A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells
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感染与免疫杂志 2006年第5期
Departments of Microbiology/Immunology Medicine, Northwestern University, Chicago, Illinois 60611
ABSTRACT
ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.
INTRODUCTION
The gram-negative bacterium Pseudomonas aeruginosa is the fifth most frequently isolated nosocomial pathogen and is responsible for 10% of all hospital-acquired infections (4). The severity of disease caused by this bacterium is mediated in part by its type III secretion system and the four known effector proteins secreted by it, ExoS, ExoT, ExoU, and ExoY (15). Following translocation into host cells, these proteins utilize a variety of enzymatic activities and molecular interactions to disrupt normal cellular physiology, which results in bacterial persistence and tissue damage.
The evidence for a significant pathogenic role in disease is particularly compelling for ExoU (41, 48). Strains of P. aeruginosa that naturally secreted ExoU were more virulent than strains that did not in a mouse model of pneumonia (53), and disruption of the exoU gene resulted in decreased virulence (12, 20). Furthermore, transformation of the exoU gene into strains that did not normally harbor it increased virulence (1). An analysis of isogenic mutants indicated that ExoU was the most toxic of the four P. aeruginosa effector proteins in an animal model of acute pneumonia (32, 54). Secretion of ExoU was associated with increased mortality, increased bacterial burden in the lungs, and increased dissemination (54). Studies of human patients with naturally occurring P. aeruginosa infections supported these conclusions (18, 45). For example, hospital-acquired pneumonia patients infected with ExoU-secreting isolates had poorer outcomes than patients infected with nonsecreting isolates (18). Thus, secretion of ExoU confers upon P. aeruginosa strains the ability to cause especially severe disease.
ExoU's in vivo virulence has been associated with in vitro cytotoxicity (37, 50, 53). (Here "cytotoxicity" refers to cytolytic cell death.) In cell culture assays, ExoU-mediated killing is not limited to one or a few cell types. Rather, ExoU cytotoxicity has been observed in cell lines derived from macrophages, epithelial cells, and fibroblasts (5, 10, 12, 14, 19, 26, 30, 49, 51). This suggests that delivery of ExoU and its mechanism of cytotoxicity involve factors that are common to many cell types. In addition, ExoU is quite potent. Half-maximal killing requires only 300 to 600 molecules per cell (40), which explains why several groups of workers were unable to detect ExoU in eukaryotic cells using either green fluorescent protein (GFP) tags or immunoblot analysis (11, 40, 48). The membranes of intoxicated cells become disrupted, but features of apoptosis, such as DNA fragmentation, are not observed, indicating that death occurs by necrosis (5, 11, 12, 19, 20). Furthermore, expression of ExoU in CHO cells was sufficient for cytotoxicity, indicating that once inside a mammalian cell, ExoU does not require the presence of other bacterial proteins (11, 48).
To date, two domains of ExoU have been identified. Based on studies of other effector proteins, the N-terminal 100 amino acids is hypothesized to target this toxin to the type III secretion machinery (23). Residues 107 to 357 encode a patatin-like phospholipase A2 (PLA2) domain (40, 49). Patatin is a major storage protein of potatoes (16, 59) that has PLA2 activity which is used for protection under conditions of stress or infection (22, 46, 56). Patatin-like proteins possess a Ser-Asp catalytic dyad and an oxyanion hole essential for catalysis (22, 46). A sequence comparison revealed that the N terminus of ExoU possesses motifs similar to those of patatin and that disruption of these motifs eliminates ExoU's PLA2 activity (40, 42, 49). Cell culture and animal models of infection demonstrated that the PLA2 activity of ExoU is an integral part of its ability to kill eukaryotic cells and cause disease (37, 40, 48).
Although it has been established that ExoU's phospholipase activity is crucial for pathogenesis, the details of the mechanism by which this toxin kills mammalian cells are unclear. In this study, we showed that the C-terminal half of ExoU contains a localization domain that targets ExoU to the plasma membrane. Such targeting of this phospholipase to a region of the cell rich in phospholipids may be an important step in the process by which ExoU kills cells.
MATERIALS AND METHODS
Bacterial strains, media, and plasmids. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strain XL1-Blue and Top10 cells were used in cloning experiments and grown in Luria-Bertani (LB) broth. When necessary, the medium was supplemented with ampicillin at a concentration of 50 to 100 μg/ml. P. aeruginosa strains PA103UT/S142A and PA103UT/LS608 were grown in LB broth. HeLa cervical carcinoma cells were grown in Eagle's minimal essential medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) in the presence of 5% CO2 at 37°C. 3T3 fibroblast cells stably transfected with pSwitch (Invitrogen, Carlsbad, CA) were maintained in Dulbecco's minimal essential medium (Invitrogen) supplemented with 10% FBS and 50 μg/ml hygromycin B (Roche Applied Science, Indianapolis, IN) in the presence of 5% CO2 at 37°C.
ExoU-expressing transfection constructs. Most of the transfection constructs expressing GFP-ExoU fusion proteins were generated previously (42); the exceptions were pGFP-ExoU-LS604 and pGFP-ExoU-LS619 (Table 1), which were generated as follows. An AgeI restriction endonuclease site was introduced 5' of the exoU gene and a KpnI site was introduced 3' of the exoU gene by PCR amplification of the appropriate exoU allele (42), using upstream primer ExoU-AgeI-5 and downstream primer ExoU-KpnI-3 (Table 2). DNA was amplified using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 62°C for 30 s, and 72°C for 90 s for 35 cycles and then 72°C for 10 min. The amplified products and the mammalian transfection vector pCDNA 3.1 NT-GFP (Invitrogen) (referred to below as pGFP) were digested with AgeI and KpnI (New England Biolabs) and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue competent cells. Transformants were checked for correct insertion by restriction endonuclease digestion with AgeI and KpnI, and the exoU gene in each construct was verified by nucleotide sequencing.
A construct expressing an untagged variant of ExoU containing an alanine substitution for the catalytic serine residue 142 (referred to as ExoU-S142A) was constructed as follows. An AgeI-KpnI fragment of pGFP-ExoU-S142A containing only the mutated exoU allele was treated with T4 polymerase, ligated into EcoRV-digested pCDNA(–) (Invitrogen), and transformed into Top10 cells. The correct orientation of the exoU gene was verified by restriction digestion.
Transfection constructs used for a deletion analysis aimed at identifying the N-terminal boundary of the ExoU localization domain were generated as follows. exoU gene fragments corresponding to amino acids 450 to 687, 500 to 687, 550 to 687, 600 to 687, and 650 to 687 were generated by PCR amplification of pExoU (43) with primers that introduced an AgeI restriction site immediately upstream of the exoU fragment and a KpnI restriction site approximately 300 bp downstream of the exoU fragment. The downstream primer for each construct was GFP750KpnI-3, and the upstream primers were GFP450AgeI-5, GFP500AgeI-5, GFP550AgeI-5, GFP600AgeI-5, and GFP650AgeI-5 (Table 2). The original stop codon after residue 687 was left intact. Amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 62°C for 30 s, and 72°C for 2 min for 35 cycles and then 72°C for 10 min. The amplified products and pGFP were digested with KpnI and NotI and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into Top10 cells, which generated transfection constructs that encoded ExoU fragments with N-terminal GFP tags. The integrity of the exoU allele in each construct was verified by nucleotide sequencing.
Transfection constructs used for a deletion analysis aimed at identifying the C-terminal boundary of the ExoU localization domain were generated as follows. exoU alleles encoding C-terminally truncated variants of ExoU were PCR amplified using the C1 and C2 vectors as templates (43) and primers that introduced an AgeI restriction site immediately upstream of the exoU gene and a KpnI restriction site and stop codon immediately downstream of the exoU gene. The upstream primer used for this was ExoU-AgeI-5, and the downstream primers were U623-KpnI-3 and U656-KpnI-3 (Table 2). Amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 64°C for 30 s, and 72°C for 3.5 min for 35 cycles and then 72°C for 10 min. The amplified products and pGFP were digested with AgeI and KpnI and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue cells to generate N-terminally GFP-tagged ExoU variants with 31 and 54 amino acid truncations (referred to as GFP-ExoU657-687 and GFP-ExoU624-687, respectively). The integrity of the exoU allele in each construct was verified by nucleotide sequencing.
Transient transfections. One day prior to transfection, 1.5 x 104 HeLa or 3T3 cells were seeded onto 12-mm coverslips in a 24-well non-tissue-culture-treated plate. The coverslips were treated with 10 μg/ml rat tail collagen type I (Upstate Biotechnology, Lake Placid, NY) prior to 3T3 seeding. Transient transfections were performed by adding 25 μl of prewarmed serum-free medium, 0.75 μl FuGENE 6 (Roche Applied Science), and 0.5 μg DNA to each well of cells. Alternatively, HeLa cells in 100-mm tissue culture plates were transiently transfected at 50 to 70% confluence by addition of 600 μl serum-free medium, 18 μl FuGENE 6, and 6 μg DNA to each plate of cells. Following transfection, cells were incubated at 37°C in the presence of 5% CO2 for 12 to 24 h prior to examination by fluorescence microscopy.
P. aeruginosa infection of HeLa cells. HeLa cells were grown almost to confluence on coverslips in 24-well cell culture plates. PA103 strains were grown overnight at 37°C in LB broth without shaking. The equivalent of 108 CFU of bacteria from this culture was then diluted into 1 ml of LB broth and incubated without shaking for 1.5 h at 37°C. Bacteria were then pelleted by centrifugation and resuspended in 1 ml of serum-free modified Eagle's medium. Each well containing HeLa cells was infected with 100 μl of bacterial culture. Centrifugation at 500 x g for 10 min was used to pellet the bacteria onto the HeLa cells and synchronize the infections. Following coincubation for 3 h at 37°C in the presence of 5% CO2, cells were examined for the presence and localization of ExoU by fluorescence microscopy.
Fluorescence microscopy. In transfection studies, HeLa cells were fixed with 3.7% formaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 20 min, and this was followed by quenching of autofluorescence with 50 mM NH4Cl. In experiments in which antibodies were not used, cells were incubated with 10 μg/ml Hoechst 33342 stain (Molecular Probes, Eugene, OR) for 3 min to visualize the nucleus, after which coverslips were placed on the slides with 1 drop of DABCO (Fluka, Switzerland) and sealed with nail polish. Otherwise, the following manipulations were performed prior to the addition of Hoechst stain. Cells were blocked and permeabilized with 0.1% (vol/vol) saponin-10% (vol/vol) FBS in phosphate-buffered saline (PBS) (blocking-permeabilization buffer) for 30 min and then incubated for 30 min with polyclonal ExoU antiserum (20) diluted 1:1,000 in blocking-permeabilization buffer. The cells were then incubated for 30 min with rhodamine-conjugated anti-rabbit secondary antibody (ICN/Cappel, Aurora, OH) diluted 1:200 in blocking-permeabilization buffer. The cells were viewed using an Olympus IX inverted fluorescence microscope with standard filter sets and a Photometrix cooled charge-coupled device camera (CH350/LCCD) driven by DeltaVision software (Applied Precision Inc., Seattle, WA). Images were deconvolved using DeltaVision software. Single Z sections are shown in the figures. Experiments without permeabilization were performed as described above except that the saponin treatment was omitted. In concanavalin A (ConA) experiments, cells were first incubated at 4°C for 15 min to inhibit endocytosis. Alexa Fluor 647-conjugated ConA (Molecular Probes) was then added to unfixed cells at a concentration of 100 μl/ml for 10 min at 4°C. Cells were visualized immediately after the coverslips were placed on slides with 1 drop of ice-cold PBS and sealed with nail polish.
