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Division of Immunology, Infection and Inflammation, University of Glasgow, Western Infirmary, Glasgow G11 6NT, UK
Correspondence
Tom J Evans
t.j.evans{at}udcf.gla.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Cornelis & Van Gijsegem, 2000). The bacteria elaborate a multi-subunit apparatus, the injectisome, which acts as a conduit for secreted toxins. The toxins secreted by these type III secretion systems (TTSSs) usually act to subvert or modify normal eukaryotic cellular processes, to favour survival of the invading pathogen (Cornelis, 2002; Mota & Cornelis, 2005).
In many cases, a particular biochemical activity has been identified for individual type III secreted toxins. However, how this activity then translates into the observed phenotypic effects of the toxin on cells is often much less clear. Part of the problem in analysing toxin action in mammalian cells is the difficulty in identifying the crucial target of toxin action. Alternative models of infection have thus been developed to study toxin action in a tractable genetic system. One such model uses the yeast Saccharomyces cerevisiae (Lesser & Miller, 2001). Although a simple eukaryote with 6591 ORFs, there is significant conservation of fundamental cellular processes between yeast and human cells. Indeed, up to 30 % of positionally cloned genes implicated in human disease have yeast homologues (Foury, 1997). S. cerevisiae is easily transformed with DNA, undergoes homologous replication with high efficiency, and its complete gene sequence is known and well annotated. Importantly, its cell cycle is extremely well understood, and cells can be readily synchronized at specific stages.
The human pathogen Pseudomonas aeruginosa is an important cause of nosocomial infection in those with impaired immune function, in patients with cystic fibrosis, and in those with burns (Chastre & Fagon, 2002; Lyczak et al., 2002; Richards et al., 1999). It secretes four toxins by a TTSS: ExoS, ExoT, ExoU and ExoY (Barbieri & Sun, 2004; Finck-Barbancon et al., 1997; Frank, 1997; Hauser et al., 1998; Yahr et al., 1996, 1998). ExoS can induce a cytotoxic effect (Olson et al., 1999), and is associated with the ability to produce lung damage and poor outcome from P. aeruginosa infection (Roy-Burman et al., 2001). ExoS is a bifunctional toxin, with N-terminal GTPase-activating protein (GAP) activity, and a C-terminal ADP ribosyl transferase (ADPRT) domain (Barbieri, 2000). The latter domain has been more clearly linked with the cytotoxic effects of ExoS, and has the ability to inhibit DNA synthesis in cultured cells (Fraylick et al., 2001). The GAP domain of ExoS had no effect on DNA synthesis. Many targets of the ExoS ADPRT domain action have been identified, including members of the small GTPase family proteins, such as Ras, Rab, Ral and Rap, as well as vimentin, and the GAP domain within its own N-terminal region (Aktories et al., 2000; Barbieri & Sun, 2004). These studies have demonstrated the effects of ExoS on the activation and interaction of small GTPases with downstream effectors. Targeting of Ras pathways, in particular, would provide a link between the actions of ExoS and inhibiting cell proliferation (Henriksson et al., 2000, 2002; McGuffie et al., 1998). However, it is not clear which of the small GTPase pathways is crucial in the cytotoxic effects of ExoS. Moreover, ADP ribosylation of Ras can be prevented with no effect on the cytotoxicity of ExoS (Pederson et al., 2002). In addition, in epithelial cells, ADP-ribosylation by ExoS can lead to activation of the small GTPase Rac (Rocha et al., 2005), which could act to increase cell proliferation through p21-activated kinase activity.
Given that the mechanisms of ExoS action in mammalian cells remain unclear, we sought to develop an alternative simpler system to analyse ExoS action using the yeast S. cerevisiae. We describe here a model system for the expression of ExoS in S. cerevisiae under tetracycline regulation. Using this system, we demonstrate that ExoS inhibits yeast cell growth, an effect largely dependent on the activity of the ADPRT domain. ExoS expression produces an unusual effect on actin within yeast cells, resulting in large cortical patches and thickened cables, again dependent on ExoS ADPRT activity. ExoS increased the numbers of mating projections formed following treatment of haploid yeast cells with mating pheromone. Following release of yeast cells from growth arrest with mating factor, ExoS prevented DNA replication, and resulted in unusual elongated projections with expanded tips. These studies demonstrate a unique phenotype of S. cerevisiae following ExoS expression, and suggest previously unsuspected targets for ExoS action within the cell.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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S. cerevisiae strains.
