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Infection and Immunity, July 2003, p. 4144-4150, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.4144-4150.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Pseudomonas aeruginosa ExoU, a Toxin Transported by the Type III Secretion System, Kills Saccharomyces cerevisiae

Shira D. P. Rabin1 and Alan R. Hauser1,2*

Departments of Microbiology/Immunology,1 Medicine, Northwestern University, Chicago, Illinois 606112

Received 24 February 2003/ Accepted 16 April 2003


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ABSTRACT
 
ExoU, a protein transported by the type III secretion system of Pseudomonas aeruginosa, is an important cytotoxin, though its mechanism of action is unclear. Here we show that the intracellular expression of ExoU is cytotoxic to Saccharomyces cerevisiae. Furthermore, internal amino- and carboxyl-terminal deletions confirmed that regions of ExoU previously shown to be essential for killing mammalian cells were also required for killing yeast cells. These findings indicate that S. cerevisiae is a useful model organism for the study of ExoU.


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TEXT
 
Pseudomonas aeruginosa is an important cause of hospital-acquired infections, frequently complicating the clinical course for individuals who require mechanical ventilation, undergo intravascular or urinary catheterization, or have surgical wounds. Many isolates of P. aeruginosa are resistant to commonly used antibiotics (2) and, consequently, infections are often fatal. Although much is known about this gram-negative bacterium, a better understanding of P. aeruginosa pathogenesis is necessary to develop alternative strategies for treatment.

P. aeruginosa uses a type III secretion system to deliver toxins directly into eukaryotic cells (reviewed in reference 12). Although the genes encoding the P. aeruginosa type III secretion apparatus are similar to corresponding genes in bacteria such as Yersinia, Salmonella, and Shigella spp., the effector proteins transported by this apparatus are distinct. Thus far, four P. aeruginosa effector proteins have been identified: ExoS, ExoT, ExoU (also called PepA), and ExoY. Characterization of ExoU in particular has been the focus of much investigation.

ExoU was initially identified by its cytotoxic activity towards mammalian cells. Apodaca et al. noted that some P. aeruginosa strains were capable of rapidly killing epithelial cells following cocultivation in vitro (3), suggesting that these strains expressed a cytotoxic factor. The potential importance of this observation was highlighted by the finding that cytotoxic isolates were more virulent in animal models of pneumonia (18, 22). Fleiszig et al. and Finck-Barbançon et al. noted that cytotoxic clinical isolates of P. aeruginosa expressed a 74-kDa protein whereas noncytotoxic isolates did not (9, 11). They subsequently characterized this protein, which they named ExoU, and showed that it was secreted by the type III system and was essential for cytotoxicity. Independently, Engel and colleagues identified the same protein, which they named PepA, by screening a transposon insertion library of a cytotoxic P. aeruginosa strain for mutants defective in the ability to kill mammalian cells (15, 17). To date, ExoU secretion has been associated with the death of epithelial cells, macrophages, fibroblasts, Chinese hamster ovary (CHO) cells, and even amoebae (5, 6, 9, 10, 14, 15, 20, 21).

The in vitro cytotoxic activity of ExoU is of clinical significance. Although only one-third of clinical isolates from acute infections harbor the exoU gene (7), disruption of this gene results in decreased virulence in animal models of acute pneumonia (9, 15). In addition, transformation with an exoU-containing plasmid converted some strains that did not naturally harbor the exoU gene to cytotoxic and virulent phenotypes (1). Furthermore, patients with hospital-acquired pneumonia caused by ExoU-secreting isolates of P. aeruginosa had worse clinical outcomes than patients infected with isolates harboring inactive type III secretion systems, suggesting that this protein may contribute to disease severity in humans (13).

ExoU does not have significant similarity to any characterized proteins, and little is known about its mechanism of action. In an effort to better understand the manner by which ExoU kills eukaryotic cells, Finck-Barbançon et al. performed a structure-function analysis of this protein using a CHO cell transfection system (8). These investigators found that deletions in the amino terminus or the carboxyl terminus eliminated the cytotoxic activity of ExoU. In addition, a central portion of the protein was also shown to be important. Based upon these findings, they proposed a tethering model whereby the amino and carboxyl termini of ExoU each binds distinct eukaryotic factors, while the central region brings these factors into close proximity to result in cell death.