In P. aeruginosa infection studies, HeLa cells were washed with PBS, fixed with 3.7% formaldehyde for 15 min, and quenched with 50 mM NH4Cl. The cells were treated with 2 μl/ml Alexa Fluor 647-conjugated ConA for 5 min and permeabilized with 0.5% (vol/vol) Triton X-100 (Sigma, St. Louis, Mo.) in PBS for 20 min. The cells were then blocked for 30 min in Image-iT FX signal enhancer buffer (Molecular Probes) and incubated for 1 h with polyclonal ExoU antibody (1:1,000 dilution) in 0.4% (vol/vol) fish gelatin (Sigma) in PBS. The cells were then incubated for 1 h with Alexa Fluor 555 (Cy3) goat anti-rabbit antibody (Molecular Probes) at a dilution of 1:250 (vol/vol) in 0.4% (vol/vol) fish gelatin in PBS. Finally, the cells were incubated for 3 min with Hoechst 33342 stain. Coverslips were placed on slides with 1 drop of DABCO and sealed with nail polish. The cells were viewed with a Leica DMIREZ microscope powered by a 100-W mercury lamp and equipped with a Hamamatsu ORCA-ER camera. Pictures were taken at a magnification of x1,000 using the Openlab 4.0.4 software and were deconvolved using the Volocity 3.6.1 software.
Cell fractionation. For each condition, five 100-mm plates containing HeLa cells were transfected with pGFP-ExoU-S142A as described above. Twelve hours after transfection, the cells were washed and incubated with PBS at 4°C for 25 min while they were gently rocked. Cells were scraped into 25 ml PBS, collected by centrifugation, and resuspended in 1 ml ice-cold 5 mM Tris (pH 7.8). After incubation on ice for 10 min, the cells were lysed by passage 30 times through a 21.5-gauge needle. Intact organelles and nuclei were removed by centrifugation at 10,000 x g at 4°C for 10 min. Supernatants were added to polyallomar tubes (2 by 0.5 in.; Beckman, Palo Alto, CA), and ultracentrifugation was performed with a Beckman model L8-80 M centrifuge at 32,000 x g for 1 h at 4°C. The supernatant, corresponding to cell cytoplasm, and the pellet, corresponding to membranes, were collected. The pellet was resuspended in 1 ml of 0.1% (vol/vol) Triton X-100 supplemented with a protease inhibitor cocktail (Complete Mini protease inhibitor pellet; Roche Applied Science). After addition of 5x sodium dodecyl sulfate (SDS) protein electrophoresis sample buffer, proteins were boiled for 10 min, electrophoresed through a 10% (wt/vol) SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. An immunoblot analysis was then performed as previously described (53), using rabbit polyclonal ExoU antiserum (1:6,000) (20), mouse monoclonal pan-cadherin antibodies (1:2,000) (Abcam, Cambridge, MA), and mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (1:20,000) (Abcam) diluted in blocking buffer (5% [wt/vol] dry milk and 0.05% [vol/vol] Tween 20 in PBS). Goat anti-rabbit or goat anti-mouse immunoglobulin G horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:5,000 in blocking buffer were used as secondary antibodies.
Bioinformatics analysis. Residues 550 to 687 of ExoU were analyzed for the presence of informative sequences using a number of approaches. Homology to known proteins and the presence of motifs were determined using the Basic Local Alignment Search Tool (BLAST) to search the Non-Redundant Protein Sequence Database and the Conserved Domain Database (2, 3, 35). A search for putative transmembrane domains was performed using the TMHMM algorithm (29). The Simple Architecture Modular Research Tool (SMART) algorithm (33) was used to search for the following phosphoinositide-binding motifs: C2, PH, FYVE, ENTH, and PX. Patterns representative of coiled-coil domains were identified using COILS (34).
Kinetics of ExoU localization. Inducible expression of ExoU in 3T3 cells was performed using the GeneSwitch approach (Invitrogen), as described by the manufacturer. Briefly, GFP-tagged ExoU-S142A was expressed in 3T3 cells by ligating the appropriate exoU allele into the pGene/V5-His B vector. This was accomplished by PCR amplification of the exoU allele using pGFP-ExoU-S142A (42) as a template. Upstream primer GFP-5 and downstream primer ExoU-3 were designed to incorporate a KpnI restriction site immediately upstream of the gfp gene and a NotI restriction site immediately downstream of the exoU gene (Table 2). PCR amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 64°C for 30 s, and 72°C for 4 min for 35 cycles and then 72°C for 10 min. The amplified products and pGene/V5-His B were digested with KpnI and NotI and were purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue competent cells. The resulting construct was designated pGene-GFP-ExoU-S142A. The integrity of the exoU fragment in each construct was verified by nucleotide sequencing.
pGene-GFP-ExoU-S142A was transiently transfected into a stable 3T3 cell line harboring pSwitch, a second GeneSwitch vector required for inducible expression. ExoU expression was induced by addition of 10–8 M mifepristone 24 h after transfection. At 0, 1, 2, 3, and 4 h postinduction, cells were fixed with 3.7% (vol/vol) formaldehyde in PBS for 20 min. Cells were incubated with 10 μg/ml Hoechst stain for 3 min to allow visualization of the nucleus. Coverslips were then placed on slides with 1 drop of DABCO and sealed with nail polish. The cells were viewed using an Olympus IX inverted fluorescence microscope and deconvolved as described above.
RESULTS
ExoU localizes to the cell periphery of transfected HeLa cells. To determine the subcellular localization of ExoU, we attempted to detect intact ExoU fused to GFP and expressed in HeLa cells by transfection. However, ExoU was not detectable by fluorescence microscopy or immunoblot analysis in these experiments, although there was evidence of cytotoxicity. These data suggested that small amounts of ExoU, below the limit of detection, were sufficient to lyse cells and thus prevent visualization. Note that previous attempts to detect wild-type ExoU in eukaryotic cells either by microscopy or immunoblot analysis were also unsuccessful (11, 40, 42, 43, 49).
Since the cytotoxicity of ExoU prevented successful visualization of this toxin, subcellular localization was examined using a catalytically inactive form of the toxin. ExoU having an alanine substitution in place of the putative catalytic serine residue (referred to as ExoU-S142A) was used for this purpose. This variant of ExoU, which had previously been shown to lack PLA2 activity and to be noncytotoxic (40, 42, 48), was tagged with GFP (referred to as GFP-ExoU-S142A) and expressed in HeLa cells by transient transfection. Approximately 18 h later, the cells were fixed, incubated with Hoechst stain, and visualized by fluorescence microscopy. Whereas the GFP control was diffusely green throughout the cytoplasm, GFP-ExoU-S142A was localized predominantly at the cell periphery in a punctate fashion, although some fluorescence was also observed in the cell interior (Fig. 1A). To ensure that this pattern was not an artifact of the GFP tag, polyclonal ExoU antiserum was used to visualize ExoU-S142A lacking a GFP tag. Similar to the GFP fusion protein, ExoU-S142A lacking GFP was visualized at the cell periphery (Fig. 1B). These data indicate that ExoU-S142A localizes to the cell periphery.
To better characterize the peripheral localization of ExoU relative to the HeLa cell plasma membrane, colocalization experiments were performed using the lectin ConA, which binds carbohydrate residues on the cell surface and is thus used to visualize the plasma membrane. Following transfection with the GFP-ExoU-S142A-expressing construct, HeLa cells were incubated with Alexa Fluor 647-conjugated ConA and visualized by fluorescence microscopy. The GFP-ExoU-S142A and ConA fluorescence patterns were very similar (Fig. 1C), indicating that ExoU was at or adjacent to the plasma membrane.
ExoU is in the membrane fraction of transfected HeLa cells. To better characterize the subcellular localization of ExoU, lysates from transfected HeLa cells were fractionated by differential centrifugation. Pellet fractions, containing plasma and organelle membranes, and supernatant fractions, containing cytosolic components, were volume adjusted and electrophoresed on an SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and exposed to polyclonal ExoU antiserum. The relative purity of each fraction was confirmed using pan-cadherin as a marker for the cell membrane (57) and GAPDH as a marker for the cytosol (57). Although some GFP-S142A-ExoU was found in the cytosolic fraction, the majority of this protein was in the membrane fraction (Fig. 2). These experiments were done without a cross-linking reagent, indicating that S142A-ExoU was strongly associated with the plasma membrane. This association may be a high-affinity interaction with a membrane-associated factor, or ExoU itself may be inserted into the plasma membrane.
If ExoU were intercalated into the plasma membrane, one would expect it to be exposed on the surface of the cell. To experimentally examine this possibility, immunofluorescence microscopy using polyclonal ExoU antiserum was performed in the presence or absence of the permeabilizing detergent saponin. A fluorescent signal was absent when the membranes were intact, indicating that ExoU is not externally exposed (Fig. 3). Similar results were obtained using anti-GFP antibodies (data not shown). These data indicate that ExoU is not surface exposed and therefore is unlikely to be inserted through the plasma membrane.
C terminus of ExoU is necessary for localization to the plasma membrane. Next, we attempted to identify the portion of ExoU that was necessary for localization to the plasma membrane. To accomplish this, we utilized a previously generated panel of constructs that encoded ExoU variants having a substitution or insertion in each of five regions of ExoU required for cytotoxicity (42): ExoU-G112W (region 1), ExoU-S142A (region 2; the noncatalytic variant used in the experiments described above), ExoU-G286W (region 3), ExoU-D344A (region 4), and ExoU-LS608 (region 5; a 5-amino-acid insertion immediately prior to N608). Regions 1, 2, 3, and 4 are in the patatin-like phospholipase domain of the N-terminal half of ExoU, whereas region 5 is located in the C-terminal half of ExoU (see Fig. 8 for the locations of these regions in ExoU). Each ExoU variant was tagged with GFP, expressed in HeLa cells, and viewed by fluorescence microscopy 18 h later. As expected, GFP alone localized to the cytoplasmic compartment of the HeLa cells (Fig. 4). Like the region 2 mutant ExoU-S142A, ExoU containing a single amino acid substitution in region 1 or 4 was observed in a punctate pattern on the circumference of the cell (Fig. 4). In contrast, the region 5 ExoU variant was found diffusely throughout the cytoplasm, similar to GFP alone (Fig. 4). These data indicated that the C terminus of ExoU is necessary for localization to the plasma membrane. A region 3 variant of ExoU was found at the cell periphery, but increased amounts were also present in the cell interior, suggesting a possible role for this region in modifying the localization process (Fig. 4).
To confirm that region 5 of ExoU is essential for localization to the plasma membrane, two additional ExoU variants were examined. ExoU-LS604 and ExoU-LS619 had 5-amino-acid insertions immediately prior to V604 and Y619, respectively, in region 5 of ExoU. Constructs encoding these variants were transfected in HeLa cells, and ExoU localization was examined by fluorescence microscopy. In both cases, ExoU was observed diffusely throughout the cytoplasm (data not shown). Thus, insertions at three different sites in region 5 of ExoU abolished targeting to the plasma membrane, confirming the importance of this portion of the toxin for appropriate localization.
C terminus of ExoU is sufficient for localization to the plasma membrane. To determine whether the C-terminal half of ExoU was sufficient for localization to the plasma membrane, the ability of this portion of the toxin to appropriately localize an unrelated protein was determined. GFP was chosen as the cargo protein for this experiment. A fusion protein consisting of the C-terminal half of ExoU (ExoU450-687) and GFP was expressed in HeLa cells by transient transfection, and localization was examined by fluorescence microscopy. Whereas GFP alone was visualized in the cell interior, the GFP-ExoU450-687 fusion protein was localized to the cell periphery (Fig. 5A). This indicated that the C-terminal half of ExoU is sufficient to target an unrelated protein to the cell periphery and therefore contains a localization domain.