The strains of S. cerevisiae used in this study were the diploid strain INVSc1 (Invitrogen), and the haploid strain BMA64-1A (Euroscarf). The genotypes of these strains are: INVSc1, his3
1/his31 leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52; and BMA64-1A, MATa ura3-52 trp12 leu2-3_112 his3-11 ade2-1 can1-100.
Maintenance and growth of S. cerevisiae.
Yeast strains were grown in yeast peptone glucose (YPD) broth containing 1 % yeast extract (Oxoid), 2 % peptone (Oxoid), and 2 % glucose. Transformed yeast cells were grown in synthetic dropout (SD) medium to maintain the selective pressure on the plasmid. For plates, 4 % agar (Oxoid) was added to the YPD and SD media. All yeast incubations were carried out at 30 °C, with shaking at 250 r.p.m. for liquid cultures. Working stock plates of yeast were kept for up to 2 months at 4 °C, and, for long-term storage, yeast strains were stored in 25 % (v/v) glycerol at –80 °C.
Construction of plasmids.
This study utilized three yeast expression vectors to examine the effect of ExoS, and the GAP and ADPRT domains of ExoS, on S. cerevisiae. The three vectors were pYES2/NT (Invitrogen), pYC2/NT (Invitrogen) and pCM252 (Euroscarf). pYES2/NT and pYC2/NT contain the URA3 gene, and pCM252 contains the TRP1 gene; therefore, S. cerevisiae cells transformed with these plasmids were selected for on SD medium lacking uracil and tryptophan, respectively. Wild-type ExoS, ExoS with the GAP domain mutated, ExoS with the ADPRT domain mutated, and ExoS with GAP and ADPRT domains mutated, were expressed from all three vectors. Table 1 summarizes how each construct was made and Table 2 lists the primers utilized.
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), and 5 % DMSO, in a total volume of 100 µl. The mixture was subjected to one denaturing cycle of 5 min at 94 °C, and then 30 cycles using the following conditions: a 45 s denaturing step at 94 °C, followed by a 45 s annealing step at 52 °C, and a 1 min extension step at 72 °C, and completed by a final extension cycle of 10 min at 72 °C. After cloning into the KpnI and XhoI sites of pYES2/NTA, the sequence of exoS was confirmed by sequencing with the T7prom, CYC1R and ExoS-Int primers (Table 2).
Site-directed mutagenesis.
The QuikChange site-directed mutagenesis kit (Stratagene) was used to incorporate the R146A and E379A-E381A mutations into ExoS in pYES2/NT-ExoS, according to the manufacturer's instructions. For each site-directed mutagenesis reaction, two complementary primers were designed (Table 2) with nucleotide mutations that would result in the desired amino acid substitutions, and would also introduce a new restriction site into pYES2/NT-ExoS. Mutations were subsequently confirmed by sequencing the constructs with the T7 prom, CYC1R and ExoS-Int primers (Table 2).
Yeast transformation.
S. cerevisiae cells were transformed with plasmids using the Frozen-EZ Yeast Transformation II kit (Zymo Research), according to the manufacturer's instructions. Briefly, competent cells were prepared by centrifuging 10 ml of mid-exponential phase yeast cells at 500 g for 4 min, then washing the pellet in 10 ml EZ1 solution, and resuspending the cells in 1 ml EZ2 solution. For transformation, 0.2–1 µg of plasmid DNA was mixed with 50 µl of competent cells, and 500 µl EZ3 solution. The transformation reaction was incubated at 30 °C for 45 min, and subjected to vigorous mixing three times during incubation. A 100 µl volume of the transformation mixture was plated onto appropriate SD agar, and allowed to grow at 30 °C for 2–4 days.
Integration of pCM242 into the LEU2 locus of the S. cerevisiae genome.
