribosylation. The results are consistent with the ADP-ribosylation of Rac1 by TTS-ExoS, leading to membrane-associated, active GTP-bound Rac1.
Effect of ExoS GAP and ADPRT domains on Rac1 function. With the recognition that both ExoS GAP and ADPRT domains target Rac1, we next used ExoS GAP and ADPRT mutants to examine how the function of ExoS was coordinated in eukaryotic cells. For these analyses, WT ExoS, ExoS with mutant GAP (R146A), ExoS with mutant ADPRT (E379A/E381A), or ExoS with mutant GAP and ADPRT activities (R146A/E379A/E381A) were compared for their effects on Rac1 function when TTS translocated by strain PA103UT.
(i) Cellular localization of Rac1. To monitor how ExoS GAP and ADPRT domains contributed to the cellular localization of Rac1 in association with its ADP-ribosylation by TTS-ExoS, HT-29 and T-24 human epithelial cells were cocultured with strains expressing ExoS and ExoS mutants for 2, 4, or 6 h. Cells were then fractionated, and Rac1 localization to the cytosolic or membrane fractions was compared. After a 2-h coculture period (before Rac1 ADP-ribosylation), Rac1 remained primarily in the cytosolic fraction of both HT-29 and T-24 cells, as it did with control, nonbacterially treated cells (0) (Fig. 4). After a 6-h coculture period, Rac1 ADP-ribosylation was evident in cells exposed to strains expressing ADPRT-active ExoS (ExoS and R146A-ExoS GAP mutant), and Rac1 ADP-ribosylation was associated with its relocalization to the membrane faction. No relocalization of Rac1 was detected in association with ExoS-GAP activity. The finding that membrane localization required an active ADPRT domain and occurred in the presence or absence of active GAP highlights the independent nature of the effect of ExoS ADPRT activity on Rac1 localization.
(ii) Activation of Rac1. An effect of ExoS GAP activity on eukaryotic cell function has previously been recognized in studies of J774A.1 macrophages (45). Rac1 is not ADP-ribosylated by ExoS in J774A.1 cells (refer to Fig. 1), and in this cell line ExoS GAP activity is linked to Rac1 inactivation and the antiphagocytic function of ExoS (45). To directly compare the effects of ExoS GAP and ADPRT domains on Rac1 activation in relation to Rac1 ADP-ribosylation, Rac1 interaction with PAK1 was examined after exposure of J774A.1 and HT-29 cells to bacterial strains expressing ExoS GAP or ADPRT mutants. As shown in Fig. 5, levels of active Rac1 increased in HT-29 cells in response to ADP-ribosylation by ExoS (seen in ExoS and R146A-ExoS GAP mutant treated cells). In J774A.1 cells, where Rac1 is not ADP-ribosylated, the levels of active Rac1 increased in response to the ExoS GAP mutant and not in response to ExoS ADPRT activity. The increase in active Rac1 in J774A.1 cells treated with the ExoS GAP mutant is predicted to relate to the inability of mutant ExoS-GAP to downregulate Rac1 activity. As previously noted (45) and as also evident in Fig. 5, less activated Rac1 was detected in J774A.1 cells treated with the ExoS-R146A/E378A/E381A mutant than with the ExoS-R146A GAP mutant, although both strains lack active GAP (compare 146 and 146/379/81 treated J774A.1 cells). These comparisons provide evidence of a form of functional interaction between the ExoS ADPRT and GAP domains, which is reflected in indirect effects of ExoS ADPRT activity on Rac1 activation. We conclude from these studies that the targeting of ExoS ADPRT activity to Rac1 in HT-29 cells leads to Rac1 activation, and this coincides with the inability of ExoS GAP to effectively inactivate Rac1. When ExoS ADPRT activity does not target Rac1, as in J774A.1 cells, ExoS GAP effectively downregulates Rac1 activity.
Cellular localization of Rac1 in response to ExoS ADPRT activity. To gain further understanding of cellular mechanisms that might influence the differential targeting of TTS-ExoS GAP and ADPRT activity to Rac1, immunofluorescence studies were performed in HT-29 and J774A.1 cells stained for endogenous Rac1 in conjunction with bacteria expressing WT or mutant forms of ExoS. In the absence of bacteria (0), Rac1 was found to be diffusely distributed in the cytosol in HT-29 cells. However, in J774A.1 cells Rac1 was found to be more localized to the plasma membrane, with diffuse, weaker staining in the cytosol (Fig. 6). After a 2.5-h exposure of HT-29 cells to strain PA103UT expressing ADPRT-active ExoS (ExoS or R146A-GAP mutant), intense Rac1 staining was apparent at the site of bacterial contact. Rac1 also appeared to further accumulate in HT-29 cells in the vicinity of bacterial contact after a more prolonged, 5-h exposure to bacteria expressing ADPRT-active ExoS. No increased intensity or accumulation of Rac1 was evident in HT-29 cells exposed to strain PA103UT expressing E379A/E381A-ADPRT inactive ExoS after a 2.5- or 5-h coculture period. In examining J774A.1 macrophages, no Rac1 accumulation was evident at the site of contact with bacteria expressing ADPRT-active or E379A/E381A-ADPRT-inactive ExoS. However, Rac1-associated membrane ruffling was evident in J774A.1 cells in the vicinity of bacteria expressing the ExoS-R146A GAP mutant. These results are consistent with previous studies which found that exposure of J774A.1 cells to an ExoS-R146A GAP mutant led to increased membrane ruffling in association with Rac1 activation (45).
Together, these studies identify differences in the cellular distribution of Rac1 in HT-29 and J774A.1 macrophages in association with their differences in the targeting of TTS-ExoS GAP and ADPRT activities. In HT-29 cells, Rac1 resides primarily within the cytosol and relocalizes to the site of bacterial contact in association with ADPRT-active ExoS and Rac1 ADP-ribosylation. In J774A.1 cells, which maintain a high steady-state levels of plasma membrane associated Rac1, Rac1 does not accumulate at the site of bacterial contact, nor is Rac1 ADP-ribosylated. The identification of differences in the cellular localization of Rac1 in association with its ADP-ribosylation by TTS-ExoS provides further insight into possible mechanisms used by host cells to differentially coordinate and target ExoS GAP and ADPRT activity to Rac1.