The localization domain of ExoU lies in residues 550 to 687. Next, the boundaries of the localization domain were defined more precisely. To determine the N-terminal boundary of the localization domain, a deletion analysis was performed in which residues 650 to 687, 600 to 687, 550 to 687, and 500 to 687 were each fused with GFP. Fusion proteins were expressed in HeLa cells, and localization was examined by fluorescence microscopy. GFP fused to residues 450 to 687, 500 to 687, or 550 to 687 of ExoU was targeted to the cell periphery (Fig. 5). Fluorescence was detected both in the cell interior and at the periphery when GFP was fused to residues 600 to 687 and only in the cell interior when GFP was fused to residues 650 to 687 (Fig. 5). These data indicated that the N-terminal boundary of the localization domain is between residues 550 and 600 of ExoU.
To determine the C-terminal boundary of the localization domain, two C-terminal deletion variants of ExoU were fused to GFP, expressed in HeLa cells, and viewed by fluorescence microscopy. ExoU657-687 consists of amino acids 1 to 656 and is missing the last 31 amino acids of ExoU, whereas ExoU624-687 consists of amino acids 1 to 623 and is missing the last 64 amino acids. Both ExoU variants localized to the cell interior and not the cell periphery, indicating that the C-terminal boundary of the localization domain is after amino acid 656 (Fig. 5). Together with the data described above, these results indicate that the localization domain is between residues 550 and 687, within the C-terminal 138 amino acids of ExoU.
The kinetics of ExoU localization are consistent with those of cytotoxicity. ExoU-mediated killing is quite rapid and occurs within the first 3 h of infection when P. aeruginosa is coincubated with eukaryotic cells in vitro (5). We therefore wished to investigate whether localization to the plasma membrane occurred in a similar time frame, which would be consistent with a role for localization in killing. To accomplish this, ExoU was expressed in 3T3 cells using the mifepristone-inducible GeneSwitch expression system. This approach uses a positive feedback system to express a gene of interest in mammalian cells following the addition of mifepristone to the culture medium. An allele encoding GFP-tagged ExoU-S142A was cloned into the GeneSwitch vector, which was then transfected into 3T3 cells expressing regulatory components of the GeneSwitch system. Eighteen to twenty-four hours after transfection, mifepristone was added to the cells to induce GFP-ExoU-S142A expression. The cells were incubated with mifepristone for 0, 1, 2, 3, or 4 h and then fixed and viewed by fluorescence microscopy. No fluorescence was detectable prior to the addition of mifepristone, but by 1 h postinduction, GFP-ExoU-S142A was already at the periphery of the cells (Fig. 6). GFP-ExoU-S142A was still present primarily along the circumference of the cells after 2 h. Interestingly, by 3 to 4 h postinduction, GFP-ExoU-S142A was accumulating internally as well as at the cell periphery (Fig. 6). These data indicate that ExoU localizes to the cell periphery within 1 h after expression from a transfected construct. Furthermore, they indicate that ExoU peripheral localization is unlikely to be an artifact of overexpression, since it occurs at early times when little ExoU has accumulated in cells. The fluorescence in the cell interior at 3 and 4 h may have been due to either endocytosis of ExoU into the cell from the plasma membrane or overexpression at these later times leading to nonspecific protein aggregation.
The localization domain is required for appropriate targeting of ExoU following translocation into cells by the P. aeruginosa type III secretion system. To ensure that membrane targeting was not an artifact of transfection, we determined the localization of ExoU following translocation into host cells by the P. aeruginosa type III secretion system. PA103UT, a P. aeruginosa strain with in-frame deletions in its endogenous exoU and exoT genes, was used for these studies because it has an intact type III secretion system but does not secrete any known effector proteins. Single copies of mutated exoU alleles under the control of their endogenous promoters were inserted into the chromosomal attB site of PA103UT. HeLa cells were coincubated with these P. aeruginosa strains for 3 h, and ExoU localization was examined by immunofluorescence microscopy. Labeled ConA was used to visualize the HeLa cell plasma membranes.
Infection with PA103UT/S142A, which secreted catalytically inactive ExoU-S142A, resulted in peripheral localization of the toxin, similar to the pattern observed following transfection (Fig. 7A). In contrast, predominantly cytoplasmic localization was seen with PA103UT/LS608, which secreted the ExoU-LS608 variant of ExoU that contained a 5-amino-acid insertion in the localization domain (Fig. 7B). Visualization of uninfected cells confirmed that the observed signal was indeed due to ExoU (Fig. 7C).
DISCUSSION
In this study, the subcellular localization of ExoU was examined to increase our understanding of the mechanism by which this toxin kills cells. Microscopy studies showed that ExoU rapidly localized to the cell periphery. Together with the results of fractionation experiments, these findings indicated that ExoU localized either to the plasma membrane or to a structure associated with the plasma membrane. This is in agreement with work of Phillips et al., who found peripheral localization of ExoU in syringe-loaded cells (40). Colocalization studies with the lectin ConA indicated that there is a very close association between ExoU and the plasma membrane, although ExoU was not exposed on the cell surface. Deletional analysis indicated that the localization domain of ExoU, which was both necessary and sufficient for targeting to the plasma membrane, was in residues 550 to 687. Localization occurred within 1 h of ExoU expression in transfected cells. Although the kinetics of localization in transfected cells may differ from those in cells in which ExoU is injected by type III secretion, it is interesting that the entire process of ExoU secretion, translocation, and killing occurs in 1.5 to 3 h (5). Thus, rapid localization to the membrane would be expected if this were an essential step in ExoU-mediated cytotoxicity.
Targeting to specific subcellular compartments is an emerging theme among type III effector proteins. Although some of these proteins localize to the nucleus (e.g., YopM of Yersinia [6, 55]), mitochondria (e.g., SopA and SipB of Salmonella [21, 31]), Lamp1-positive vesicles (e.g., SifA of Salmonella), or other intracellular vesicles of host cells (e.g., YopE of Yersinia [28]), many effector proteins target the plasma membrane or structures closely associated with it. The Salmonella type III-secreted proteins SopE, SopE2, SopB, SptP, SipA, SipC, and SspC all localize to the plasma membrane (8, 52, 58), as do YpkA of Yersinia (17) and Tir of enteropathogenic E. coli (27). For many of these effector proteins, localization to the plasma membrane has been shown to be important for function. For example, following localization to the plasma membrane, SipA and SipC cooperate to cause actin polymerization and bundling at the cell surface, augmenting the ruffling necessary for the internalization of Salmonella cells (8, 36, 61). Likewise, Tir inserts into the plasma membrane so that an internal domain of the protein is exposed on the cell surface and accessible for binding by its bacterial ligand, intimin (9, 27). In this regard, the localization of the phospholipase ExoU to the plasma membrane may have pathogenic relevance since this subcellular location is rich in phospholipids.
Deletional analysis indicated that the localization domain of ExoU was between residues 550 and 687, but a closer examination of this region did not yield clues regarding the mechanism of targeting. Database searches detected no homology between this region and any characterized protein or motif. Nor did this portion of ExoU contain predicted transmembrane domains. These findings and our inability to detect ExoU exposed on the surface of transfected cells make insertion through the plasma membrane, like the insertion that occurs with Tir (9, 27), unlikely. Sites for prenylation or acylation, such as those identified in the Salmonella effector protein SifA (7, 44), were not found. Nor was there similarity to characterized proteins such as actin-binding proteins, which would suggest targeting to the actin-rich cell cortex immediately adjacent to the plasma membrane. This is the mechanism by which the YpkA localization domain, which exhibits homology with the actin bundling protein coronin, targets this Yersinia effector protein to the cell periphery (17, 25). The phosphoinositide-binding domains, such as PH, PX, and ENTH, are a family of motifs that target proteins to membrane inositol phospholipids and play an important role in a diverse array of cellular processes (24). However, no such domains were present in the C-terminal portion of ExoU. A search for coiled-coil domains yielded only very low or no probability matches. It is possible that residues 550 to 687 of ExoU indirectly move this toxin to the cell periphery by binding a second factor that itself targets the plasma membrane. For example, SopE and SptP require targeting of their binding partner, the host cell small GTPase Cdc42, to the plasma membrane for their own membrane localization (8). Although no host proteins that bind to ExoU have been identified yet, it is known that this toxin requires a host cell cofactor for activation of its PLA2 activity (40, 47, 49). This factor may play a role in both activating ExoU and targeting ExoU to the plasma membrane. Alternatively, localization to the cell periphery may occur by a novel mechanism.
Recent reports have indicated that ExoS also contains a localization domain that targets this toxin to membranes (38, 39). Although both ExoS and ExoU are secreted by the same P. aeruginosa type III system, their localization domains have little in common. The localization domain of ExoS is located in residues 51 to 72 of the N terminus, whereas the localization domain of ExoU is between residues 550 and 687 of the C terminus (28, 38). Thus, the localization domain of ExoS is both smaller and in a different part of the protein than the corresponding domain of ExoU. Furthermore, there is no sequence homology between these domains. It is unclear whether ExoU and ExoS localize to the same subcellular compartments. Although ExoS is initially targeted to the plasma membrane, it subsequently localizes to the Golgi-endoplasmic reticulum of the host cell (28, 60). ExoU is primarily targeted to the plasma membrane, although localization to the cell interior also occurs at later times.
Our findings further clarify a domain structure for ExoU (Fig. 8). As is the case with other type III effector proteins (23), the extreme N terminus likely contains information required to direct ExoU through the type III secretion and translocation apparatus. Consistent with this is the observation that residues 3 to 123 were required for binding of ExoU's chaperone, SpcU (13). Immediately adjacent to the secretion/translocation domain is the patatin-like PLA2 domain from residue 107 to residue 357. This domain is required for the phospholipase activity of ExoU. Substitution of one of several critical amino acids in this domain, including the putative catalytic serine S142 and catalytic aspartate D344, abrogates PLA2 activity and cytotoxicity in vitro (40, 42, 48), as well as virulence in vivo (37). Finally, in this paper we describe a localization domain at the C terminus of ExoU, in residues 550 to 687. This domain is required for targeting to the plasma membrane. Interestingly, previous work demonstrated that portions of this domain were also required for cytotoxicity and PLA2 activity (11, 20, 40, 42, 43). Other important regions of ExoU, including a site for binding of the activating host cofactor, have yet to be identified.
Together, the results of these studies suggest a model for the mechanism of ExoU cytotoxicity. First, information carried within the secretion/translocation domain of ExoU is used to inject this toxin into mammalian cells through the type III secretion needle. Once ExoU is within the host cell, this domain is dispensable for subsequent steps in the killing process (43). ExoU is then rapidly targeted to the cell membrane in a process that requires the localization domain in the C-terminal half of the toxin. The finding that membrane localization is essential for cytotoxicity is consistent with previous reports that residues 550 to 687 of ExoU are also necessary for cytotoxicity (11, 20, 40, 42, 43). During or after this localization, ExoU interacts with a host cell factor that activates the phospholipase activity of the patatin-like domain, explaining the requirement for the localization domain for PLA2 activity. This activity then causes cell death by utilizing plasma membrane phospholipids as substrates. Death may occur by direct disruption of the plasma membrane integrity or indirectly by perturbation of signaling cascades involving phospholipids, such as the prostaglandin pathway. Further work is necessary to test this model and determine whether there are additional steps in the mechanism by which ExoU kills host cells.
ACKNOWLEDGMENTS
We thank Everett Roark, Parwez Nawabi, Christiaan van Ooij, Kasturi Haldar, and Ciara Shaver for helpful technical suggestions and assistance. We also thank Kasturi Haldar for use of her microscope.
This work was supported by the NIH (grants AI053674 and AI065615 to A.R.H.).
Present address: Center for Oral Health & Systemic Disease, School of Dentistry, University of Louisville, Louisville, KY 40292.
REFERENCES
1. Allewelt, M., F. T. Coleman, M. Grout, G. P. Priebe, and G. B. Pier. 2000. Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread. Infect. Immun. 68:3998-4004.