For the present study, we wished to determine whether Saccharomyces cerevisiae was a useful model organism for the investigation of ExoU cytotoxicity. To accomplish this, we examined whether ExoU was cytotoxic when expressed in yeast cells and whether regions of this protein necessary for the killing of mammalian cells were also essential for lethality in yeast. The ability to use S. cerevisiae as a model organism would greatly facilitate future efforts to determine ExoU's mechanism of action and to analyze the structure-function relationships of this toxin.

The exoU gene was placed under the control of the GAL1 galactose-inducible promoter of the low-copy-number yeast/bacterial vector pYC2/NT A (Invitrogen, Carlsbad, Calif.) by ligation of a 2.3-kb SspI-AgeI exoU-containing fragment of plasmid pAH808 (G. S. Schulert, H. Feltman, S. D. P. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser, submitted for publication) into EcoRI-digested and blunted pYC2/NT A. (pYC2/NT A will henceforth be referred to as pVector.) The resulting construct, designated pExoU, encoded ExoU with amino-terminal His and Xpress epitope tags. It was transformed into the bacterial strain XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lacIqZ {Delta}M15 Tn10 {Tetr}]; Stratagene, La Jolla, Calif.), which was maintained in Luria-Bertani medium supplemented with ampicillin (final concentration, 50 µg/ml). The S. cerevisiae strain INVSc1 (his3{Delta}1/his3{Delta}1 leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52; Invitrogen) was used to test for ExoU-mediated cytotoxicity. Yeast cells were grown in yeast peptone dextrose medium (1% yeast extract, 2% peptone, 2% glucose) or synthetic complete (SC) medium lacking uracil (0.17% yeast nitrogen base, 0.5% NH4SO4, 40 mg of Leu per liter, 10 mg of Ade per liter, 20 mg of Trp per liter, 50 mg of Phe per liter, 100 mg of Glu per liter, 100 mg of Asp per liter, 200 mg of Thr per liter, 20 mg of Arg per liter, 20 mg of Met per liter, 30 mg of Ile per liter, 30 mg of Lys per liter, 150 mg of Val per liter, 30 mg of Tyr per liter, 20 mg of His per liter, 20 mg of Pro per liter, 40 mg of Cys per liter, 400 mg of Ser per liter [pH = 7.0]) to maintain the plasmid. Unless otherwise specified, SC medium supplemented with 2% glucose (SC-Glc) was used to repress the GAL1 promoter and decrease ExoU expression, whereas SC medium supplemented with 2% galactose (SC-Gal) was used to induce the GAL1 promoter and increase ExoU expression.

ExoU is lethal to S. cerevisiae. To determine whether ExoU was cytotoxic to yeast cells, S. cerevisiae strain INVSc1 was transformed with pExoU or pVector using the Frozen-EZ Yeast Transformation II protocol (ZymoResearch, Orange, Calif.) and grown on SC-Glc at 30°C for 3 days. Individual colonies containing pExoU were then streaked onto both repressing SC-Glc agar and inducing SC-Gal agar (Fig. 1). INVSc1 transformed with pVector was used as a control. Thousands of INVSc1(pVector) colonies grew on plates supplemented with either glucose or galactose (Fig. 1). Likewise, abundant growth of INVSc1(pExoU) was observed on SC-Glc agar, which repressed expression of the exoU gene. However, growth of INVSc1(pExoU) was not observed on galactose-containing agar, which induced expression of the exoU gene. These results indicate that ExoU is extremely toxic to S. cerevisiae. However, we could not distinguish between rapid cell death and growth inhibition from these data.



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  		FIG. 1. ExoU is toxic to yeast. Yeast strain INVSc1 transformed with pVector or pExoU was streaked onto inducing (SC-Gal) or repressing (SC-Glc) agar and incubated at 30°C for 3 days.