DISCUSSION
Many microbial pathogens utilize eukaryotic cell cytoskeletal structure in their strategy of pathogenesis. Host cytoskeletal rearrangement has also proven to be a common mechanism of bacterial TTS effectors. TTS is induced upon bacterial contact with the host cell and leads to the formation of a needle-like structure that translocates bacterial effectors that affect host cell function. Consistent with the contact-mediated nature of TTS induction, evidence supports the involvement of TTS in the establishment of the infectious process of many gram-negative bacteria, including P. aeruginosa (6, 8, 19, 31, 34, 46). Although the genes regulating TTS and encoding the needle-structure are conserved among gram-negative bacteria, TTS effectors are bacterium specific (23). The diversity observed among TTS effectors attests to the ability of bacteria to evolve or acquire effectors that accommodate their specific lifestyle within a particular eukaryotic niche.
TTS effectors differ in structure and function from classic bacterial AB toxins, such as diphtheria toxin, in essentially two ways. First, TTS effectors lack cell receptor binding regions that allow their direct internalization into eukaryotic cells. Second, multiple TTS effectors can be translocated into eukaryotic cells, where they can function in a coordinate manner. This again differs from classic bacterial toxins that generally exert independent effects on host cells. The coordinated function of TTS effectors became evident during studies of Salmonella enterica serovar Typhimurium TTS effectors SopE, SopB, and SptP. In the salmonella infectious process, SopE and SopB stimulate Cdc42 and Rac activation, leading to actin cytoskeletal alterations that allow bacterial internalization (16, 48). This is followed by the inactivation of Cdc42 and Rac1 by SptP GAP activity, allowing host cell recovery and Salmonella intracellular growth to proceed (15).
The bifunctionality of ExoS, and the recognized potential for its GAP and ADPRT domains to target the same eukaryotic LMWG proteins, identifies ExoS as a model to study how the functions of a single TTS effector might be coordinated within the host cell. This potential can be further developed based on the finding that the coordinate function of ExoS GAP and ADPRT activities varies in different cell lines (45, 47), and that the GAP and ADPRT activities of ExoS can both target Rac1 but exert opposite effects on Rac1 function.
Using Rac1 to monitor ExoS GAP and ADPRT function within eukaryotic cells, the ADP-ribosylation of Rac1 by ExoS was detected in human cell lines, such as HT-29 cells, but not in rodent or macrophage cell lines and was associated with Rac1 membrane localization and activation. In cell lines where Rac1 was ADP-ribosylated, ExoS GAP exerted limited detectable effects on Rac1 function. These results indicate the potential of ExoS ADPRT activity to override the effects of ExoS GAP activity on Rac1 function in cell lines where Rac1 is ADP-ribosylated. In examining the molecular mechanism of Rac1 activation by ExoS ADPRT activity, R66 and R68 were identified as preferred sites of ExoS ADP-ribosylation in in vitro reactions, with R66 appearing to be the most preferred site. These residues reside within the Switch II domain of Rac1 in a region that can affect GAP-mediated Rac1 inactivation. The shift in Rac1 mobility detected by SDS-PAGE upon ADP-ribosylation by ExoS is characteristically less than that observed for other LMWG-protein substrates of ExoS ADPRT activity, such as Ras and RalA, where preferred sites of ExoS ADP-ribosylation are within the Switch I region (11, 12, 17). Two-dimensional electrophoresis analyses support that Rac1 is likely ADP-ribosylated at only one site upon its TTS-mediated translocation into the host cell (12). The single modification of Rac1 by TTS-ExoS within eukaryotic cells is also evident in the decreased shift in mobility of Rac1 ADP-ribosylated by ExoS ex vivo versus in vitro and implicates a cellular bias in the actual site of ADP-ribosylation by ExoS within the host cell. Although the site of ExoS ADP-ribosylation of Rac1 within the cell was not directly determined in these studies, the increased levels of active Rac1 detected in response to ExoS ADPRT activity are consistent with ExoS ADP-ribosylating Rac1 at either R66 or R68 and this leading to an interference of GAP-mediated inactivation of Rac1.
Insight into cellular differences in Rac1 ADP-ribosylation was gained through immunofluorescence studies monitoring endogenous Rac1 localization relative to bacterial contact and TTS translocation of ADPRT-active ExoS. Rac1 ADP-ribosylation occurs as a later event in the P. aeruginosa TTS-mediated infectious process (12). This delay suggests an initial inaccessibility of Rac1 to TTS-ExoS, which can be explained by the normal cytosolic localization. In HT-29 cells, where Rac1 is ADP-ribosylated by ExoS, Rac1 was diffusely distributed in the cytosol prior to exposure to bacteria. Upon contact with bacteria expressing ADPRT-active ExoS, Rac1 localized to the site of bacterial contact. In J774A.1 cells, where Rac1 is not ADP-ribosylated by ExoS, intense Rac1 staining at the plasma membrane was evident prior to exposure to bacteria. Rac1 staining at the plasma membrane intensified upon prolonged exposure of J774.1 cells to bacteria but not at the site of bacterial contact and independently of ExoS ADPRT activity. Evidence of ExoS-GAP accessibility to Rac1 was indirectly evident in J774A.1 cells exposed to an ExoS-GAP mutant by the enhanced lamellopodia formation at the site of bacterial contact, in association with increased levels of active (not inactivated) Rac1. The identification of differences in the steady-state localization of Rac1, and differences in the movement of Rac1 in response to TTS-ExoS in HT-29 and J774A.1 cells supports the potential for host cell properties that affect Rac1 localization to influence the targeting of ExoS ADPRT activity to Rac1.
Our findings on the effects of ExoS GAP and ADPRT activities on Rac1 function have allowed the development of a model to explain the coordinate and differential targeting of ExoS to Rac1 in host cells. This model predicts that cellular properties that direct or position Rac1 at the plasma membrane favor its initial accessibility to TTS-ExoS GAP, which results in Rac1 inactivation, as occurs in J774A.1 macrophages. In cells where Rac1 is not efficiently positioned at the plasma membrane, TTS-ExoS gains access to cytosolic Rac1 by using host cell trafficking mechanisms. In this context, ExoS-ADPRT activity targets Rac1 and causes Rac1 activation, as occurs in HT-29 cells. The model also predicts that the coordinate function of the ExoS GAP and ADPRT relates to GAP activity targeting plasma membrane-associated Rac1, whereas ExoS ADPRT activity targets cytosolic Rac1. The unknown factors of this model are (i) cellular properties that position Rac1 at the plasma membrane to facilitate its accessibility to ExoS-GAP and (ii), alternatively, host cell mechanisms that allow ExoS-ADPRT activity to be trafficked to cytosolic Rac1. However, it is known that these properties are integral to cell function, since ExoS ADP-ribosylation of Rac1 is host cell dependent and is an unalterable phenotype.