2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
4. American Thoracic Society. 1996. Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies. A consensus statement, American Thoracic Society, November 1995. Am. J. Respir. Crit. Care Med. 153:1711-1725.
5. Apodaca, G., M. Bomsel, R. Lindstedt, J. Engel, D. Frank, K. Mostov, and J. Wiener-Kronish. 1995. Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation defective host cells are resistant to bacterial killing. Infect. Immun. 63:1541-1551.
6. Benabdillah, R., L. J. Mota, S. Lutzelschwab, E. Demoinet, and G. R. Cornelis. 2004. Identification of a nuclear targeting signal in YopM from Yersinia spp. Microb. Pathog. 36:247-261.
7. Boucrot, E., C. R. Beuzon, D. W. Holden, J. P. Gorvel, and S. Meresse. 2003. Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function. J. Biol. Chem. 278:14196-14202.
8. Cain, R. J., R. D. Hayward, and V. Koronakis. 2004. The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay. Mol. Microbiol. 54:887-904.
9. de Grado, M., A. Abe, A. Gauthier, O. Steele-Mortimer, R. DeVinney, and B. B. Finlay. 1999. Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli. Cell. Microbiol. 1:7-17.
10. Evans, D. J., T. C. Kuo, M. Kwong, R. Van, and S. M. Fleiszig. 2002. Mutation of csk, encoding the C-terminal Src kinase, reduces Pseudomonas aeruginosa internalization by mammalian cells and enhances bacterial cytotoxicity. Microb. Pathog. 33:135-143.
11. Finck-Barbanon, V., and D. W. Frank. 2001. Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU. J. Bacteriol. 183:4330-4344.
12. Finck-Barbanon, V., J. Goranson, L. Zhu, T. Sawa, J. P. Wiener-Kronish, S. M. J. Fleiszig, C. Wu, L. Mende-Mueller, and D. Frank. 1997. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol. Microbiol. 25:547-557.
13. Finck-Barbanon, V., T. L. Yahr, and D. W. Frank. 1998. Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin. J. Bacteriol. 180:6224-6231.
14. Fleiszig, S. M. J., T. S. Zaidi, M. J. Preston, M. Grout, D. J. Evans, and G. B. Pier. 1996. Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 64:2288-2294.
15. Frank, D. W. 1997. The exoenzyme S regulon of Pseudomonas aeruginosa. Mol. Microbiol. 26:621-629.
16. Ganal, M. W., M. W. Bonierbale, M. S. Roeder, W. D. Park, and S. D. Tanksley. 1991. Genetic and physical mapping of the patatin genes in potato and tomato. Mol. Gen. Genet. 225:501-509.
17. Hakansson, S., E. Galyov, R. Rosqvist, and H. Wolf-Watz. 1996. The Yersinia Ypk Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol. Microbiol. 20:593-603.
18. Hauser, A. R., E. Cobb, M. Bodí, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30:521-528.
19. Hauser, A. R., and J. N. Engel. 1999. Pseudomonas aeruginosa induces type III secretion-mediated apoptosis of macrophages and epithelial cells. Infect. Immun. 67:5530-5537.
20. Hauser, A. R., P. J. Kang, and J. Engel. 1998. PepA, a novel secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol. Microbiol. 27:807-818.
21. Hernandez, L. D., M. Pypaert, R. A. Flavell, and J. E. Galan. 2003. A Salmonella protein causes macrophage cell death by inducing autophagy. J. Cell Biol. 163:1123-1131.
22. Hirschberg, H. J., J. W. Simons, N. Dekker, and M. R. Egmond. 2001. Cloning, expression, purification and characterization of patatin, a novel phospholipase A. Eur. J. Biochem. 268:5037-5044.
23. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:279-433.
24. Itoh, T., and T. Takenawa. 2002. Phosphoinositide-binding domains: functional units for temporal and spatial regulation of intracellular signalling. Cell. Signal. 14:733-743.
25. Juris, S. J., A. E. Rudolph, D. Huddler, K. Orth, and J. E. Dixon. 2000. A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption. Proc. Natl. Acad. Sci. USA 97:9431-9436.
26. Kang, P. J., A. R. Hauser, G. Apodaca, S. Fleiszig, J. Wiener-Kronish, K. Mostov, and J. N. Engel. 1997. Identification of Pseudomonas aeruginosa genes required for epithelial cell injury. Mol. Microbiol. 24:1249-1262.
27. Kenny, B., R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B. Finlay. 1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91:511-520.
28. Krall, R., Y. Zhang, and J. T. Barbieri. 2004. Intracellular membrane localization of Pseudomonas ExoS and Yersinia YopE in mammalian cells. J. Biol. Chem. 279:2747-2753.
29. Krogh, A., B. Larsson, G. von Heijne, and E. L. Sonnhammer. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305:567-580.
30. Kurahashi, K., O. Kajikawa, T. Sawa, M. Ohara, M. A. Gropper, D. W. Frank, T. R. Martin, and J. P. Wiener-Kronish. 1999. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J. Clin. Investig. 104:743-750.
31. Layton, A. N., P. J. Brown, and E. E. Galyov. 2005. The Salmonella translocated effector SopA is targeted to the mitochondria of infected cells. J. Bacteriol. 187:3565-3571.
32. Lee, V. T., R. S. Smith, B. Tummler, and S. Lory. 2005. Activities of Pseudomonas aeruginosa effectors secreted by the type III secretion system in vitro and during infection. Infect. Immun. 73:1695-1705.
33. Letunic, I., R. R. Copley, S. Schmidt, F. D. Ciccarelli, T. Doerks, J. Schultz, C. P. Ponting, and P. Bork. 2004. SMART 4.0: towards genomic data integration. Nucleic Acids Res. 32:D142-D144.
34. Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science 252:1162-1164.
35. Marchler-Bauer, A., J. B. Anderson, P. F. Cherukuri, C. DeWeese-Scott, L. Y. Geer, M. Gwadz, S. He, D. I. Hurwitz, J. D. Jackson, Z. Ke, C. J. Lanczycki, C. A. Liebert, C. Liu, F. Lu, G. H. Marchler, M. Mullokandov, B. A. Shoemaker, V. Simonyan, J. S. Song, P. A. Thiessen, R. A. Yamashita, J. J. Yin, D. Zhang, and S. H. Bryant. 2005. CDD: a conserved domain database for protein classification. Nucleic Acids Res. 33:D192-D196.
36. McGhie, E. J., R. D. Hayward, and V. Koronakis. 2001. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin. EMBO J. 20:2131-2139.
37. Pankhaniya, R. R., M. Tamura, L. R. Allmond, K. Moriyama, T. Ajayi, J. P. Wiener-Kronish, and T. Sawa. 2004. Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU. Crit. Care Med. 32:2293-2299.
38. Pederson, K. J., R. Krall, M. J. Riese, and J. T. Barbieri. 2002. Intracellular localization modulates targeting of ExoS, a type III cytotoxin, to eukaryotic signalling proteins. Mol. Microbiol. 46:1381-1390.
39. Pederson, K. J., S. Pal, A. J. Vallis, D. W. Frank, and J. T. Barbieri. 2000. Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells. Mol. Microbiol. 37:287-299.
40. Phillips, R. M., D. A. Six, E. A. Dennis, and P. Ghosh. 2003. In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J. Biol. Chem. 278:41326-41332.
41. Rabin, S. D. P., and A. R. Hauser. 2004. ExoU: a cytotoxin delivered by the type III secretion system of Pseudomonas aeruginosa, p. 69-90. In R. Schaffrath and M. Schmitt (ed.), Microbial protein toxins. Springer-Verlag, Heidelberg, Germany.
42. Rabin, S. D. P., and A. R. Hauser. 2005. Functional regions of the Pseudomonas aeruginosa cytotoxin ExoU. Infect. Immun. 73:573-582.
43. Rabin, S. D. P., and A. R. Hauser. 2003. Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae. Infect. Immun. 71:4144-4150.
44. Reinicke, A. T., J. L. Hutchinson, A. I. Magee, P. Mastroeni, J. Trowsdale, and A. P. Kelly. 2005. A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery. J. Biol. Chem. 280:14620-14627.
45. Roy-Burman, A., R. H. Savel, S. Racine, B. L. Swanson, N. S. Revadigar, J. Fujimoto, T. Sawa, D. W. Frank, and J. P. Wiener-Kronish. 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183:1767-1774.
46. Rydel, T. J., J. M. Williams, E. Krieger, F. Moshiri, W. C. Stallings, S. M. Brown, J. C. Pershing, J. P. Purcell, and M. F. Alibhai. 2003. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad. Biochemistry 42:6696-6708.
47. Sato, H., J. B. Feix, C. J. Hillard, and D. W. Frank. 2005. Characterization of phospholipase activity of the Pseudomonas aeruginosa type III cytotoxin, ExoU. J. Bacteriol. 187:1192-1195.
48. Sato, H., and D. W. Frank. 2004. ExoU is a potent intracellular phospholipase. Mol. Microbiol. 53:1279-1290.
49. Sato, H., D. W. Frank, C. J. Hillard, J. B. Feix, R. R. Pankhaniya, K. Moriyama, V. Finck-Barbancon, A. Buchaklian, M. Lei, R. M. Long, J. Wiener-Kronish, and T. Sawa. 2003. The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J. 22:2959-2969.
50. Sawa, T., M. Ohara, K. Kurahashi, S. S. Twining, D. Frank, D. B. Doroques, T. Long, M. A. Gropper, and J. P. Wiener-Kronish. 1998. In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections. Infect. Immun. 66:3242-3249.
51. Sawa, T., T. Yahr, M. Ohara, K. Kurahashi, M. A. Gropper, J. P. Wiener-Kronish, and D. W. Frank. 1999. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat. Med. 5:392-398.
52. Scherer, C. A., E. Cooper, and S. I. Miller. 2000. The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection. Mol. Microbiol. 37:1133-1145.
53. Schulert, G. S., H. Feltman, S. D. P. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser. 2003. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J. Infect. Dis. 188:1695-1706.
54. Shaver, C. M., and A. R. Hauser. 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect. Immun. 72:6969-6977.
55. Skrzypek, E., T. Myers-Morales, S. W. Whiteheart, and S. C. Straley. 2003. Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eukaryotic cells. Infect. Immun. 71:937-947.
56. Strickland, J. A., G. L. Orr, and T. A. Walsh. 1995. Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers. Plant Physiol. 109:667-674.
57. Takeichi, M. 1990. Cadherins: a molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59:237-252.
58. Terebiznik, M. R., O. V. Vieira, S. L. Marcus, A. Slade, C. M. Yip, W. S. Trimble, T. Meyer, B. B. Finlay, and S. Grinstein. 2002. Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella. Nat. Cell Biol. 4:766-773.
59. Vancanneyt, G., U. Sonnewald, R. Hofgen, and L. Willmitzer. 1989. Expression of a patatin-like protein in the anthers of potato and sweet pepper flowers. Plant Cell 1:533-540.
60. Zhang, Y., and J. T. Barbieri. 2005. A leucine-rich motif targets Pseudomonas aeruginosa ExoS within mammalian cells. Infect. Immun. 73:7938-7945.
61. Zhou, D., M. S. Mooseker, and J. E. Galan. 1999. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283:2092-2095.(Shira D. P. Rabin, Jeffre)
ABSTRACT
ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.
INTRODUCTION
The gram-negative bacterium Pseudomonas aeruginosa is the fifth most frequently isolated nosocomial pathogen and is responsible for 10% of all hospital-acquired infections (4). The severity of disease caused by this bacterium is mediated in part by its type III secretion system and the four known effector proteins secreted by it, ExoS, ExoT, ExoU, and ExoY (15). Following translocation into host cells, these proteins utilize a variety of enzymatic activities and molecular interactions to disrupt normal cellular physiology, which results in bacterial persistence and tissue damage.