The pCM242 plasmid was integrated into the genome of INVSc1 and BMA64-1A by homologous recombination between the functional LEU2 gene in pCM242, and the mutated LEU2 locus in the yeast chromosomes. The pCM242 plasmid was digested with EcoRV, and purified using the QIAquick PCR purification kit (Qiagen), according to the manufacturer's instructions. The EcoRV-linearized pCM242 was transformed into INVSc1 or BMA64-1A, and the yeast cells that had successfully integrated the plasmid were selected on SD agar lacking Leu, with glucose added (SD–Leu+glucose).
Growth assays.
For the plate growth assays, overnight cultures of yeast grown in SD medium lacking Leu and Trp, with glucose added (SD–Leu–Trp+glucose), were diluted in PBS (10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.47 mM KH2PO4) to an OD600 of 1. These cultures were serially 10-fold diluted four times in PBS, and 5 µl of each dilution was spotted onto SD–Leu–Trp+glucose agar supplemented with doxycycline as indicated. The plates were incubated at 30 °C for 4 days before being photographed using a Kodak DX4530 digital camera.
For the liquid growth assay, cultures of INVSc1(pCM242), transformed with either pCM252 or pCM252-ExoS, were grown overnight in SD–Leu–Trp+glucose. These cultures were diluted in fresh media to an OD600 of 0.3. After 4 h growth, doxycycline was added at a final concentration of 2 µg ml–1. OD600 readings were taken every hour to assess the growth of the cultures.
Analysis of ExoS protein expression.
ExoS and individual domain mutants with the Xpress expression tag were constructed in pCM252, as outlined in Table 1. Plasmids were transformed into INVSc1(pCM242), and protein expression was induced by the addition of 2 µg doxycycline ml–1, as described above. Four hours after doxycycline addition, 1.5 ml of yeast culture was centrifuged at 3000 g for 5 min at 4 °C, and the pellet was resuspended in Y-PER (Pierce) at 5 µl (mg pellet)–1, vortexed, and agitated by rotation at room temperature for 20 min. An equal volume of SDS loading buffer was added, and the samples were denatured by heating at 100 °C for 3 min. After centrifugation, equal volumes were separated on SDS-polyacrylamide gels, and immunoblotted with anti-Xpress antibody (Invitrogen) and ECL detection regents (Amersham).
Fluorescence analysis of yeast actin.
Cultures of INVSc1(pCM242) containing a pCM252-based plasmid were grown and induced as described for the liquid growth assay. Four hours after the addition of doxycycline, the S. cerevisiae cells were fixed by adding 5 % formaldehyde to 5 ml of culture, and incubating for 30 min at room temperature, with occasional inversion. The cells were centrifuged at 1500 g for 5 min, and the pellets were washed three times in PBS. The pellets were resuspended in 200–800 µl PBS (to roughly normalize cell density), and 150 µl aliquots were washed in 1 ml solution B (100 mM K2HPO4, 100mM KH2PO4 and 1.2 M sorbitol). Each pellet was incubated in 1 ml solution B, containing 0.2 % 2-mercaptoethanol and 2 µg lyticase ml–1, at 37 °C for 30 min, to permeabilize the yeast. After permeabilization, the cells were centrifuged at 500 g, and washed once in 1 ml solution B. To stain for actin, the fixed and permeabilized S. cerevisiae were incubated in 50 µl solution B, containing 0.4 U Alexa Fluor 488 Phalloidin (Invitrogen), at 37 °C overnight, in the dark. The cells were washed three times in 1x PBS before being spread onto a Poly Prep slide (Sigma-Aldrich), and allowed to dry for 10 min. A drop of Vectashield (Vector Laboratories) was used to mount the yeast under a coverslip. The slides were viewed using a Nikon Eclipse E600 microscope with a Nikon pan Fluor x100 lens, and captured using an Optronics digital camera and MagnaFire software (Meyor Instruments).
Effects of latrunculin A on yeast actin cytoskeleton.
INVSc1(pCM242) with pCM252 alone, or expressing ExoS, was induced to express ExoS with 2.0 µg doxycycline ml–1 for 4 h, as described above. Samples were removed for actin staining (time 0), and latrunculin A (Biomol) was added at 200 µM. Samples of 1 ml were removed at various times after addition of latrunculin A, fixed by addition of formaldehyde to 5 % final concentration, washed three times in PBS, and then stained for actin with 0.4 U Alexa Fluor 488 phalloidin, in a final volume of 100 µl PBS, at 37 °C overnight in the dark, with shaking. Cells were washed three times with PBS, spread on poly L-lysine coverslips, and analysed by a Zeiss Confocal microscope and associated software. Since, in this protocol, not every cell stained for actin, quantification of the effects of latrunculin A on yeast actin was normalized to the numbers of cells showing actin staining of their cytoskeleton prior to the addition of latrunculin A (time 0).