Gaining an understanding of how bacterial proteins manipulate Rho-family proteins has not only provided insight into mechanisms of pathogenesis but has also provided tools to study eukaryotic cell signaling processes that affect cell structure, migration, phagocytosis, and adhesion. Studies of the effects of ExoS on Rac1 have similarly provided insight into how the activity of a bifunctional TTS effector can be coordinated within the host cell and also into how host cell properties can differentially influence Rac1 localization and function.
ACKNOWLEDGMENTS
We thank Dara Frank (Medical College of Wisconsin, Milwaukee) for providing the P. aeruginosa strains used in these studies and Jennifer Meredith and Timothy Vincent for valuable contributions to this project.
This study was supported by Public Health Services grant NIH-NIAID 45569, by the Medical University of South Carolina Institutional Research Funds, and by the Mary C. Babb Cancer Center Foundation Fund 2V882 from West Virginia University.
C.L.R., E.A.R., and D.M.V. contributed equally to this study.
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Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina 29425
ABSTRACT
Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.
INTRODUCTION
Pseudomonas aeruginosa is a ubiquitous, environmentally beneficial bacterium that can adapt to become a highly virulent opportunistic pathogen in compromised individuals. The versatility and pathogenicity of P. aeruginosa are multifactorial and relate to its ability to respond to its environment by the regulated production of a variety of cell-associated and extracellular products. The establishment of infection begins with the adherence of P. aeruginosa to host cells through type IV pili or nonpilus adherence mechanisms (42, 50, 52). After colonization, the organism ensures its survival in the host through the secretion of virulence factors, including exotoxin A (24), hemolysins (40), elastases LasA and LasB (2, 20, 21), and pigments (53). In addition to secreted virulence factors, P. aeruginosa is able to directly affect eukaryotic cell function through the contact-dependent translocation of effector proteins by the type III secretion system (59). Four P. aeruginosa-type III secretory (TTS) effector proteins have been identified, ExoS, ExoT, ExoU, and ExoY, each affecting eukaryotic cell function differently. Notably, the TTS system is found in both clinical and environmental P. aeruginosa isolates, suggesting an essential role of TTS in P. aeruginosa overall growth and survival (8, 9, 49). The integral relationship between P. aeruginosa TTS effectors and eukaryotic cell function is also evident in the requirement of eukaryotic cofactors for the enzymatic function of ExoS, ExoT, ExoU, and ExoY (4, 14, 51, 60).
ExoS is the most extensively studied of the TTS effectors. Structure-function analyses identified ExoS as a bifunctional toxin, including separate domains with GTPase-activating protein (GAP) and carboxy-terminal ADP-ribosyltransferase (ADPRT) activities (18, 25, 26). In vitro studies and cell culture models of infection found the GAP activity of ExoS to target low-molecular-weight G (LMWG) proteins in the Rho family that regulate cytoskeletal structure (27, 41). Arginine at position 146 (R146) was identified as essential for ExoS GAP activity (18), and in three-dimensional structural analysis R146 was found to function as an arginine finger in stabilizing the transition state of the Rac-GTPase reaction (58). The ADPRT activity of ExoS also targets LMWG proteins but preferentially targets Ras superfamily proteins that are integral to cell signaling pathways (3, 5, 11, 12, 22, 33). In vitro kinetic analyses identified two residues involved in the ADPRT reaction of ExoS. Glutamic acid at position 379 (E379) contributes to the transfer of ADP-ribose to target substrates, and glutamic acid at position 381 (E381) functions as a catalytic residue (43).
Eukaryotic cell culture models of infection found TTS-translocated ExoS to have multiple and cell line-dependent effects on cell function. ExoS affects the cell growth, morphology, and adherence of epithelial and fibroblastic cell lines and also exerts antiphagocytic effects on macrophages (13, 38, 39). When ExoS GAP or ADPRT mutants were used to assess the role of each domain on HT-29 epithelial cell function, ADPRT activity was found to be required for effects of ExoS on cell growth and morphology, while minimal effects of ExoS GAP activity were detected (10). In studies of J774A.1 macrophages, ExoS GAP activity was found to exert an antiphagocytic function, whereas ExoS ADPRT activity was again linked to effects on cell morphology and adherence (45). The identification of effects of ExoS ADPRT activity in some cell lines and effects of ExoS GAP in other cell lines led us to explore how ExoS GAP and ADPRT functions were being coordinated and targeted within the host cell.
In examining the mechanism for the effects of ExoS ADPRT activity on cell morphology, a cell-line-dependent hierarchy of LMWG protein substrate modification was detected (12, 47). In human epithelial cells, early targets of ExoS ADPRT activity included plasma membrane-associated LMWG proteins, Ras and RalA, followed by membrane-associated Rab5, then Rab8, Rab11, and Rab7, and finally Rac1 and Cdc42 (12). Notably, the sequential targeting of ExoS ADPRT activity to substrates in human epithelial cells mimicked a vesicular retrograde trafficking process. In linking substrate modification to alterations in cell morphology, Ras and RalA are known to indirectly affect cytoskeletal structure, whereas Rho family proteins, such as Rac1, provide a more direct link for effects of ExoS on cytoskeletal structure. The recent identification of the non-G-protein substrates of ExoS ADPRT activity, the Ezrin/Radixin/Moesin (ERM) family of proteins, provides another mechanism by which ExoS can affect cytoskeleton dynamics (32). Notably, in studies of ExoS substrate specificity, it was recognized that ExoS GAP and ADPRT activity can both target Rac1, identifying Rac1 as a candidate protein for studying how the domain function of ExoS might be coordinated within the eukaryotic cell.