The evidence for a significant pathogenic role in disease is particularly compelling for ExoU (41, 48). Strains of P. aeruginosa that naturally secreted ExoU were more virulent than strains that did not in a mouse model of pneumonia (53), and disruption of the exoU gene resulted in decreased virulence (12, 20). Furthermore, transformation of the exoU gene into strains that did not normally harbor it increased virulence (1). An analysis of isogenic mutants indicated that ExoU was the most toxic of the four P. aeruginosa effector proteins in an animal model of acute pneumonia (32, 54). Secretion of ExoU was associated with increased mortality, increased bacterial burden in the lungs, and increased dissemination (54). Studies of human patients with naturally occurring P. aeruginosa infections supported these conclusions (18, 45). For example, hospital-acquired pneumonia patients infected with ExoU-secreting isolates had poorer outcomes than patients infected with nonsecreting isolates (18). Thus, secretion of ExoU confers upon P. aeruginosa strains the ability to cause especially severe disease.
ExoU's in vivo virulence has been associated with in vitro cytotoxicity (37, 50, 53). (Here "cytotoxicity" refers to cytolytic cell death.) In cell culture assays, ExoU-mediated killing is not limited to one or a few cell types. Rather, ExoU cytotoxicity has been observed in cell lines derived from macrophages, epithelial cells, and fibroblasts (5, 10, 12, 14, 19, 26, 30, 49, 51). This suggests that delivery of ExoU and its mechanism of cytotoxicity involve factors that are common to many cell types. In addition, ExoU is quite potent. Half-maximal killing requires only 300 to 600 molecules per cell (40), which explains why several groups of workers were unable to detect ExoU in eukaryotic cells using either green fluorescent protein (GFP) tags or immunoblot analysis (11, 40, 48). The membranes of intoxicated cells become disrupted, but features of apoptosis, such as DNA fragmentation, are not observed, indicating that death occurs by necrosis (5, 11, 12, 19, 20). Furthermore, expression of ExoU in CHO cells was sufficient for cytotoxicity, indicating that once inside a mammalian cell, ExoU does not require the presence of other bacterial proteins (11, 48).
To date, two domains of ExoU have been identified. Based on studies of other effector proteins, the N-terminal 100 amino acids is hypothesized to target this toxin to the type III secretion machinery (23). Residues 107 to 357 encode a patatin-like phospholipase A2 (PLA2) domain (40, 49). Patatin is a major storage protein of potatoes (16, 59) that has PLA2 activity which is used for protection under conditions of stress or infection (22, 46, 56). Patatin-like proteins possess a Ser-Asp catalytic dyad and an oxyanion hole essential for catalysis (22, 46). A sequence comparison revealed that the N terminus of ExoU possesses motifs similar to those of patatin and that disruption of these motifs eliminates ExoU's PLA2 activity (40, 42, 49). Cell culture and animal models of infection demonstrated that the PLA2 activity of ExoU is an integral part of its ability to kill eukaryotic cells and cause disease (37, 40, 48).
Although it has been established that ExoU's phospholipase activity is crucial for pathogenesis, the details of the mechanism by which this toxin kills mammalian cells are unclear. In this study, we showed that the C-terminal half of ExoU contains a localization domain that targets ExoU to the plasma membrane. Such targeting of this phospholipase to a region of the cell rich in phospholipids may be an important step in the process by which ExoU kills cells.
MATERIALS AND METHODS
Bacterial strains, media, and plasmids. Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strain XL1-Blue and Top10 cells were used in cloning experiments and grown in Luria-Bertani (LB) broth. When necessary, the medium was supplemented with ampicillin at a concentration of 50 to 100 μg/ml. P. aeruginosa strains PA103UT/S142A and PA103UT/LS608 were grown in LB broth. HeLa cervical carcinoma cells were grown in Eagle's minimal essential medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) in the presence of 5% CO2 at 37°C. 3T3 fibroblast cells stably transfected with pSwitch (Invitrogen, Carlsbad, CA) were maintained in Dulbecco's minimal essential medium (Invitrogen) supplemented with 10% FBS and 50 μg/ml hygromycin B (Roche Applied Science, Indianapolis, IN) in the presence of 5% CO2 at 37°C.
ExoU-expressing transfection constructs. Most of the transfection constructs expressing GFP-ExoU fusion proteins were generated previously (42); the exceptions were pGFP-ExoU-LS604 and pGFP-ExoU-LS619 (Table 1), which were generated as follows. An AgeI restriction endonuclease site was introduced 5' of the exoU gene and a KpnI site was introduced 3' of the exoU gene by PCR amplification of the appropriate exoU allele (42), using upstream primer ExoU-AgeI-5 and downstream primer ExoU-KpnI-3 (Table 2). DNA was amplified using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 62°C for 30 s, and 72°C for 90 s for 35 cycles and then 72°C for 10 min. The amplified products and the mammalian transfection vector pCDNA 3.1 NT-GFP (Invitrogen) (referred to below as pGFP) were digested with AgeI and KpnI (New England Biolabs) and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue competent cells. Transformants were checked for correct insertion by restriction endonuclease digestion with AgeI and KpnI, and the exoU gene in each construct was verified by nucleotide sequencing.
A construct expressing an untagged variant of ExoU containing an alanine substitution for the catalytic serine residue 142 (referred to as ExoU-S142A) was constructed as follows. An AgeI-KpnI fragment of pGFP-ExoU-S142A containing only the mutated exoU allele was treated with T4 polymerase, ligated into EcoRV-digested pCDNA(–) (Invitrogen), and transformed into Top10 cells. The correct orientation of the exoU gene was verified by restriction digestion.
Transfection constructs used for a deletion analysis aimed at identifying the N-terminal boundary of the ExoU localization domain were generated as follows. exoU gene fragments corresponding to amino acids 450 to 687, 500 to 687, 550 to 687, 600 to 687, and 650 to 687 were generated by PCR amplification of pExoU (43) with primers that introduced an AgeI restriction site immediately upstream of the exoU fragment and a KpnI restriction site approximately 300 bp downstream of the exoU fragment. The downstream primer for each construct was GFP750KpnI-3, and the upstream primers were GFP450AgeI-5, GFP500AgeI-5, GFP550AgeI-5, GFP600AgeI-5, and GFP650AgeI-5 (Table 2). The original stop codon after residue 687 was left intact. Amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 62°C for 30 s, and 72°C for 2 min for 35 cycles and then 72°C for 10 min. The amplified products and pGFP were digested with KpnI and NotI and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into Top10 cells, which generated transfection constructs that encoded ExoU fragments with N-terminal GFP tags. The integrity of the exoU allele in each construct was verified by nucleotide sequencing.
Transfection constructs used for a deletion analysis aimed at identifying the C-terminal boundary of the ExoU localization domain were generated as follows. exoU alleles encoding C-terminally truncated variants of ExoU were PCR amplified using the C1 and C2 vectors as templates (43) and primers that introduced an AgeI restriction site immediately upstream of the exoU gene and a KpnI restriction site and stop codon immediately downstream of the exoU gene. The upstream primer used for this was ExoU-AgeI-5, and the downstream primers were U623-KpnI-3 and U656-KpnI-3 (Table 2). Amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 64°C for 30 s, and 72°C for 3.5 min for 35 cycles and then 72°C for 10 min. The amplified products and pGFP were digested with AgeI and KpnI and purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue cells to generate N-terminally GFP-tagged ExoU variants with 31 and 54 amino acid truncations (referred to as GFP-ExoU657-687 and GFP-ExoU624-687, respectively). The integrity of the exoU allele in each construct was verified by nucleotide sequencing.
Transient transfections. One day prior to transfection, 1.5 x 104 HeLa or 3T3 cells were seeded onto 12-mm coverslips in a 24-well non-tissue-culture-treated plate. The coverslips were treated with 10 μg/ml rat tail collagen type I (Upstate Biotechnology, Lake Placid, NY) prior to 3T3 seeding. Transient transfections were performed by adding 25 μl of prewarmed serum-free medium, 0.75 μl FuGENE 6 (Roche Applied Science), and 0.5 μg DNA to each well of cells. Alternatively, HeLa cells in 100-mm tissue culture plates were transiently transfected at 50 to 70% confluence by addition of 600 μl serum-free medium, 18 μl FuGENE 6, and 6 μg DNA to each plate of cells. Following transfection, cells were incubated at 37°C in the presence of 5% CO2 for 12 to 24 h prior to examination by fluorescence microscopy.
P. aeruginosa infection of HeLa cells. HeLa cells were grown almost to confluence on coverslips in 24-well cell culture plates. PA103 strains were grown overnight at 37°C in LB broth without shaking. The equivalent of 108 CFU of bacteria from this culture was then diluted into 1 ml of LB broth and incubated without shaking for 1.5 h at 37°C. Bacteria were then pelleted by centrifugation and resuspended in 1 ml of serum-free modified Eagle's medium. Each well containing HeLa cells was infected with 100 μl of bacterial culture. Centrifugation at 500 x g for 10 min was used to pellet the bacteria onto the HeLa cells and synchronize the infections. Following coincubation for 3 h at 37°C in the presence of 5% CO2, cells were examined for the presence and localization of ExoU by fluorescence microscopy.
Fluorescence microscopy. In transfection studies, HeLa cells were fixed with 3.7% formaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 20 min, and this was followed by quenching of autofluorescence with 50 mM NH4Cl. In experiments in which antibodies were not used, cells were incubated with 10 μg/ml Hoechst 33342 stain (Molecular Probes, Eugene, OR) for 3 min to visualize the nucleus, after which coverslips were placed on the slides with 1 drop of DABCO (Fluka, Switzerland) and sealed with nail polish. Otherwise, the following manipulations were performed prior to the addition of Hoechst stain. Cells were blocked and permeabilized with 0.1% (vol/vol) saponin-10% (vol/vol) FBS in phosphate-buffered saline (PBS) (blocking-permeabilization buffer) for 30 min and then incubated for 30 min with polyclonal ExoU antiserum (20) diluted 1:1,000 in blocking-permeabilization buffer. The cells were then incubated for 30 min with rhodamine-conjugated anti-rabbit secondary antibody (ICN/Cappel, Aurora, OH) diluted 1:200 in blocking-permeabilization buffer. The cells were viewed using an Olympus IX inverted fluorescence microscope with standard filter sets and a Photometrix cooled charge-coupled device camera (CH350/LCCD) driven by DeltaVision software (Applied Precision Inc., Seattle, WA). Images were deconvolved using DeltaVision software. Single Z sections are shown in the figures. Experiments without permeabilization were performed as described above except that the saponin treatment was omitted. In concanavalin A (ConA) experiments, cells were first incubated at 4°C for 15 min to inhibit endocytosis. Alexa Fluor 647-conjugated ConA (Molecular Probes) was then added to unfixed cells at a concentration of 100 μl/ml for 10 min at 4°C. Cells were visualized immediately after the coverslips were placed on slides with 1 drop of ice-cold PBS and sealed with nail polish.
In P. aeruginosa infection studies, HeLa cells were washed with PBS, fixed with 3.7% formaldehyde for 15 min, and quenched with 50 mM NH4Cl. The cells were treated with 2 μl/ml Alexa Fluor 647-conjugated ConA for 5 min and permeabilized with 0.5% (vol/vol) Triton X-100 (Sigma, St. Louis, Mo.) in PBS for 20 min. The cells were then blocked for 30 min in Image-iT FX signal enhancer buffer (Molecular Probes) and incubated for 1 h with polyclonal ExoU antibody (1:1,000 dilution) in 0.4% (vol/vol) fish gelatin (Sigma) in PBS. The cells were then incubated for 1 h with Alexa Fluor 555 (Cy3) goat anti-rabbit antibody (Molecular Probes) at a dilution of 1:250 (vol/vol) in 0.4% (vol/vol) fish gelatin in PBS. Finally, the cells were incubated for 3 min with Hoechst 33342 stain. Coverslips were placed on slides with 1 drop of DABCO and sealed with nail polish. The cells were viewed with a Leica DMIREZ microscope powered by a 100-W mercury lamp and equipped with a Hamamatsu ORCA-ER camera. Pictures were taken at a magnification of x1,000 using the Openlab 4.0.4 software and were deconvolved using the Volocity 3.6.1 software.