Synchronization of S. cerevisiae.
The haploid MATa strain BMA64-1A, containing the integrated pCM242 plasmid and a pCM252-based plasmid, was grown to mid-exponential phase in SD–Leu–Trp+glucose. The cultures were diluted to an OD600 of 0.2, and arrested in G1 with 20 µg 
-factor ml–1 (Zymo Research). Two hours after addition of the -factor, ExoS or mutant ExoS was induced by adding 2 µg doxycycline ml–1. The yeast were released from -factor-arrest an hour after the doxycycline was added, by centrifuging the cells at 1500 g for 5 min, then washing twice in pre-warmed SD–Leu–Trp+glucose containing 2 µg doxycycline ml–1. The cells were resuspended in pre-warmed SD–Leu–Trp+glucose containing 2 µg doxycycline ml–1, and cells were fixed for immunofluorescence or flow cytometry analysis every 30 min.
Flow cytometry analysis.
Synchronized BMA64-1A(pCM242), containing a pCM252-based plasmid, was prepared for flow cytometry analysis. The cells from 1 ml of culture were harvested by centrifugation at 500 g for 5 min, and the pellet was resuspended in 1.5 ml double-distilled water (DDW). The yeast cells were fixed by adding 3.5 ml 95 % ethanol, and incubating overnight at 4 °C. The cells were centrifuged at 500 g for 5 min, and washed in 1 ml DDW. The RNA was degraded by incubating the yeast with 0.5 ml 2 mg RNase A ml–1 in 50 mM Tris/HCl, pH 8.0, for 1–2 h at 37 °C. The cells were centrifuged at 500 g for 5 min, and the pellet was resuspended in 200 µl 5 mg pepsin ml–1 and 0.45 % conc. HCl, and incubated for 30–60 min at 37 °C to degrade the proteins. The yeast cells were harvested by centrifugation at 500 g for 5 min, and the pellet was resuspended in 0.5 ml 1x propidium iodide (PI) solution (180 mM NaCl, 70 mM MgCl2, 75 µM PI, 100 mM Tris/HCl, pH 7.5). The yeast cells were incubated in the 1x PI solution overnight at 4 °C to stain the DNA. A 50 µl aliquot of cells was diluted in 0.1x PI solution diluted in 50 mM Tris/HCl, pH 7.5, for flow cytometry analysis. Samples were analysed on a FACscan flow cytometer using CellQuest software to obtain and analyse the data (BD Biosciences).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Rabin & Hauser, 2003), so we concluded that ExoS must be more toxic to yeast than these other TTSS toxins.
To overcome this problem, we expressed ExoS under the control of a tetracycline-regulated activator–repressor dual system (Belli et al., 1998). This required chromosomal integration of an expression plasmid (pCM242) for the wild-type tetracycline repressor tetR fused to Ssn6, a component of the general repressor complex in yeast. We generated this chromosomal integration at the LEU2 locus of the S. cerevisiae strain INVSc1, yielding the strain INVSc1(pCM242). We then transformed this strain with ExoS constructs in the expression plasmid pCM252. This vector expresses inserted genes under the control of the tetracycline-responsive tetO promoter, and is maintained episomally. It also expresses a fusion protein of tetR'-VP16 transactivator that will only bind and activate the tetO promoter in the presence of tetracycline. This system provides very tight control of transcription of exogenous genes under the control of tetracycline (Belli et al., 1998). In the absence of tetracycline, the TetR–Ssn6 fusion protein binds to the tetO promoter constitutively, and potently inhibits transcription. In the presence of tetracycline, this repression is lifted, and the TetR'-VP16 transactivator can bind to the tetO promoter, resulting in high-level expression of the introduced gene.