Rac1 is a member of the Rho family of LMWG proteins involved in the reorganization of actin at the plasma membrane, leading to membrane ruffling and the formation of lamellipodia and focal complexes (36, 44). Like all LMWG proteins, the activation state of Rac1 depends upon guanine nucleotide binding. Guanine nucleotide exchange factors (GEFs) and GAPs modulate the nucleotide-bound state of LMWG proteins via binding to the Switch I and Switch II regions (57). GEFs catalyze the exchange of GDP for GTP, and GAPs activate the intrinsic GTPase activity of LMWG proteins. The Switch domains form a maneuverable pocket cleft that undergoes a conformational change in response to GTP binding and GTP hydrolysis. This structural movement alters the ability of LMWG proteins to interact with and signal through downstream cellular effectors. GTP bound Rac1 associates with the membrane through an isoprenylated C terminus. Upon the inactivation of Rac1 by GAP, RhoGDI, a guanine dissociation inhibitor, dissociates GDP-bound Rac1 from the membrane and sequesters it in a GDP-bound state in the cytosol (30, 35, 37).
The present study examines the effects of ExoS ADPRT and GAP activities on Rac1, using the differential effects of the two domains on Rac1 function, and cell line differences in Rac1 ADP-ribosylation to examine how ExoS activity is coordinated within eukaryotic cells. The ADP-ribosylation of Rac1 in human epithelial cell lines was found to be associated with Rac1 activation and membrane localization. Conversely, in cell lines where Rac1 was not ADP-ribosylated, ExoS GAP functioned to maintain Rac1 in an inactive form. The studies highlight the potential of host cell properties to dictate and coordinate the function of the ExoS GAP and ADPRT domains.
MATERIALS AND METHODS
Bacterial strains and culture conditions. P. aeruginosa strains 388 (25), 388exoS (an ExoS isogenic mutant of strain 388 [28]), and strain PA103exoUexoT::Tc (PA103UT; a derivative of strain PA103 that lacks production of known TTS effectors [56]) were provided by Dara Frank (Medical College of Wisconsin, Milwaukee). Strain PA103UT was used to express pUCP plasmid-encoded ExoS, an R146A ExoS-GAP mutant, an E379A/E381A ExoS-ADPRT mutant, or a GAP-ADPRT inactive R146A/E379A/E381A ExoS mutant, which have been previously described (10). P. aeruginosa strains were grown in ExoS induction medium (TSBD-N) (25) for 16 h prior to coculture with eukaryotic cells, as previously described (39).
Eukaryotic cell culture. All cell lines, with the exception of RHEC and WI-38, were obtained from the American Type Cell Culture Collection (Manassas, VA). RHEC cells were provided by Carwile LeRoy, and WI-38 cells were provided by Daohong Zhou; both are investigators from the Medical University of South Carolina (Charleston). RHEC and WI-38 cells were cultured in Dulbecco modified Eagle medium. Other cell lines were cultured in media specified by the American Type Culture Collection. All media were supplemented with 10% fetal bovine serum, 100 U of penicillin/ml, and 100 μg of streptomycin/ml (Gibco-BRL, Gaithersburg, MD). Cells were cultured at 37°C in 5% CO2 to 95% air.
Coculture of eukaryotic cells with bacteria. For bacterial infection, eukaryotic cells were seeded at a density of 105 cells/ml and grown for 24, 48, or 72 h, depending on the cell line, to 60 to 90% confluence. Medium was removed, and bacteria were diluted based on an optical density at 590 nm of ca. 107 CFU/ml in cell line-specific medium containing 0.6% bovine serum albumin (BSA). Bacteria were added to eukaryotic cells at a multiplicity of infection of 30 to 100, and cells were cocultured for 2 to 6 h as indicated. All experiments included eukaryotic cells cultured in medium containing 0.6% BSA, but without bacteria, as a control.
Analysis of ADP-ribosylation of LMWG proteins. Ex vivo ADP-ribosylation of endogenous Rac1 by TTS-ExoS was assessed based on altered protein mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses after the coculture of eukaryotic cells with bacteria, as previously described (12). To examine Rac1 ADP-ribosylation, cells were lysed in Laemmli electrophoresis buffer (29), resolved by SDS-12% PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) (55), and probed with a mouse monoclonal antibody to Rac1 (BD-Transduction Laboratories, Lexington, KY), followed by the addition of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (Fc specific; Sigma). Rac1 was visualized by enhanced chemiluminescence (Amersham Pharmacia, Arlington Heights, IL).
In vitro ADP-ribosylation of Rac1 was performed with 10 μg of purified Rac1 (12) loaded with 10 μM GTPS, according to the procedure of Antonny et al. (1). For ADP-ribosylation reactions, 1 μg of Rac1-GTPS was incubated for 30 to 60 min at 25°C in 200 mM Tris acetate (pH 6.0) containing the indicated amount of purified His-tagged ExoS, 10 mM NAD, 1 mM MgCl2, and a source of the eukaryotic ExoS ADPRT cofactor—either 10 μl of wheat germ extract or 1 μM 14-3-3 protein (Upstate Biotechnology, Lake Placid, NY)—in a final reaction volume of 50 μl.
To monitor the incorporation of ADP-ribose into Rac1 and Rac1 mutants (see below), 2 μCi of nicotinamide adenine [adenylate-32P]dinucleotide (32P-NAD; 200 mCi/mmol; Amersham Biosciences) was added to 15-μl ADPRT reaction mixtures containing 200 mM Tris acetate, 100 μM NAD, 150 ng of His-Rac1 or His-Rac1 mutant, 250 nM 14-3-3, and either 0 or 5 nM purified His-ExoS. The reaction was terminated with 4x Laemmli sample buffer and heating for 3 min at 95°C. Reactions were resolved by SDS-PAGE, analyzed by autoradiography, and relative differences in the incorporation of radioactivity into Rac1 proteins were quantified by densitometry.
Analysis of the site of ADP-ribosylation by ExoS on Rac1. To examine the preferred site of ExoS ADP-ribosylation on Rac1, each of the nine arginine residues of Rac1 (R66, R68, R94, R102, R120, R163, R174, R185, and R187), potential targets of ExoS ADP-ribosylation, was mutated to a lysine. Mutations were introduced into Rac1 cloned into pET15b (12) by using the QuikChange PCR-based site-directed mutagenesis system (Strategene, La Jolla, CA). The primers designed to introduce specific mutations along with diagnostic restriction sites, are listed in Table 1. Rac1 proteins were expressed and purified as His-tagged proteins, by using previously described methods (7, 12).