Cell fractionation. For each condition, five 100-mm plates containing HeLa cells were transfected with pGFP-ExoU-S142A as described above. Twelve hours after transfection, the cells were washed and incubated with PBS at 4°C for 25 min while they were gently rocked. Cells were scraped into 25 ml PBS, collected by centrifugation, and resuspended in 1 ml ice-cold 5 mM Tris (pH 7.8). After incubation on ice for 10 min, the cells were lysed by passage 30 times through a 21.5-gauge needle. Intact organelles and nuclei were removed by centrifugation at 10,000 x g at 4°C for 10 min. Supernatants were added to polyallomar tubes (2 by 0.5 in.; Beckman, Palo Alto, CA), and ultracentrifugation was performed with a Beckman model L8-80 M centrifuge at 32,000 x g for 1 h at 4°C. The supernatant, corresponding to cell cytoplasm, and the pellet, corresponding to membranes, were collected. The pellet was resuspended in 1 ml of 0.1% (vol/vol) Triton X-100 supplemented with a protease inhibitor cocktail (Complete Mini protease inhibitor pellet; Roche Applied Science). After addition of 5x sodium dodecyl sulfate (SDS) protein electrophoresis sample buffer, proteins were boiled for 10 min, electrophoresed through a 10% (wt/vol) SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. An immunoblot analysis was then performed as previously described (53), using rabbit polyclonal ExoU antiserum (1:6,000) (20), mouse monoclonal pan-cadherin antibodies (1:2,000) (Abcam, Cambridge, MA), and mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (1:20,000) (Abcam) diluted in blocking buffer (5% [wt/vol] dry milk and 0.05% [vol/vol] Tween 20 in PBS). Goat anti-rabbit or goat anti-mouse immunoglobulin G horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:5,000 in blocking buffer were used as secondary antibodies.
Bioinformatics analysis. Residues 550 to 687 of ExoU were analyzed for the presence of informative sequences using a number of approaches. Homology to known proteins and the presence of motifs were determined using the Basic Local Alignment Search Tool (BLAST) to search the Non-Redundant Protein Sequence Database and the Conserved Domain Database (2, 3, 35). A search for putative transmembrane domains was performed using the TMHMM algorithm (29). The Simple Architecture Modular Research Tool (SMART) algorithm (33) was used to search for the following phosphoinositide-binding motifs: C2, PH, FYVE, ENTH, and PX. Patterns representative of coiled-coil domains were identified using COILS (34).
Kinetics of ExoU localization. Inducible expression of ExoU in 3T3 cells was performed using the GeneSwitch approach (Invitrogen), as described by the manufacturer. Briefly, GFP-tagged ExoU-S142A was expressed in 3T3 cells by ligating the appropriate exoU allele into the pGene/V5-His B vector. This was accomplished by PCR amplification of the exoU allele using pGFP-ExoU-S142A (42) as a template. Upstream primer GFP-5 and downstream primer ExoU-3 were designed to incorporate a KpnI restriction site immediately upstream of the gfp gene and a NotI restriction site immediately downstream of the exoU gene (Table 2). PCR amplification was performed using the following parameters: 95°C for 2 min, followed by 95°C for 30 s, 64°C for 30 s, and 72°C for 4 min for 35 cycles and then 72°C for 10 min. The amplified products and pGene/V5-His B were digested with KpnI and NotI and were purified by electrophoresis through a 0.8% (wt/vol) agarose gel. The two DNA fragments were ligated together and transformed into XL1-Blue competent cells. The resulting construct was designated pGene-GFP-ExoU-S142A. The integrity of the exoU fragment in each construct was verified by nucleotide sequencing.
pGene-GFP-ExoU-S142A was transiently transfected into a stable 3T3 cell line harboring pSwitch, a second GeneSwitch vector required for inducible expression. ExoU expression was induced by addition of 10–8 M mifepristone 24 h after transfection. At 0, 1, 2, 3, and 4 h postinduction, cells were fixed with 3.7% (vol/vol) formaldehyde in PBS for 20 min. Cells were incubated with 10 μg/ml Hoechst stain for 3 min to allow visualization of the nucleus. Coverslips were then placed on slides with 1 drop of DABCO and sealed with nail polish. The cells were viewed using an Olympus IX inverted fluorescence microscope and deconvolved as described above.
RESULTS
ExoU localizes to the cell periphery of transfected HeLa cells. To determine the subcellular localization of ExoU, we attempted to detect intact ExoU fused to GFP and expressed in HeLa cells by transfection. However, ExoU was not detectable by fluorescence microscopy or immunoblot analysis in these experiments, although there was evidence of cytotoxicity. These data suggested that small amounts of ExoU, below the limit of detection, were sufficient to lyse cells and thus prevent visualization. Note that previous attempts to detect wild-type ExoU in eukaryotic cells either by microscopy or immunoblot analysis were also unsuccessful (11, 40, 42, 43, 49).
Since the cytotoxicity of ExoU prevented successful visualization of this toxin, subcellular localization was examined using a catalytically inactive form of the toxin. ExoU having an alanine substitution in place of the putative catalytic serine residue (referred to as ExoU-S142A) was used for this purpose. This variant of ExoU, which had previously been shown to lack PLA2 activity and to be noncytotoxic (40, 42, 48), was tagged with GFP (referred to as GFP-ExoU-S142A) and expressed in HeLa cells by transient transfection. Approximately 18 h later, the cells were fixed, incubated with Hoechst stain, and visualized by fluorescence microscopy. Whereas the GFP control was diffusely green throughout the cytoplasm, GFP-ExoU-S142A was localized predominantly at the cell periphery in a punctate fashion, although some fluorescence was also observed in the cell interior (Fig. 1A). To ensure that this pattern was not an artifact of the GFP tag, polyclonal ExoU antiserum was used to visualize ExoU-S142A lacking a GFP tag. Similar to the GFP fusion protein, ExoU-S142A lacking GFP was visualized at the cell periphery (Fig. 1B). These data indicate that ExoU-S142A localizes to the cell periphery.
To better characterize the peripheral localization of ExoU relative to the HeLa cell plasma membrane, colocalization experiments were performed using the lectin ConA, which binds carbohydrate residues on the cell surface and is thus used to visualize the plasma membrane. Following transfection with the GFP-ExoU-S142A-expressing construct, HeLa cells were incubated with Alexa Fluor 647-conjugated ConA and visualized by fluorescence microscopy. The GFP-ExoU-S142A and ConA fluorescence patterns were very similar (Fig. 1C), indicating that ExoU was at or adjacent to the plasma membrane.
ExoU is in the membrane fraction of transfected HeLa cells. To better characterize the subcellular localization of ExoU, lysates from transfected HeLa cells were fractionated by differential centrifugation. Pellet fractions, containing plasma and organelle membranes, and supernatant fractions, containing cytosolic components, were volume adjusted and electrophoresed on an SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and exposed to polyclonal ExoU antiserum. The relative purity of each fraction was confirmed using pan-cadherin as a marker for the cell membrane (57) and GAPDH as a marker for the cytosol (57). Although some GFP-S142A-ExoU was found in the cytosolic fraction, the majority of this protein was in the membrane fraction (Fig. 2). These experiments were done without a cross-linking reagent, indicating that S142A-ExoU was strongly associated with the plasma membrane. This association may be a high-affinity interaction with a membrane-associated factor, or ExoU itself may be inserted into the plasma membrane.
If ExoU were intercalated into the plasma membrane, one would expect it to be exposed on the surface of the cell. To experimentally examine this possibility, immunofluorescence microscopy using polyclonal ExoU antiserum was performed in the presence or absence of the permeabilizing detergent saponin. A fluorescent signal was absent when the membranes were intact, indicating that ExoU is not externally exposed (Fig. 3). Similar results were obtained using anti-GFP antibodies (data not shown). These data indicate that ExoU is not surface exposed and therefore is unlikely to be inserted through the plasma membrane.
C terminus of ExoU is necessary for localization to the plasma membrane. Next, we attempted to identify the portion of ExoU that was necessary for localization to the plasma membrane. To accomplish this, we utilized a previously generated panel of constructs that encoded ExoU variants having a substitution or insertion in each of five regions of ExoU required for cytotoxicity (42): ExoU-G112W (region 1), ExoU-S142A (region 2; the noncatalytic variant used in the experiments described above), ExoU-G286W (region 3), ExoU-D344A (region 4), and ExoU-LS608 (region 5; a 5-amino-acid insertion immediately prior to N608). Regions 1, 2, 3, and 4 are in the patatin-like phospholipase domain of the N-terminal half of ExoU, whereas region 5 is located in the C-terminal half of ExoU (see Fig. 8 for the locations of these regions in ExoU). Each ExoU variant was tagged with GFP, expressed in HeLa cells, and viewed by fluorescence microscopy 18 h later. As expected, GFP alone localized to the cytoplasmic compartment of the HeLa cells (Fig. 4). Like the region 2 mutant ExoU-S142A, ExoU containing a single amino acid substitution in region 1 or 4 was observed in a punctate pattern on the circumference of the cell (Fig. 4). In contrast, the region 5 ExoU variant was found diffusely throughout the cytoplasm, similar to GFP alone (Fig. 4). These data indicated that the C terminus of ExoU is necessary for localization to the plasma membrane. A region 3 variant of ExoU was found at the cell periphery, but increased amounts were also present in the cell interior, suggesting a possible role for this region in modifying the localization process (Fig. 4).
To confirm that region 5 of ExoU is essential for localization to the plasma membrane, two additional ExoU variants were examined. ExoU-LS604 and ExoU-LS619 had 5-amino-acid insertions immediately prior to V604 and Y619, respectively, in region 5 of ExoU. Constructs encoding these variants were transfected in HeLa cells, and ExoU localization was examined by fluorescence microscopy. In both cases, ExoU was observed diffusely throughout the cytoplasm (data not shown). Thus, insertions at three different sites in region 5 of ExoU abolished targeting to the plasma membrane, confirming the importance of this portion of the toxin for appropriate localization.
C terminus of ExoU is sufficient for localization to the plasma membrane. To determine whether the C-terminal half of ExoU was sufficient for localization to the plasma membrane, the ability of this portion of the toxin to appropriately localize an unrelated protein was determined. GFP was chosen as the cargo protein for this experiment. A fusion protein consisting of the C-terminal half of ExoU (ExoU450-687) and GFP was expressed in HeLa cells by transient transfection, and localization was examined by fluorescence microscopy. Whereas GFP alone was visualized in the cell interior, the GFP-ExoU450-687 fusion protein was localized to the cell periphery (Fig. 5A). This indicated that the C-terminal half of ExoU is sufficient to target an unrelated protein to the cell periphery and therefore contains a localization domain.
The localization domain of ExoU lies in residues 550 to 687. Next, the boundaries of the localization domain were defined more precisely. To determine the N-terminal boundary of the localization domain, a deletion analysis was performed in which residues 650 to 687, 600 to 687, 550 to 687, and 500 to 687 were each fused with GFP. Fusion proteins were expressed in HeLa cells, and localization was examined by fluorescence microscopy. GFP fused to residues 450 to 687, 500 to 687, or 550 to 687 of ExoU was targeted to the cell periphery (Fig. 5). Fluorescence was detected both in the cell interior and at the periphery when GFP was fused to residues 600 to 687 and only in the cell interior when GFP was fused to residues 650 to 687 (Fig. 5). These data indicated that the N-terminal boundary of the localization domain is between residues 550 and 600 of ExoU.