Using this system, we were able successfully to introduce the exoS gene and a variety of mutants into S. cerevisiae, and express them under the control of tetracycline. We transformed INVSc1(pCM242) with pCM252 alone, or pCM252 containing wild-type ExoS (ExoS), ExoS with the active site of the GAP domain mutated (GAPM), ExoS with the ADPRT active sites mutated (ADPRTM), or ExoS with both these alterations (GAPM+ADPRTM). In the absence of tetracycline, none of these constructs produced any effect on yeast growth, as assayed by spotting serial dilutions of each strain on agar (Fig. 1). Virtually identical numbers of each transformed strain were detected in this assay. When these dilutions of each strain were spotted on plates containing 0.1 µg tetracycline ml–1, yeast transformed with pCM252 containing wild-type ExoS were profoundly growth-inhibited compared with yeast transformed with plasmid alone. Further increases in tetracycline concentration did not produce appreciable increases in this growth inhibitory effect (Fig. 1). Yeast transformed with the GAPM mutant were similarly profoundly growth inhibited by 0.1 µg tetracycline ml–1, with no growth observable beyond the first yeast dilution spotted. Transformation with the ADPRTM mutant was much less toxic towards the yeast, with detectable growth inhibition only seen when the concentration of tetracycline was greater than 1 µg ml–1 (Fig. 1). The double mutant GAPM+ADPRTM showed no toxicity compared with yeast transformed with vector pCM252 alone. Thus, ExoS shows clear toxicity towards S. cerevisiae in this system, with virtually all of this effect being dependent on an intact ADPRT domain. The GAP domain did have some toxicity, however, as the ADPRTM mutant did inhibit yeast growth when expression levels were elevated by increased tetracycline concentrations. This inhibitory effect of ExoS was also observed in liquid culture following induction of ExoS expression by the addition of tetracycline (Fig. 2).
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). This did not differ from untransformed yeast (data not shown). Following induction of ExoS, there was a marked change in the distribution of filamentous actin within the yeast cells. Patches of actin were much larger and brighter than those seen in the cells transformed with pCM252 alone (Fig. 3B). The images shown in Fig. 3 were each photographed with the same settings to allow direct comparison of actin intensity. Many of these large actin patches aggregated in areas of the cell. The number of cells showing buds did not appear to differ from yeast transformed with vector alone. However, the degree of polarization of actin was disrupted. Although actin patches did tend to accumulate within the nascent daughter cell, patches were still visible within the mother in cells expressing ExoS. In addition, actin cables were thickened and disorganized, and did not exhibit the normal polarized distribution along the mother–daughter axis. This is seen in the cell boxed in Fig. 3(B), and it is enlarged in Fig. 3(C). A disorganized actin cable is shown by an arrowhead.
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). Yeast expressing ExoS containing the functioning ADPRT domain alone had the same alterations in actin as seen with the wild-type ExoS (Fig. 3D, GAPM). Expression of ExoS with a mutation of the ADPRT domain produced no discernible difference in actin staining from control cells (ADPRTM, Fig. 3E, compared with pCM252, Fig. 3A). Expression of ExoS with mutation of both the ADPRT and GAP domains (Fig. 3F) also gave an identical pattern of actin staining to the vector control (Fig. 3A). Thus, we concluded that the changes in yeast actin produced following expression of ExoS resulted from the activity of the ADPRT domain.
Expression of ExoS and ExoS mutant proteins
To be certain that the lack of effect of the ExoS ADPRT domain mutant was not due to a lack of expression of this protein, we analysed the levels of expression of ExoS and the different domain mutants 4 h after induction in yeast, the time at which the effects upon the actin cytoskeleton were determined in Fig. 3. As shown in Fig. 4, we did not detect any expression of the intact ExoS or GAPM ExoS at this time point, as we would expect, given the high toxicity of both these forms of ExoS to the yeast (Fig. 1). However, we were able to detect both the ADPRTM and ADPRTM+GAPM ExoS proteins (Fig. 4) in amounts that reflected their relative toxicity within the cell; levels of both proteins were sustained for up to 24 h (data not shown). Thus, ExoS with only the GAP domain active (ADPRTM) was expressed within yeast at a time when no effect on actin organization was detected.