To differentiate between the two possibilities, S. cerevisiae grown under inducing and repressing conditions was microscopically examined after incubation with the live-dead yeast stain FUN-1 and the yeast cell wall stain Calcofluor (Molecular Probes, Eugene, Oreg.). In metabolically active cells, FUN-1 is cleaved and concentrated into vacuolar structures to yield a red punctate pattern under fluorescence microscopy. In contrast, FUN-1 remains uncleaved and evenly distributed throughout dead cells to yield diffuse green staining. INVSc1(pVector) was diluted to 1 x 107 CFU/ml in either inducing or repressing medium, whereas INVSc1(pExoU) was diluted to 1 x 107 CFU/ml in repressing medium and 3 x 107 CFU/ml in inducing medium. The cultures were allowed to grow for 4 h and then resuspended in 10 mM HEPES (pH 7.2) supplemented with either 2% glucose or 2% galactose. Each culture was protected from light during a 30-min incubation at 30°C with 10 µM FUN-1 and 25 µM Calcofluor. Finally, 4 x 105 cells from each sample were allowed to attach to coverslips coated with 0.1% poly-L-lysine in 5% NaHCO3 and were examined by use of fluorescence microscopy. Metabolically active cells were seen with INVSc1(pVector) grown under both repressing and inducing conditions and with INVSc1(pExoU) grown under repressing conditions (Fig. 2). In contrast, most INVSc1(pExoU) cells grown under inducing conditions exhibited the diffuse green fluorescent staining associated with dead cells. Additionally, some yeast cells had no intracellular fluorescence (Fig. 2), suggesting that they represented dead yeast cells that had extruded their cellular contents in the final stages of death. From these data, then, we conclude that the expression of ExoU is lethal to S. cerevisiae.



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  		FIG. 2. Microscopic examination of yeast expressing ExoU. INVSc1(pVector) and INVSc1(pExoU) were grown under repressing conditions for 18 h, transferred to either repressing SC-Glc or inducing SC-Gal medium, and stained with Calcofluor and FUN-1. In metabolically active yeast cells, FUN-1 is cleaved and fluoresces red within vacuolar structures, while dead but intact yeast cells have a diffuse green fluorescence. Calcofluor causes yeast cell walls to fluoresce blue. The blue shells seen when INVSc1(pExoU) was grown under inducing conditions likely represent dead yeast cells that have extruded their cellular contents.

Minimal amounts of ExoU kill S. cerevisiae. To determine whether minimal expression of ExoU was sufficient to kill S. cerevisiae, we titrated ExoU expression by varying the amount of repressing sugar (glucose) or inducing sugar (galactose) in agar plates. For these experiments, INVSc1(pVector) and INVSc1(pExoU) were grown for 18 h in repressing SC-Glc medium. The cultures were diluted to an optical density at 600 nm (OD600) of 1.0, and serial dilutions were inoculated onto agar plates with differing amounts of glucose or galactose. Total sugar was kept constant at 2% by supplementing with raffinose, which does not affect GAL1 promoter activity. Colonies were counted following incubation at 30°C for 4 days. While the number of INVSc1(pVector) colonies remained constant at approximately 8 x 106 CFU/ml regardless of the amount and type of sugar, the number of INVSc1(pExoU) colonies steadily decreased as repression of the exoU gene decreased and induction increased (Fig. 3). The toxicity of ExoU was so great that even under conditions that partially repressed the GAL1 promoter, fewer INVSc1(pExoU) colonies were noted than INVSc1(pVector) colonies. These data indicate that extremely small amounts of ExoU are sufficient to kill S. cerevisiae. Additionally, these results suggest that ExoU-mediated toxicity is due to a specific action of ExoU and not to nonspecific protein accumulation.



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  		FIG. 3. Minimal amounts of ExoU are sufficient for lethality. ExoU expression was controlled by varying the concentration of repressing sugar (glucose) or inducing sugar (galactose) in agar plates. Total sugar was kept constant at 2% by supplementing with raffinose, which does not affect GAL1 promoter activity. INVSc1(pVector) and INVSc1(pExoU) were grown under repressing conditions for 18 h, diluted to an OD600 of 1.0, and inoculated onto agar plates. Colonies were counted after incubation at 30°C for 4 days. Note that the small number of viable colonies observed under inducing conditions is likely due to spontaneous suppressor mutations. Data were pooled from two separate experiments, each of which was done in triplicate.