To determine the C-terminal boundary of the localization domain, two C-terminal deletion variants of ExoU were fused to GFP, expressed in HeLa cells, and viewed by fluorescence microscopy. ExoU657-687 consists of amino acids 1 to 656 and is missing the last 31 amino acids of ExoU, whereas ExoU624-687 consists of amino acids 1 to 623 and is missing the last 64 amino acids. Both ExoU variants localized to the cell interior and not the cell periphery, indicating that the C-terminal boundary of the localization domain is after amino acid 656 (Fig. 5). Together with the data described above, these results indicate that the localization domain is between residues 550 and 687, within the C-terminal 138 amino acids of ExoU.
The kinetics of ExoU localization are consistent with those of cytotoxicity. ExoU-mediated killing is quite rapid and occurs within the first 3 h of infection when P. aeruginosa is coincubated with eukaryotic cells in vitro (5). We therefore wished to investigate whether localization to the plasma membrane occurred in a similar time frame, which would be consistent with a role for localization in killing. To accomplish this, ExoU was expressed in 3T3 cells using the mifepristone-inducible GeneSwitch expression system. This approach uses a positive feedback system to express a gene of interest in mammalian cells following the addition of mifepristone to the culture medium. An allele encoding GFP-tagged ExoU-S142A was cloned into the GeneSwitch vector, which was then transfected into 3T3 cells expressing regulatory components of the GeneSwitch system. Eighteen to twenty-four hours after transfection, mifepristone was added to the cells to induce GFP-ExoU-S142A expression. The cells were incubated with mifepristone for 0, 1, 2, 3, or 4 h and then fixed and viewed by fluorescence microscopy. No fluorescence was detectable prior to the addition of mifepristone, but by 1 h postinduction, GFP-ExoU-S142A was already at the periphery of the cells (Fig. 6). GFP-ExoU-S142A was still present primarily along the circumference of the cells after 2 h. Interestingly, by 3 to 4 h postinduction, GFP-ExoU-S142A was accumulating internally as well as at the cell periphery (Fig. 6). These data indicate that ExoU localizes to the cell periphery within 1 h after expression from a transfected construct. Furthermore, they indicate that ExoU peripheral localization is unlikely to be an artifact of overexpression, since it occurs at early times when little ExoU has accumulated in cells. The fluorescence in the cell interior at 3 and 4 h may have been due to either endocytosis of ExoU into the cell from the plasma membrane or overexpression at these later times leading to nonspecific protein aggregation.
The localization domain is required for appropriate targeting of ExoU following translocation into cells by the P. aeruginosa type III secretion system. To ensure that membrane targeting was not an artifact of transfection, we determined the localization of ExoU following translocation into host cells by the P. aeruginosa type III secretion system. PA103UT, a P. aeruginosa strain with in-frame deletions in its endogenous exoU and exoT genes, was used for these studies because it has an intact type III secretion system but does not secrete any known effector proteins. Single copies of mutated exoU alleles under the control of their endogenous promoters were inserted into the chromosomal attB site of PA103UT. HeLa cells were coincubated with these P. aeruginosa strains for 3 h, and ExoU localization was examined by immunofluorescence microscopy. Labeled ConA was used to visualize the HeLa cell plasma membranes.
Infection with PA103UT/S142A, which secreted catalytically inactive ExoU-S142A, resulted in peripheral localization of the toxin, similar to the pattern observed following transfection (Fig. 7A). In contrast, predominantly cytoplasmic localization was seen with PA103UT/LS608, which secreted the ExoU-LS608 variant of ExoU that contained a 5-amino-acid insertion in the localization domain (Fig. 7B). Visualization of uninfected cells confirmed that the observed signal was indeed due to ExoU (Fig. 7C).
DISCUSSION
In this study, the subcellular localization of ExoU was examined to increase our understanding of the mechanism by which this toxin kills cells. Microscopy studies showed that ExoU rapidly localized to the cell periphery. Together with the results of fractionation experiments, these findings indicated that ExoU localized either to the plasma membrane or to a structure associated with the plasma membrane. This is in agreement with work of Phillips et al., who found peripheral localization of ExoU in syringe-loaded cells (40). Colocalization studies with the lectin ConA indicated that there is a very close association between ExoU and the plasma membrane, although ExoU was not exposed on the cell surface. Deletional analysis indicated that the localization domain of ExoU, which was both necessary and sufficient for targeting to the plasma membrane, was in residues 550 to 687. Localization occurred within 1 h of ExoU expression in transfected cells. Although the kinetics of localization in transfected cells may differ from those in cells in which ExoU is injected by type III secretion, it is interesting that the entire process of ExoU secretion, translocation, and killing occurs in 1.5 to 3 h (5). Thus, rapid localization to the membrane would be expected if this were an essential step in ExoU-mediated cytotoxicity.
Targeting to specific subcellular compartments is an emerging theme among type III effector proteins. Although some of these proteins localize to the nucleus (e.g., YopM of Yersinia [6, 55]), mitochondria (e.g., SopA and SipB of Salmonella [21, 31]), Lamp1-positive vesicles (e.g., SifA of Salmonella), or other intracellular vesicles of host cells (e.g., YopE of Yersinia [28]), many effector proteins target the plasma membrane or structures closely associated with it. The Salmonella type III-secreted proteins SopE, SopE2, SopB, SptP, SipA, SipC, and SspC all localize to the plasma membrane (8, 52, 58), as do YpkA of Yersinia (17) and Tir of enteropathogenic E. coli (27). For many of these effector proteins, localization to the plasma membrane has been shown to be important for function. For example, following localization to the plasma membrane, SipA and SipC cooperate to cause actin polymerization and bundling at the cell surface, augmenting the ruffling necessary for the internalization of Salmonella cells (8, 36, 61). Likewise, Tir inserts into the plasma membrane so that an internal domain of the protein is exposed on the cell surface and accessible for binding by its bacterial ligand, intimin (9, 27). In this regard, the localization of the phospholipase ExoU to the plasma membrane may have pathogenic relevance since this subcellular location is rich in phospholipids.
Deletional analysis indicated that the localization domain of ExoU was between residues 550 and 687, but a closer examination of this region did not yield clues regarding the mechanism of targeting. Database searches detected no homology between this region and any characterized protein or motif. Nor did this portion of ExoU contain predicted transmembrane domains. These findings and our inability to detect ExoU exposed on the surface of transfected cells make insertion through the plasma membrane, like the insertion that occurs with Tir (9, 27), unlikely. Sites for prenylation or acylation, such as those identified in the Salmonella effector protein SifA (7, 44), were not found. Nor was there similarity to characterized proteins such as actin-binding proteins, which would suggest targeting to the actin-rich cell cortex immediately adjacent to the plasma membrane. This is the mechanism by which the YpkA localization domain, which exhibits homology with the actin bundling protein coronin, targets this Yersinia effector protein to the cell periphery (17, 25). The phosphoinositide-binding domains, such as PH, PX, and ENTH, are a family of motifs that target proteins to membrane inositol phospholipids and play an important role in a diverse array of cellular processes (24). However, no such domains were present in the C-terminal portion of ExoU. A search for coiled-coil domains yielded only very low or no probability matches. It is possible that residues 550 to 687 of ExoU indirectly move this toxin to the cell periphery by binding a second factor that itself targets the plasma membrane. For example, SopE and SptP require targeting of their binding partner, the host cell small GTPase Cdc42, to the plasma membrane for their own membrane localization (8). Although no host proteins that bind to ExoU have been identified yet, it is known that this toxin requires a host cell cofactor for activation of its PLA2 activity (40, 47, 49). This factor may play a role in both activating ExoU and targeting ExoU to the plasma membrane. Alternatively, localization to the cell periphery may occur by a novel mechanism.
Recent reports have indicated that ExoS also contains a localization domain that targets this toxin to membranes (38, 39). Although both ExoS and ExoU are secreted by the same P. aeruginosa type III system, their localization domains have little in common. The localization domain of ExoS is located in residues 51 to 72 of the N terminus, whereas the localization domain of ExoU is between residues 550 and 687 of the C terminus (28, 38). Thus, the localization domain of ExoS is both smaller and in a different part of the protein than the corresponding domain of ExoU. Furthermore, there is no sequence homology between these domains. It is unclear whether ExoU and ExoS localize to the same subcellular compartments. Although ExoS is initially targeted to the plasma membrane, it subsequently localizes to the Golgi-endoplasmic reticulum of the host cell (28, 60). ExoU is primarily targeted to the plasma membrane, although localization to the cell interior also occurs at later times.
Our findings further clarify a domain structure for ExoU (Fig. 8). As is the case with other type III effector proteins (23), the extreme N terminus likely contains information required to direct ExoU through the type III secretion and translocation apparatus. Consistent with this is the observation that residues 3 to 123 were required for binding of ExoU's chaperone, SpcU (13). Immediately adjacent to the secretion/translocation domain is the patatin-like PLA2 domain from residue 107 to residue 357. This domain is required for the phospholipase activity of ExoU. Substitution of one of several critical amino acids in this domain, including the putative catalytic serine S142 and catalytic aspartate D344, abrogates PLA2 activity and cytotoxicity in vitro (40, 42, 48), as well as virulence in vivo (37). Finally, in this paper we describe a localization domain at the C terminus of ExoU, in residues 550 to 687. This domain is required for targeting to the plasma membrane. Interestingly, previous work demonstrated that portions of this domain were also required for cytotoxicity and PLA2 activity (11, 20, 40, 42, 43). Other important regions of ExoU, including a site for binding of the activating host cofactor, have yet to be identified.
Together, the results of these studies suggest a model for the mechanism of ExoU cytotoxicity. First, information carried within the secretion/translocation domain of ExoU is used to inject this toxin into mammalian cells through the type III secretion needle. Once ExoU is within the host cell, this domain is dispensable for subsequent steps in the killing process (43). ExoU is then rapidly targeted to the cell membrane in a process that requires the localization domain in the C-terminal half of the toxin. The finding that membrane localization is essential for cytotoxicity is consistent with previous reports that residues 550 to 687 of ExoU are also necessary for cytotoxicity (11, 20, 40, 42, 43). During or after this localization, ExoU interacts with a host cell factor that activates the phospholipase activity of the patatin-like domain, explaining the requirement for the localization domain for PLA2 activity. This activity then causes cell death by utilizing plasma membrane phospholipids as substrates. Death may occur by direct disruption of the plasma membrane integrity or indirectly by perturbation of signaling cascades involving phospholipids, such as the prostaglandin pathway. Further work is necessary to test this model and determine whether there are additional steps in the mechanism by which ExoU kills host cells.
ACKNOWLEDGMENTS
We thank Everett Roark, Parwez Nawabi, Christiaan van Ooij, Kasturi Haldar, and Ciara Shaver for helpful technical suggestions and assistance. We also thank Kasturi Haldar for use of her microscope.
This work was supported by the NIH (grants AI053674 and AI065615 to A.R.H.).
Present address: Center for Oral Health & Systemic Disease, School of Dentistry, University of Louisville, Louisville, KY 40292.
REFERENCES
1. Allewelt, M., F. T. Coleman, M. Grout, G. P. Priebe, and G. B. Pier. 2000. Acquisition of expression of the Pseudomonas aeruginosa ExoU cytotoxin leads to increased bacterial virulence in a murine model of acute pneumonia and systemic spread. Infect. Immun. 68:3998-4004.
2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
3. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.
4. American Thoracic Society. 1996. Hospital-acquired pneumonia in adults: diagnosis, assessment of severity, initial antimicrobial therapy, and preventive strategies. A consensus statement, American Thoracic Society, November 1995. Am. J. Respir. Crit. Care Med. 153:1711-1725.