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). To explore this possibility, we utilized the effects of the drug latrunculin A on the yeast actin cytoskeleton. This drug sequesters actin monomers, preventing new filamentous actin formation (Ayscough et al., 1997), although not affecting filament disassembly that occurs within minutes in S. cerevisiae (Ayscough et al., 1997). Thus, factors that lead to stabilization of actin filaments result in actin structures that are relatively resistant to latrunculin-A-induced actin depolymerization. Fig. 5 shows the effects of ExoS expression on the stability of cortical actin patches in the presence of latrunculin A. ExoS expression dramatically stabilized the actin cortical patches that disappeared in wild-type cells following just 1 min treatment with latrunculin A (Fig. 5). Thus, the changes in actin organization following ExoS expression appear to result from stabilization of actin filaments by ExoS.
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-factor, which arrests haploid yeast cells of mating type MATa just prior to START in the G1 phase of the cell cycle. We therefore constructed the tetracycline-inducible activator–repressor system in the haploid strain BMA64-1A by stable integration of the Tet-repressor plasmid pCM242, and expression of ExoS in the pCM252 vector. We confirmed that, in this haploid strain, ExoS expression inhibited yeast growth at 0.1 µg doxycycline ml–1, as seen with ExoS in the diploid INVSc1 strain (data not shown). We then studied this haploid strain BMA64-1A(pCM242) arrested using -factor, and transformed with either the pCM252 vector containing ExoS, or the pCM252 vector alone as a control, as described in Methods.
First, we examined the effect of ExoS expression on the ability of yeast to replicate their DNA in the first S phase following mating-factor growth arrest. The BMA64-1A(pCM242) strain was transformed with either pCM252 or pCM252-ExoS, growth arrested for 2 h with -factor, and induced with doxycycline in the continuing presence of -factor, for another 1 h. The -factor was then removed, and the DNA content of the yeast was then followed for the next 3 h. At time 0, yeast transformed with vector alone had a haploid content of DNA (Fig. 6, pCM242). Sixty minutes after mating-factor release, a very small number of the pCM242-transformed yeast had replicated their DNA, but, at 90 min, the majority of the cells had undergone DNA replication. At 120 and 150 min following -factor release, a significant number of yeast still had a diploid DNA content, but, at 180 min, virtually all of the yeast cells had divided and had returned to a haploid DNA content (Fig. 6, pCM242). The pattern of DNA replication in cells expressing ExoS was dramatically different. Following -factor release, only about 15 % of the yeast cells replicated their DNA at the peak times of 120 and 150 min (Fig. 6, ExoS). At 180 min, the diploid peak had subsided, suggested that those cells that did replicate their DNA eventually went on to divide (Fig. 6, ExoS). However, the majority of cells expressing ExoS did not replicate their DNA.
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-factor clearly showed that the ADPRT domain was entirely responsible for this inhibition of DNA replication (Fig. 6).
Next, we examined the distribution of filamentous actin within the yeast using fluorescent phalloidin. Following -factor arrest, yeast form mating projections or ‘schmoos’, and these did not differ in form or actin distribution between cells expressing ExoS or vector controls (Fig. 7, time 0). However, in cells expressing ExoS, we found that there was an increase in the number of yeast cells expressing more than one mating projection, as can be seen in Fig. 7 (pCM252-ExoS) at time 0. We quantified this difference, as shown in Fig. 8, and showed that this was significantly different between yeast transformed with pCM252 alone, and those expressing ExoS (P<0.001, 
2 test). Following removal of -factor, yeast transformed with vector alone showed a normal polarization of actin at a nascent bud at 30–60 min after -factor release. At 90 min, polarized buds were larger, and continued to increase in size up to 120 min after -factor removal (Fig. 7, pCM252). In contrast, cells expressing ExoS had a grossly abnormal morphology following -factor release. Initial polarization of the actin cytoskeleton was not inhibited at 30 min following -factor removal; indeed, it often appeared rather more marked than in the control cells transformed with vector alone (Fig. 7). However, rather than the small nascent buds apparent in the control cells, the expression of ExoS produced polarized actin within a markedly elongated projection. This became more pronounced at later times following -factor removal. Thus, at the 60 and 120 min time points, the projections were longer, and had a terminal bud-like swelling (Fig. 7). However, there was virtually no sign of actin accumulating at the neck of the process, and many of the cells showed the large bright staining actin patches already seen in non-synchronized cells expressing ExoS (Fig. 2).