Mutational analysis of ExoU. Seven amino-terminal, internal, and carboxyl-terminal deletions were made to determine which regions of ExoU were important for toxicity in S. cerevisiae (Fig. 4). pExoU was digested with Acc65I and SacII (N1), AvrII and SmaI with an inserted SmaI linker (N2), or SacII and SmaI (I1). In each case, the ends of the remaining construct were religated to obtain in-frame deletions within the exoU gene. Two carboxyl-terminal deletions were constructed by PCR amplification of pExoU using a forward primer that effectively inserted a SalI site (C1) or an MscI site (C2) immediately 5' of the exoU stop codon and a reverse primer that hybridized to a native BsrGI restriction site located approximately 1.5 kb downstream of the exoU gene (C1 forward, 5'-AAAAAAAAAGTCGACTGATTGATACATGGC-3'; C2 forward, 5'-AAAAAAAAATGGCCACTGATTGATACATGGC-3'; reverse, 5'-AAAAAATGTACAGAAAAAAAAGAAAA-3'). The resulting 550-bp DNA fragments and pExoU were digested with SalI and BsrGI for C1 or MscI and BsrGI for C2 and then ligated together. Note that the exoU gene has native internal SalI and MscI sites. The exoU allele encoding C1 contained a stop codon 93 bp upstream of the original stop codon, and the allele encoding C2 contained a histidine immediately preceding a stop codon 192 bp upstream of the original stop codon. Each encoded a truncated form of ExoU that lacks the carboxyl-terminal 31 or 64 amino acids, respectively. An additional carboxyl-terminal mutation (C3) was constructed by amplifying the first 900 nucleotides of the exoU gene from pExoU using a forward primer with an inserted KpnI site and a reverse primer with an inserted SacI site, stop codon, and BamHI site (forward, 5'-AAAAAAAAAGGTACCCATGCATATCCAATCG-3'; reverse, 5'-AAAAAAAAAAGGATCCCTCAGAGCTCGGCATTGAACACCAC-3').pVector and the approximately 900-bp DNA fragment were each digested with KpnI and BamHI and then ligated. Thus, the protein with the C3 mutation contained 300 amino acids and resulted in a 387-amino-acid carboxyl-terminal deletion. Finally, we constructed an internal deletion mutation (I2) by amplifying the nucleic acid sequence encoding amino acids 343 to 687 from pExoU with a forward primer containing an inserted SacI site and a reverse primer with an inserted NotI site and a stop codon (forward, 5'-AAAAAAAAAGAGCTCGATGGCGGGGTGATG-3'; reverse, 5'-AAAAAAAAAGCGGCCGCAGTCATGTGAACTCCTT-3'). The resulting DNA fragment and the plasmid encoding C3 were each digested with SacI and NotI and ligated together to yield a construct that encoded I2, which lacks amino acids 301 to 342. Nucleic acid sequencing was performed on all amplified sequences and ligation junctions to ensure that the expected protein was encoded by each construct.



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  		FIG. 4. Mutational analysis of ExoU. Wild-type and mutated forms of ExoU containing amino-terminal His and Xpress epitope tags were expressed in yeast cells and their cytotoxic activities were determined by streaking the yeast cells onto inducing (SC-Gal) agar. Deleted segments of the protein are indicated, as are the restriction endonucleases that were used to generate the deletions. To the right, the viability of yeast transformed with the mutated exoU alleles and grown on inducing agar is indicated. The C1 mutant had a partially cytotoxic phenotype.