5. Apodaca, G., M. Bomsel, R. Lindstedt, J. Engel, D. Frank, K. Mostov, and J. Wiener-Kronish. 1995. Characterization of Pseudomonas aeruginosa-induced MDCK cell injury: glycosylation defective host cells are resistant to bacterial killing. Infect. Immun. 63:1541-1551.
6. Benabdillah, R., L. J. Mota, S. Lutzelschwab, E. Demoinet, and G. R. Cornelis. 2004. Identification of a nuclear targeting signal in YopM from Yersinia spp. Microb. Pathog. 36:247-261.
7. Boucrot, E., C. R. Beuzon, D. W. Holden, J. P. Gorvel, and S. Meresse. 2003. Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function. J. Biol. Chem. 278:14196-14202.
8. Cain, R. J., R. D. Hayward, and V. Koronakis. 2004. The target cell plasma membrane is a critical interface for Salmonella cell entry effector-host interplay. Mol. Microbiol. 54:887-904.
9. de Grado, M., A. Abe, A. Gauthier, O. Steele-Mortimer, R. DeVinney, and B. B. Finlay. 1999. Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli. Cell. Microbiol. 1:7-17.
10. Evans, D. J., T. C. Kuo, M. Kwong, R. Van, and S. M. Fleiszig. 2002. Mutation of csk, encoding the C-terminal Src kinase, reduces Pseudomonas aeruginosa internalization by mammalian cells and enhances bacterial cytotoxicity. Microb. Pathog. 33:135-143.
11. Finck-Barbanon, V., and D. W. Frank. 2001. Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU. J. Bacteriol. 183:4330-4344.
12. Finck-Barbanon, V., J. Goranson, L. Zhu, T. Sawa, J. P. Wiener-Kronish, S. M. J. Fleiszig, C. Wu, L. Mende-Mueller, and D. Frank. 1997. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol. Microbiol. 25:547-557.
13. Finck-Barbanon, V., T. L. Yahr, and D. W. Frank. 1998. Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin. J. Bacteriol. 180:6224-6231.
14. Fleiszig, S. M. J., T. S. Zaidi, M. J. Preston, M. Grout, D. J. Evans, and G. B. Pier. 1996. Relationship between cytotoxicity and corneal epithelial cell invasion by clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 64:2288-2294.
15. Frank, D. W. 1997. The exoenzyme S regulon of Pseudomonas aeruginosa. Mol. Microbiol. 26:621-629.
16. Ganal, M. W., M. W. Bonierbale, M. S. Roeder, W. D. Park, and S. D. Tanksley. 1991. Genetic and physical mapping of the patatin genes in potato and tomato. Mol. Gen. Genet. 225:501-509.
17. Hakansson, S., E. Galyov, R. Rosqvist, and H. Wolf-Watz. 1996. The Yersinia Ypk Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol. Microbiol. 20:593-603.
18. Hauser, A. R., E. Cobb, M. Bodí, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30:521-528.
19. Hauser, A. R., and J. N. Engel. 1999. Pseudomonas aeruginosa induces type III secretion-mediated apoptosis of macrophages and epithelial cells. Infect. Immun. 67:5530-5537.
20. Hauser, A. R., P. J. Kang, and J. Engel. 1998. PepA, a novel secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence. Mol. Microbiol. 27:807-818.
21. Hernandez, L. D., M. Pypaert, R. A. Flavell, and J. E. Galan. 2003. A Salmonella protein causes macrophage cell death by inducing autophagy. J. Cell Biol. 163:1123-1131.
22. Hirschberg, H. J., J. W. Simons, N. Dekker, and M. R. Egmond. 2001. Cloning, expression, purification and characterization of patatin, a novel phospholipase A. Eur. J. Biochem. 268:5037-5044.
23. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:279-433.
24. Itoh, T., and T. Takenawa. 2002. Phosphoinositide-binding domains: functional units for temporal and spatial regulation of intracellular signalling. Cell. Signal. 14:733-743.
25. Juris, S. J., A. E. Rudolph, D. Huddler, K. Orth, and J. E. Dixon. 2000. A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption. Proc. Natl. Acad. Sci. USA 97:9431-9436.
26. Kang, P. J., A. R. Hauser, G. Apodaca, S. Fleiszig, J. Wiener-Kronish, K. Mostov, and J. N. Engel. 1997. Identification of Pseudomonas aeruginosa genes required for epithelial cell injury. Mol. Microbiol. 24:1249-1262.
27. Kenny, B., R. DeVinney, M. Stein, D. J. Reinscheid, E. A. Frey, and B. B. Finlay. 1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91:511-520.
28. Krall, R., Y. Zhang, and J. T. Barbieri. 2004. Intracellular membrane localization of Pseudomonas ExoS and Yersinia YopE in mammalian cells. J. Biol. Chem. 279:2747-2753.
29. Krogh, A., B. Larsson, G. von Heijne, and E. L. Sonnhammer. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305:567-580.
30. Kurahashi, K., O. Kajikawa, T. Sawa, M. Ohara, M. A. Gropper, D. W. Frank, T. R. Martin, and J. P. Wiener-Kronish. 1999. Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia. J. Clin. Investig. 104:743-750.
31. Layton, A. N., P. J. Brown, and E. E. Galyov. 2005. The Salmonella translocated effector SopA is targeted to the mitochondria of infected cells. J. Bacteriol. 187:3565-3571.
32. Lee, V. T., R. S. Smith, B. Tummler, and S. Lory. 2005. Activities of Pseudomonas aeruginosa effectors secreted by the type III secretion system in vitro and during infection. Infect. Immun. 73:1695-1705.
33. Letunic, I., R. R. Copley, S. Schmidt, F. D. Ciccarelli, T. Doerks, J. Schultz, C. P. Ponting, and P. Bork. 2004. SMART 4.0: towards genomic data integration. Nucleic Acids Res. 32:D142-D144.
34. Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science 252:1162-1164.
35. Marchler-Bauer, A., J. B. Anderson, P. F. Cherukuri, C. DeWeese-Scott, L. Y. Geer, M. Gwadz, S. He, D. I. Hurwitz, J. D. Jackson, Z. Ke, C. J. Lanczycki, C. A. Liebert, C. Liu, F. Lu, G. H. Marchler, M. Mullokandov, B. A. Shoemaker, V. Simonyan, J. S. Song, P. A. Thiessen, R. A. Yamashita, J. J. Yin, D. Zhang, and S. H. Bryant. 2005. CDD: a conserved domain database for protein classification. Nucleic Acids Res. 33:D192-D196.
36. McGhie, E. J., R. D. Hayward, and V. Koronakis. 2001. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin. EMBO J. 20:2131-2139.
37. Pankhaniya, R. R., M. Tamura, L. R. Allmond, K. Moriyama, T. Ajayi, J. P. Wiener-Kronish, and T. Sawa. 2004. Pseudomonas aeruginosa causes acute lung injury via the catalytic activity of the patatin-like phospholipase domain of ExoU. Crit. Care Med. 32:2293-2299.
38. Pederson, K. J., R. Krall, M. J. Riese, and J. T. Barbieri. 2002. Intracellular localization modulates targeting of ExoS, a type III cytotoxin, to eukaryotic signalling proteins. Mol. Microbiol. 46:1381-1390.
39. Pederson, K. J., S. Pal, A. J. Vallis, D. W. Frank, and J. T. Barbieri. 2000. Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells. Mol. Microbiol. 37:287-299.
40. Phillips, R. M., D. A. Six, E. A. Dennis, and P. Ghosh. 2003. In vivo phospholipase activity of the Pseudomonas aeruginosa cytotoxin ExoU and protection of mammalian cells with phospholipase A2 inhibitors. J. Biol. Chem. 278:41326-41332.
41. Rabin, S. D. P., and A. R. Hauser. 2004. ExoU: a cytotoxin delivered by the type III secretion system of Pseudomonas aeruginosa, p. 69-90. In R. Schaffrath and M. Schmitt (ed.), Microbial protein toxins. Springer-Verlag, Heidelberg, Germany.
42. Rabin, S. D. P., and A. R. Hauser. 2005. Functional regions of the Pseudomonas aeruginosa cytotoxin ExoU. Infect. Immun. 73:573-582.
43. Rabin, S. D. P., and A. R. Hauser. 2003. Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae. Infect. Immun. 71:4144-4150.
44. Reinicke, A. T., J. L. Hutchinson, A. I. Magee, P. Mastroeni, J. Trowsdale, and A. P. Kelly. 2005. A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery. J. Biol. Chem. 280:14620-14627.
45. Roy-Burman, A., R. H. Savel, S. Racine, B. L. Swanson, N. S. Revadigar, J. Fujimoto, T. Sawa, D. W. Frank, and J. P. Wiener-Kronish. 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183:1767-1774.
46. Rydel, T. J., J. M. Williams, E. Krieger, F. Moshiri, W. C. Stallings, S. M. Brown, J. C. Pershing, J. P. Purcell, and M. F. Alibhai. 2003. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad. Biochemistry 42:6696-6708.
47. Sato, H., J. B. Feix, C. J. Hillard, and D. W. Frank. 2005. Characterization of phospholipase activity of the Pseudomonas aeruginosa type III cytotoxin, ExoU. J. Bacteriol. 187:1192-1195.
48. Sato, H., and D. W. Frank. 2004. ExoU is a potent intracellular phospholipase. Mol. Microbiol. 53:1279-1290.
49. Sato, H., D. W. Frank, C. J. Hillard, J. B. Feix, R. R. Pankhaniya, K. Moriyama, V. Finck-Barbancon, A. Buchaklian, M. Lei, R. M. Long, J. Wiener-Kronish, and T. Sawa. 2003. The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU. EMBO J. 22:2959-2969.
50. Sawa, T., M. Ohara, K. Kurahashi, S. S. Twining, D. Frank, D. B. Doroques, T. Long, M. A. Gropper, and J. P. Wiener-Kronish. 1998. In vitro cellular toxicity predicts Pseudomonas aeruginosa virulence in lung infections. Infect. Immun. 66:3242-3249.
51. Sawa, T., T. Yahr, M. Ohara, K. Kurahashi, M. A. Gropper, J. P. Wiener-Kronish, and D. W. Frank. 1999. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat. Med. 5:392-398.
52. Scherer, C. A., E. Cooper, and S. I. Miller. 2000. The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection. Mol. Microbiol. 37:1133-1145.
53. Schulert, G. S., H. Feltman, S. D. P. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser. 2003. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J. Infect. Dis. 188:1695-1706.
54. Shaver, C. M., and A. R. Hauser. 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect. Immun. 72:6969-6977.
55. Skrzypek, E., T. Myers-Morales, S. W. Whiteheart, and S. C. Straley. 2003. Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eukaryotic cells. Infect. Immun. 71:937-947.
56. Strickland, J. A., G. L. Orr, and T. A. Walsh. 1995. Inhibition of Diabrotica larval growth by patatin, the lipid acyl hydrolase from potato tubers. Plant Physiol. 109:667-674.
57. Takeichi, M. 1990. Cadherins: a molecular family important in selective cell-cell adhesion. Annu. Rev. Biochem. 59:237-252.
58. Terebiznik, M. R., O. V. Vieira, S. L. Marcus, A. Slade, C. M. Yip, W. S. Trimble, T. Meyer, B. B. Finlay, and S. Grinstein. 2002. Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella. Nat. Cell Biol. 4:766-773.
59. Vancanneyt, G., U. Sonnewald, R. Hofgen, and L. Willmitzer. 1989. Expression of a patatin-like protein in the anthers of potato and sweet pepper flowers. Plant Cell 1:533-540.
60. Zhang, Y., and J. T. Barbieri. 2005. A leucine-rich motif targets Pseudomonas aeruginosa ExoS within mammalian cells. Infect. Immun. 73:7938-7945.
61. Zhou, D., M. S. Mooseker, and J. E. Galan. 1999. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283:2092-2095.(Shira D. P. Rabin, Jeffre)