PAK1 coprecipitation (pull-down) of active Rac1 in vitro and ex vivo. GST-PAK1:GSH beads for pull-down reactions were constructed by amplifying the cDNA corresponding to the Rac/Cdc42 binding domain (amino acids 50 to 134 of PAK1) (PBD) by using the forward primer 5'-CCCGGATCCAAAAAGAAAGAGAAAGAGCGGCC-3', containing a BamHI restriction site, and the reverse primer 5'-CCCGAATTCAGCTGACTTATCTGTAAAGCTCATG-3', containing an EcoRI restriction site (restriction sites are underlined). The PCR product was digested with BamHI and EcoRI and ligated into the pGEX4T vector (Amersham Pharmacia) for expression as a glutathione S-transferase (GST) fusion protein. The construct was verified by DNA sequence analysis and transformed into E. coli BL21(DE3) for protein expression. After induction with 1 mM IPTG (isopropyl--D-thiogalactopyranoside), bacteria were lysed by using a French pressure cell (Thermo IEC, Needham Heights, MA), and cleared lysates were bound to glutathione (GSH)-agarose beads. PAK1-GST bound to GSH-agarose beads was quantified by SDS-PAGE and gel densitometry analysis with BSA as a standard.
In vitro Rac1-PAK1 pull-down (PD) reactions were performed by adding 100 ng of either Rac1-GDP, Rac1-GTPS or ADP-ribosylated Rac1-GTPS to 5 μg of PAK1-GST:GSH-agarose beads in coimmunoprecipitation (Co-IP) lysis buffer (30 mM HEPES [pH 7.5], 1% Triton X-100, 10 mM NaCl, 10% glycerol, 1 mM EGTA, 25 mM NaF, 1 mM Na3VO4, 10 mM -glycerophosphate, and protease inhibitors). The mixture was rotated for 1 h at 4°C, the beads were washed three or four times with Co-IP wash buffer (50 mM HEPES [pH 7.5], 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, 20 mM NaF), resuspended in Laemmli electrophoresis sample buffer, resolved by SDS-PAGE, and immunoblotted for Rac1 as described above.
Ex vivo Rac1-PAK1 PD reactions were performed after a 4- to 6-h coculture of eukaryotic cells with bacteria, as indicated. Eukaryotic cells were lysed in Co-IP lysis buffer for 30 min on ice, and lysates were cleared by centrifugation for 5 min at 16,000 x g at 4°C. An aliquot of cleared lysate was removed for determination of total cellular Rac1. PAK1-GST:GSH beads (10 μg) were added to the remaining lysate and the PD reaction mixture was processed and analyzed for Rac1 as described above. Cells cultured without bacteria, cells cultured with non-ExoS-producing P. aeruginosa strains (388S or PA103UT pUCP), and cells cultured with PA103UT expressing GAP-ADPRT inactive ExoS (ExoS-R146A/E379A/E381A) served as negative controls in these studies.
Cellular localization of Rac1. Eukaryotic cells were seeded in 60- or 100-mm dishes and cocultured with the indicated bacterial strain for 2 to 6 h. Bacteria were removed, and eukaryotic cells were washed in phosphate-buffered saline (PBS) prior to fractionation by using either a homogenization or a digitonin extraction procedure. In the homogenization procedure, cells were harvested by using trypsin-EDTA, the cell suspension was then centrifuged at 400 g for 5 min and washed with PBS and then homogenization medium (250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl [pH 7.4]). The pellet was resuspended in homogenization medium and lysed with 16 strokes of a Potter-Elvehjem homogenizer. The sample was centrifuged at 1,000 x g to remove whole cells and nuclear debris and then centrifuged at 116,000 x g. The resulting pellet (membrane fraction) was resuspended in homogenization medium in an amount equivalent to the supernatant (cytosolic fraction). In the digitonin extraction procedure, cells were lysed in ice-cold digitonin extraction buffer (0.01% digitonin, 10 mM PIPES, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 5 mM EDTA, and P8340 protease inhibitor cocktail [Sigma]). Lysed cells were separated into cytosolic and membrane fractions by centrifugation at 16,000 x g for 15 min at 4°C. Cytosolic and membrane fractions were equilibrated relative to volume in Laemmli sample buffer. All samples were resolved by SDS-PAGE and immunoblotted for Rac1, as described above. Similar results were obtained from both fractionation procedures.
Immunofluorescence staining. Cellular localization of endogenous Rac1 in relation to bacterial contact was examined by seeding HT-29 and J774A.1 cells on chamber slides (Nalge Nunc International, Rochester, NY) and coculturing for 2.5 or 5 h with strain PA103UT expressing ExoS or ExoS mutants. Cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, and then blocked with 1% BSA-5% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS for 30 min at room temperature. Prior to permeabilizing the cells, extracellular Pseudomonas was stained with a polyclonal guinea pig antibody to P. aeruginosa (Biogenesis, Kensington, NH), followed by incubation with anti-guinea pig Alexa Fluor 546 conjugate (Molecular Probes, Eugene, OR). Cells were then washed three times with PBS and refixed with 4% paraformaldehyde for 30 min. HT-29 cells were permeabilized and blocked for 30 min with 0.2% Triton X-100-1% BSA in PBS. J774A.1 cells were permeabilized with chilled 100% methanol for 10 min at –20°C and then blocked for 30 min at room temperature with 1% BSA-5% normal goat serum in PBS. Endogenous Rac1 was stained with an anti-Rac1 monoclonal antibody (BD Transduction Laboratories, Lexington, KY). Cells were washed with PBS and incubated with anti-mouse Alexa Fluor 488 conjugate (Molecular Probes). Slides were mounted in ProLong antifade reagent (Molecular Probes) and examined by confocal microscopy (Carl Zeiss, Inc., Jena, Germany).