Each mutated exoU allele was transformed into INVSc1 and selected on SC-Glc agar. An individual colony was streaked onto both repressing SC-Glc agar and inducing SC-Gal agar and incubated for 4 days at 30°C. A noncytotoxic phenotype was identified by the presence of growth on SC-Gal plates. The growth phenotypes are summarized in Fig. 4. ExoU lacking amino acids 1 to 82 (N1) was still cytotoxic to yeast, indicating that this portion of the protein is unnecessary for killing. However, ExoU with a deletion of amino acids 1 to 119 (N2) was no longer lethal, suggesting that amino acids 83 to 119 encompass a domain essential for cytotoxicity. These results were confirmed with I1, which contains an internal deletion of amino acids 83 to 119 and was noncytotoxic.

Deletion of the carboxyl-terminal 64 amino acids (amino acids 624 to 687) in C2 or the carboxyl-terminal 387 residues (amino acids 301 to 687) in C3 also resulted in growth of yeast cells on both SC-Glc and SC-Gal agar. This indicates that the carboxyl 64 amino acids of ExoU contain a domain necessary for lethality. Interestingly, removal of amino acids 657 to 687 in C1, which produced a truncated form of ExoU missing the carboxyl-terminal 31 amino acids, resulted in a partial cytotoxic phenotype (data not shown). Yeast cells expressing C1 were viable but showed decreased colony numbers under inducing conditions relative to those shown under repressing conditions. When yeast cells were diluted to an OD600 of 1.0 and inoculated onto SC-Glc and SC-Gal plates, 9.5 x 106 and 8.0 x 106 CFU of INVSc1(pVector) per ml grew on repressing and inducing agar, respectively, whereas 7.5 x 106 CFU of INVSc1 transformed with the C1-encoding plasmid per ml grew on repressing medium. In contrast, only 3.1 x 106 CFU of INVSc1 transformed with C1-encoding plasmid per ml were observed on SC-Gal, which induces expression of the exoU gene. The difference in growth of INVSc1 harboring the C1-encoding plasmid on inducing medium compared to that on repressing medium was significant [P < 0.02, normalized to the difference in growth of INVSc1(pVector); two-tailed t test]. Removal of the last 31 amino acids of ExoU may have resulted in partial loss of cytotoxic activity because this area is important for modulation of cytotoxicity or is immediately adjacent to a region necessary for cytotoxicity, though other explanations are possible.

A form of ExoU containing an internal deletion was also expressed in yeast to determine whether this portion of the toxin was essential for cytotoxicity. When amino acids 301 to 342 were deleted in I2, expression of ExoU did not kill yeast. This suggests that in addition to portions of the amino and carboxyl termini, at least one internal domain of ExoU is also necessary for killing.

Immunoblot analysis of yeast cell lysates using polyclonal ExoU antisera indicated that all six noncytotoxic deletion forms of ExoU were stable in yeast (Fig. 5). In these assays, yeast cells were grown in repressing medium for 18 h, diluted to 107 cells/ml in inducing medium, and grown for an additional 4 h. Cells were lysed with 20% trichloroacetic acid and by agitation with glass beads. Precipitated material was collected, electrophoresed through sodium dodecyl sulfate-8% polyacrylamide gels, electrotransferred to nitrocellulose membranes, and exposed to polyclonal ExoU antiserum (15). Bands corresponding to each of the noncytotoxic proteins were readily apparent. Thus, it appears that the noncytotoxic phenotype associated with expression of these proteins did not result from their failure to be stably expressed, although we cannot rule out the possibility that small changes in stability partially contributed to their phenotypes.



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  		FIG. 5. Immunoblot analysis of yeast transformed with mutated exoU alleles. Yeast cells were grown under inducing conditions, diluted to 107 CFU/ml, and allowed to grow for an additional 4 h. Yeast cells were lysed with glass beads and 20% trichloroacetic acid. Proteins from cell lysates were collected, electrophoresed through a sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane, and exposed to polyclonal ExoU antiserum. Only mutated forms of ExoU that lacked cytotoxic activity are shown. Expected sizes are as follows: purified ExoU (from P. aeruginosa), 74 kDa; N2, 65 kDa; I1, 76 kDa; I2, 73 kDa; C3, 35 kDa; C2, 72 kDa; C1, 76 kDa. The faint band present in all lanes is a yeast antigen recognized nonspecifically by the ExoU antiserum.