RESULTS
Cell line dependence of ExoS ADP-ribosylation of Rac1. Rac1 ADP-ribosylation by ExoS was found to be cell line dependent. In coculture studies with the ExoS-producing strain 388 or the isogenic 388exoS mutant, Rac1 ADP-ribosylation was detected in human and simian epithelial and fibroblastic cells, after a 4- to 6-h coculture period (represented in Fig. 1A). In comparison, Rac1 ADP-ribosylation by TTS-ExoS was not detected in rodent cell lines or in macrophage or lymphocytic cell lines of either human or rodent origin (Fig. 1B), even though other LMWG proteins were ADP-ribosylated by ExoS in these cell lines (45, 47). ADP-ribosylation was assessed based on a decrease in protein mobility in SDS-PAGE analysis, which was previously determined to reflect the addition of an ADP-ribose moiety (12). Notably, Rac1 from cell lines that do not allow TTS-ExoS ADP-ribosylation can be ADP-ribosylated by ExoS in vitro, supporting the idea that in situ cellular properties, rather than intrinsic protein properties, were determining Rac1 ADP-ribosylation (12, 47).
Preferred site of ExoS ADP-ribosylation of Rac1. To further understand the effects of ExoS ADP-ribosylation on Rac1 function, residues targeted by ExoS ADPRT activity in Rac1 were explored. This analysis was approached by individually mutating the nine arginine residues in Rac1 (potential sites of ExoS ADP-ribosylation) to lysine and then examining the mutated proteins for their ability to be ADP-ribosylated by ExoS in vitro. Analyses of the efficiency of ExoS ADP-ribosylation of Rac1 (determined based on a shift in Rac1 mobility by SDS-PAGE) found the R66K and R68K Rac1 mutants to be less efficiently ADP-ribosylated by ExoS than wild-type (WT) Rac1 or the other Rac1 mutants (Fig. 2A). Using 32P-NAD to further assess ExoS ADP-ribosylation of Rac1 and Rac1 mutants, the R66K and R68K mutants were again found to incorporate radiolabeled ADP-ribose less efficiently than the other forms of Rac1. Based on densitometry measurements, the incorporation of radiolabeled ADP-ribose into R66K and R68K was calculated to be 194 ± 159 and 611 ± 230, respectively, which compared to densitometry measurements of 1,779 ± 122 for WT Rac1 and a mean densitometry measurement for the other Rac1 mutants of 2,615 ± 1,707 (range, 1,167 to 5,841). In dose-response studies, both the R66K and the R68K mutants exhibited a shift in mobility upon exposure to higher concentrations of ExoS (Fig. 2B). The results are consistent with R66 and R68 functioning as preferred but not absolute sites of ExoS ADP-ribosylation, with R66 appearing to be the most efficiently modified site.
Effect of ExoS ADP-ribosylation of Rac1 on function. (i) Interaction of ADP-ribosylated Rac1 with PAK1. R66 and R68 reside within the switch II domain of Rac1, a region that can affect GAP-mediated Rac1 inactivation. Activation of Rac1 results in structural changes in the switch I and switch II regions that allow GTP-bound Rac1 to interact with PAK1. Experimentally, Rac1 interaction with PAK1 can be used to assess Rac1 activation in pull-down reactions with PAK1-GST:GSH coupled agarose beads, as represented in Fig. 3A. Rac1 can be ADP-ribosylated by ExoS in either its GDP or GTP-bound form (Fig. 3B). In examining the effects of ExoS ADP-ribosylation of Rac1 on its interaction with PAK1, GTP (but not GDP)-bound ADP-ribosylated Rac1 was found to interact with PAK1 (Fig. 3C). The results indicate that ADP-ribosylation of Rac1 by ExoS (evident by the shift in Rac1 mobility) does not sterically interfere with Rac1 binding to PAK1.
To assess whether Rac1 ADP-ribosylation affected Rac1 activation or its ability to interact with PAK1 within eukaryotic cells, HT-29 human epithelial cells were cocultured with the ExoS-producing strain 388 or strain 388exoS, and Rac1 in cell lysates was examined for its ability to interact with PAK1-GST:GSH beads. As shown in Fig. 3D, Rac1 ADP-ribosylated by strain 388 interacted more efficiently with PAK1 than did Rac1 from cells treated with strain 388exoS or with no bacteria. The data support that ADP-ribosylation of Rac1 within eukaryotic cells by TTS-ExoS does not interfere with Rac1 interaction with PAK1 and appears to contribute to Rac1 activation. The results are consistent with ExoS ADP-ribosylating Rac1 at residue R66 or R68 interfering with GAP-mediated Rac1 inactivation, resulting in increased levels of active Rac1.
(ii) Cellular localization of ADP-ribosylated Rac1. Rac1, like other Rho-family proteins, cycles between a cytosolic, inactive GDP-bound form and a membrane-associated, active GTP-bound form (54). When cells are in steady state, Rac1 is primarily cytosolic and bound to RhoGDI. To assess the effects of TTS-ExoS on endogenous Rac1 localization, HT-29 cells were cocultured with strain 388 or 388exoS for 5 h, and cytosolic and membrane fractions were obtained by Potter-Elvehjem homogenization and centrifugation. As shown in Fig. 3E, Rac1 from cells exposed to non-ExoS-producing strain 388exoS localized primarily to the cytosol. However, upon exposure to ExoS-producing strain 388, Rac1 localized to the membrane fraction in conjunction with its ADP-ribosylation. The results are consistent with the ADP-ribosylation of Rac1 by TTS-ExoS, leading to membrane-associated, active GTP-bound Rac1.
Effect of ExoS GAP and ADPRT domains on Rac1 function. With the recognition that both ExoS GAP and ADPRT domains target Rac1, we next used ExoS GAP and ADPRT mutants to examine how the function of ExoS was coordinated in eukaryotic cells. For these analyses, WT ExoS, ExoS with mutant GAP (R146A), ExoS with mutant ADPRT (E379A/E381A), or ExoS with mutant GAP and ADPRT activities (R146A/E379A/E381A) were compared for their effects on Rac1 function when TTS translocated by strain PA103UT.
(i) Cellular localization of Rac1. To monitor how ExoS GAP and ADPRT domains contributed to the cellular localization of Rac1 in association with its ADP-ribosylation by TTS-ExoS, HT-29 and T-24 human epithelial cells were cocultured with strains expressing ExoS and ExoS mutants for 2, 4, or 6 h. Cells were then fractionated, and Rac1 localization to the cytosolic or membrane fractions was compared. After a 2-h coculture period (before Rac1 ADP-ribosylation), Rac1 remained primarily in the cytosolic fraction of both HT-29 and T-24 cells, as it did with control, nonbacterially treated cells (0) (Fig. 4). After a 6-h coculture period, Rac1 ADP-ribosylation was evident in cells exposed to strains expressing ADPRT-active ExoS (ExoS and R146A-ExoS GAP mutant), and Rac1 ADP-ribosylation was associated with its relocalization to the membrane faction. No relocalization of Rac1 was detected in association with ExoS-GAP activity. The finding that membrane localization required an active ADPRT domain and occurred in the presence or absence of active GAP highlights the independent nature of the effect of ExoS ADPRT activity on Rac1 localization.
(ii) Activation of Rac1. An effect of ExoS GAP activity on eukaryotic cell function has previously been recognized in studies of J774A.1 macrophages (45). Rac1 is not ADP-ribosylated by ExoS in J774A.1 cells (refer to Fig. 1), and in this cell line ExoS GAP activity is linked to Rac1 inactivation and the antiphagocytic function of ExoS (45). To directly compare the effects of ExoS GAP and ADPRT domains on Rac1 activation in relation to Rac1 ADP-ribosylation, Rac1 interaction with PAK1 was examined after exposure of J774A.1 and HT-29 cells to bacterial strains expressing ExoS GAP or ADPRT mutants. As shown in Fig. 5, levels of active Rac1 increased in HT-29 cells in response to ADP-ribosylation by ExoS (seen in ExoS and R146A-ExoS GAP mutant treated cells). In J774A.1 cells, where Rac1 is not ADP-ribosylated, the levels of active Rac1 increased in response to the ExoS GAP mutant and not in response to ExoS ADPRT activity. The increase in active Rac1 in J774A.1 cells treated with the ExoS GAP mutant is predicted to relate to the inability of mutant ExoS-GAP to downregulate Rac1 activity. As previously noted (45) and as also evident in Fig. 5, less activated Rac1 was detected in J774A.1 cells treated with the ExoS-R146A/E378A/E381A mutant than with the ExoS-R146A GAP mutant, although both strains lack active GAP (compare 146 and 146/379/81 treated J774A.1 cells). These comparisons provide evidence of a form of functional interaction between the ExoS ADPRT and GAP domains, which is reflected in indirect effects of ExoS ADPRT activity on Rac1 activation. We conclude from these studies that the targeting of ExoS ADPRT activity to Rac1 in HT-29 cells leads to Rac1 activation, and this coincides with the inability of ExoS GAP to effectively inactivate Rac1. When ExoS ADPRT activity does not target Rac1, as in J774A.1 cells, ExoS GAP effectively downregulates Rac1 activity.
Cellular localization of Rac1 in response to ExoS ADPRT activity. To gain further understanding of cellular mechanisms that might influence the differential targeting of TTS-ExoS GAP and ADPRT activity to Rac1, immunofluorescence studies were performed in HT-29 and J774A.1 cells stained for endogenous Rac1 in conjunction with bacteria expressing WT or mutant forms of ExoS. In the absence of bacteria (0), Rac1 was found to be diffusely distributed in the cytosol in HT-29 cells. However, in J774A.1 cells Rac1 was found to be more localized to the plasma membrane, with diffuse, weaker staining in the cytosol (Fig. 6). After a 2.5-h exposure of HT-29 cells to strain PA103UT expressing ADPRT-active ExoS (ExoS or R146A-GAP mutant), intense Rac1 staining was apparent at the site of bacterial contact. Rac1 also appeared to further accumulate in HT-29 cells in the vicinity of bacterial contact after a more prolonged, 5-h exposure to bacteria expressing ADPRT-active ExoS. No increased intensity or accumulation of Rac1 was evident in HT-29 cells exposed to strain PA103UT expressing E379A/E381A-ADPRT inactive ExoS after a 2.5- or 5-h coculture period. In examining J774A.1 macrophages, no Rac1 accumulation was evident at the site of contact with bacteria expressing ADPRT-active or E379A/E381A-ADPRT-inactive ExoS. However, Rac1-associated membrane ruffling was evident in J774A.1 cells in the vicinity of bacteria expressing the ExoS-R146A GAP mutant. These results are consistent with previous studies which found that exposure of J774A.1 cells to an ExoS-R146A GAP mutant led to increased membrane ruffling in association with Rac1 activation (45).
Together, these studies identify differences in the cellular distribution of Rac1 in HT-29 and J774A.1 macrophages in association with their differences in the targeting of TTS-ExoS GAP and ADPRT activities. In HT-29 cells, Rac1 resides primarily within the cytosol and relocalizes to the site of bacterial contact in association with ADPRT-active ExoS and Rac1 ADP-ribosylation. In J774A.1 cells, which maintain a high steady-state levels of plasma membrane associated Rac1, Rac1 does not accumulate at the site of bacterial contact, nor is Rac1 ADP-ribosylated. The identification of differences in the cellular localization of Rac1 in association with its ADP-ribosylation by TTS-ExoS provides further insight into possible mechanisms used by host cells to differentially coordinate and target ExoS GAP and ADPRT activity to Rac1.
DISCUSSION
Many microbial pathogens utilize eukaryotic cell cytoskeletal structure in their strategy of pathogenesis. Host cytoskeletal rearrangement has also proven to be a common mechanism of bacterial TTS effectors. TTS is induced upon bacterial contact with the host cell and leads to the formation of a needle-like structure that translocates bacterial effectors that affect host cell function. Consistent with the contact-mediated nature of TTS induction, evidence supports the involvement of TTS in the establishment of the infectious process of many gram-negative bacteria, including P. aeruginosa (6, 8, 19, 31, 34, 46). Although the genes regulating TTS and encoding the needle-structure are conserved among gram-negative bacteria, TTS effectors are bacterium specific (23). The diversity observed among TTS effectors attests to the ability of bacteria to evolve or acquire effectors that accommodate their specific lifestyle within a particular eukaryotic niche.
TTS effectors differ in structure and function from classic bacterial AB toxins, such as diphtheria toxin, in essentially two ways. First, TTS effectors lack cell receptor binding regions that allow their direct internalization into eukaryotic cells. Second, multiple TTS effectors can be translocated into eukaryotic cells, where they can function in a coordinate manner. This again differs from classic bacterial toxins that generally exert independent effects on host cells. The coordinated function of TTS effectors became evident during studies of Salmonella enterica serovar Typhimurium TTS effectors SopE, SopB, and SptP. In the salmonella infectious process, SopE and SopB stimulate Cdc42 and Rac activation, leading to actin cytoskeletal alterations that allow bacterial internalization (16, 48). This is followed by the inactivation of Cdc42 and Rac1 by SptP GAP activity, allowing host cell recovery and Salmonella intracellular growth to proceed (15).