Immunoblot analysis did not detect wild-type ExoU from induced INVSc1(pExoU) cell pellets (Fig. 5). One explanation for this finding is that the minimal amount of ExoU required to kill yeast may be undetectable by immunoblot analysis. This interpretation is consistent with the observation that the minimal amount of ExoU produced during growth in repressing medium is lethal to yeast (Fig. 3).

Together, these findings indicate that deletion of amino acids 83 to 119, 301 to 342, or 624 to 687 of ExoU results in loss of cytotoxicity in yeast. Thus, at least portions of each of these three regions are essential for ExoU to kill S. cerevisiae. Finck-Barbançon et al., using mutated forms of ExoU in CHO cells (8), found that deletion of all or part of any of the following three defined domains abolished ExoU-mediated cytotoxicity: amino acids 52 to 202, 300 to 352, and 580 to 687. That similar amino-terminal, internal, and carboxyl-terminal domains were essential for killing of both CHO cells and S. cerevisiae suggests that ExoU is acting by the same or a similar mechanism in yeast and mammalian cells. Therefore, we conclude that S. cerevisiae is a good model system for studying ExoU.

Proposed mechanisms for ExoU cytotoxicity must include an essential role for the amino-terminal, internal, and carboxyl-terminal portions of the toxin. As suggested by Finck-Barbançon et al., the termini of ExoU may each bind distinct eukaryotic factors while the central portion functions as a tether to bring these factors into close proximity (8). Our finding that deletion of a small portion of the internal region of ExoU results in loss of activity while still tethering the amino and carboxyl termini together argues against this model, though this may be reconciled with the tethering model if the distance between the termini is of crucial importance. Alternatively, ExoU may have an as yet unidentified enzymatic activity. In this regard, it is interesting that amino acids 107 to 357, which encompass both the amino-terminal and internal domains identified in our study, constitute a patatin domain (4, 19). Patatin domains, which have been associated with phospholipase activity in other proteins (16), have several conserved features. For example, a putative oxyanion hole in patatin (G-G-X-K/R) has been postulated to be essential for enzymatic activity (16). A similar motif (G-G-A-K, residues 112 to 115) is present within the ExoU amino-terminal domain (amino acids 83 to 119) identified in this study. In addition, alignment with the patatin domain suggests that Asp-344 of ExoU corresponds to an active site aspartate in patatin (16). Asp-344 is only two residues from the internal domain (residues 301 to 342) identified in this study. Thus, ExoU may have phospholipase activity, and deletion of the amino-terminal or internal domains of ExoU may destroy this activity, resulting in loss of cytotoxicity. However, other explanations are possible. For example, we cannot exclude the possibility that some of the identified domains mediate proper folding of ExoU.

We conclude that S. cerevisiae is a useful model organism for the study of ExoU-mediated cytotoxicity. Not only is this toxin lethal when expressed in extremely small amounts in yeast, but similar domains of ExoU are required to kill yeast and mammalian cells. Studies are under way to use this yeast model to further define domains of ExoU that are essential for activity and to elucidate the mechanism by which this toxin kills eukaryotic cells. Ideally, such information will aid in the development of novel therapies for P. aeruginosa infections.


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ACKNOWLEDGMENTS
 
We thank Ciara Martin, Grant Schulert, and Andrew Rabin for critical reading of the manuscript and Ashok Aiyar for helpful suggestions regarding experimental techniques. Additionally, we thank the laboratory of Kasturi Haldar for use of and assistance with their fluorescence microscope.

This work was supported by grants from the Schweppe Foundation (A.R.H.) and NIH (A.R.H.; grant R21 AI46543).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology/Immunology, Northwestern University, 303 East Chicago Ave., Searle 6-495, Chicago, IL 60611. Phone: (312) 503-1044. Fax: (312) 503-1339. E-mail: ahauser{at}northwestern.edu. Back

Editor: V. J. DiRita


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Infection and Immunity, July 2003, p. 4144-4150, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.4144-4150.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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