The bifunctionality of ExoS, and the recognized potential for its GAP and ADPRT domains to target the same eukaryotic LMWG proteins, identifies ExoS as a model to study how the functions of a single TTS effector might be coordinated within the host cell. This potential can be further developed based on the finding that the coordinate function of ExoS GAP and ADPRT activities varies in different cell lines (45, 47), and that the GAP and ADPRT activities of ExoS can both target Rac1 but exert opposite effects on Rac1 function.
Using Rac1 to monitor ExoS GAP and ADPRT function within eukaryotic cells, the ADP-ribosylation of Rac1 by ExoS was detected in human cell lines, such as HT-29 cells, but not in rodent or macrophage cell lines and was associated with Rac1 membrane localization and activation. In cell lines where Rac1 was ADP-ribosylated, ExoS GAP exerted limited detectable effects on Rac1 function. These results indicate the potential of ExoS ADPRT activity to override the effects of ExoS GAP activity on Rac1 function in cell lines where Rac1 is ADP-ribosylated. In examining the molecular mechanism of Rac1 activation by ExoS ADPRT activity, R66 and R68 were identified as preferred sites of ExoS ADP-ribosylation in in vitro reactions, with R66 appearing to be the most preferred site. These residues reside within the Switch II domain of Rac1 in a region that can affect GAP-mediated Rac1 inactivation. The shift in Rac1 mobility detected by SDS-PAGE upon ADP-ribosylation by ExoS is characteristically less than that observed for other LMWG-protein substrates of ExoS ADPRT activity, such as Ras and RalA, where preferred sites of ExoS ADP-ribosylation are within the Switch I region (11, 12, 17). Two-dimensional electrophoresis analyses support that Rac1 is likely ADP-ribosylated at only one site upon its TTS-mediated translocation into the host cell (12). The single modification of Rac1 by TTS-ExoS within eukaryotic cells is also evident in the decreased shift in mobility of Rac1 ADP-ribosylated by ExoS ex vivo versus in vitro and implicates a cellular bias in the actual site of ADP-ribosylation by ExoS within the host cell. Although the site of ExoS ADP-ribosylation of Rac1 within the cell was not directly determined in these studies, the increased levels of active Rac1 detected in response to ExoS ADPRT activity are consistent with ExoS ADP-ribosylating Rac1 at either R66 or R68 and this leading to an interference of GAP-mediated inactivation of Rac1.
Insight into cellular differences in Rac1 ADP-ribosylation was gained through immunofluorescence studies monitoring endogenous Rac1 localization relative to bacterial contact and TTS translocation of ADPRT-active ExoS. Rac1 ADP-ribosylation occurs as a later event in the P. aeruginosa TTS-mediated infectious process (12). This delay suggests an initial inaccessibility of Rac1 to TTS-ExoS, which can be explained by the normal cytosolic localization. In HT-29 cells, where Rac1 is ADP-ribosylated by ExoS, Rac1 was diffusely distributed in the cytosol prior to exposure to bacteria. Upon contact with bacteria expressing ADPRT-active ExoS, Rac1 localized to the site of bacterial contact. In J774A.1 cells, where Rac1 is not ADP-ribosylated by ExoS, intense Rac1 staining at the plasma membrane was evident prior to exposure to bacteria. Rac1 staining at the plasma membrane intensified upon prolonged exposure of J774.1 cells to bacteria but not at the site of bacterial contact and independently of ExoS ADPRT activity. Evidence of ExoS-GAP accessibility to Rac1 was indirectly evident in J774A.1 cells exposed to an ExoS-GAP mutant by the enhanced lamellopodia formation at the site of bacterial contact, in association with increased levels of active (not inactivated) Rac1. The identification of differences in the steady-state localization of Rac1, and differences in the movement of Rac1 in response to TTS-ExoS in HT-29 and J774A.1 cells supports the potential for host cell properties that affect Rac1 localization to influence the targeting of ExoS ADPRT activity to Rac1.
Our findings on the effects of ExoS GAP and ADPRT activities on Rac1 function have allowed the development of a model to explain the coordinate and differential targeting of ExoS to Rac1 in host cells. This model predicts that cellular properties that direct or position Rac1 at the plasma membrane favor its initial accessibility to TTS-ExoS GAP, which results in Rac1 inactivation, as occurs in J774A.1 macrophages. In cells where Rac1 is not efficiently positioned at the plasma membrane, TTS-ExoS gains access to cytosolic Rac1 by using host cell trafficking mechanisms. In this context, ExoS-ADPRT activity targets Rac1 and causes Rac1 activation, as occurs in HT-29 cells. The model also predicts that the coordinate function of the ExoS GAP and ADPRT relates to GAP activity targeting plasma membrane-associated Rac1, whereas ExoS ADPRT activity targets cytosolic Rac1. The unknown factors of this model are (i) cellular properties that position Rac1 at the plasma membrane to facilitate its accessibility to ExoS-GAP and (ii), alternatively, host cell mechanisms that allow ExoS-ADPRT activity to be trafficked to cytosolic Rac1. However, it is known that these properties are integral to cell function, since ExoS ADP-ribosylation of Rac1 is host cell dependent and is an unalterable phenotype.
Gaining an understanding of how bacterial proteins manipulate Rho-family proteins has not only provided insight into mechanisms of pathogenesis but has also provided tools to study eukaryotic cell signaling processes that affect cell structure, migration, phagocytosis, and adhesion. Studies of the effects of ExoS on Rac1 have similarly provided insight into how the activity of a bifunctional TTS effector can be coordinated within the host cell and also into how host cell properties can differentially influence Rac1 localization and function.
ACKNOWLEDGMENTS
We thank Dara Frank (Medical College of Wisconsin, Milwaukee) for providing the P. aeruginosa strains used in these studies and Jennifer Meredith and Timothy Vincent for valuable contributions to this project.
This study was supported by Public Health Services grant NIH-NIAID 45569, by the Medical University of South Carolina Institutional Research Funds, and by the Mary C. Babb Cancer Center Foundation Fund 2V882 from West Virginia University.
C.L.R., E.A.R., and D.M.V. contributed equally to this study.
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