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DISCUSSION |
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-factor, and that ExoS interfered with the normal bud development after release from -factor.
The use of this tetracycline-controlled yeast expression system offers several advantages over the glucose/galactose-based system. Firstly, the degree of repression of uninduced gene expression is greater than glucose repression of the galactose-controlled GAL1 promoter. It has been demonstrated that the tetR-Ssn6 repressor decreases expression of a lacZ control gene under the control of the tetO7 promoter to virtually undetectable levels (
0.10 Miller units of 
-galactosidase activity) (Belli et al., 1998). This is compared with the GAL1 promoter system, which only represses -galactosidase activity to 2 Miller units when repressed by growth on glucose (Belli et al., 1998). In the work reported here, tetracycline-controlled yeast expression allowed the use of S. cerevisiae to express ExoS, which could not be expressed using the glucose/galactose system because of the inhibitory effects of even tiny amounts of ExoS expression. In addition to its tight repression, the tetracycline-regulated activator–repressor dual system is also capable of inducing expression of regulated genes to high levels. For example, maximal lacZ expression levels from this system are 10-fold higher than with the MET3-based promoter system (a system that is induced by the removal of methionine), and 70 % of that achieved by the GAL1-based promoter system (Belli et al., 1998; Gari et al., 1997). Controlling gene expression by the addition (or removal) of tetracycline has another advantage over the GAL-based and MET3-based systems. The GAL- and MET3-based systems require a nutrient change to confer regulation, either a switch from glucose to galactose, or the removal of Met, and this has pleiotropic effects on yeast metabolism. Thus, it is hard to confirm which phenotypes are due to the regulated gene, and which are the consequence of the nutrient change. In contrast, doxycycline has no effect on the growth rate, cell morphology or global gene expression of S. cerevisiae, so any phenotypic change will be the result of the regulated gene (Wishart et al., 2005). Finally, the tetracycline-regulated activator–repressor dual system enables rapid gene induction without the requirement for a change of medium. It has been demonstrated that this system can induce detectable levels of protein expression by 30 min after antibiotic addition (Belli et al., 1998).
The ability of the ADPRT domain of ExoS to disrupt the actin cytoskeleton in S. cerevisiae parallels what is observed in mammalian cell culture systems, where the ADPRT domain has been shown to cause cell morphology changes in both epithelial and macrophage cell lines (Fraylick et al., 2001; Rocha et al., 2005). Although it is not clear how the ADPRT domain of ExoS leads to the observed disruption of the actin structures in S. cerevisiae, a clue to its possible mechanism of action comes from the yeast V159N actin mutant (Belmont & Drubin, 1998). When actin with a Val 159 to Asn mutation is expressed in S. cerevisiae as the sole source of actin, the resulting phenotype is very similar to that which we observed after expression of the ADPRT domain of ExoS. The V159N actin mutation results in these phenotypes because it forms actin filaments that are exceptionally stable due to their slow depolymerization (Belmont & Drubin, 1998). ATP-bound actin monomers polymerize to form filamentous actin, the ATP is then hydrolysed, and the release of Pi leads to a conformational change that destabilizes the actin filament, and promotes disassembly (Carlier, 1990). The V159N mutation results in actin that depolymerizes slowly because the filamentous actin fails to undergo a conformation change after Pi release (Belmont et al., 1999). The similarity of the V159N actin and ExoS ADPRT-domain-induced actin disruption suggests that the ADPRT domain of ExoS may also stabilize filamentous actin in some way. This is supported by experiments using latrunculin A as an inhibitor of actin polymerization (Fig. 4). Under these conditions, ExoS dramatically stabilized the actin cortical patches within the yeast, consistent with an ExoS-induced stabilization of actin filaments, as was also found for the Salmonella SspA protein when expressed in yeast (Lesser & Miller, 2001).
Our results also showed that ExoS expression led to the formation of more mating projections after -factor treatment, and the disruption of normal bud formation after -factor release. Such polarized growth in S. cerevisiae is dependent on small GTPases, such as Cdc42 (Ziman et al., 1991). When MATa yeast are treated with a high concentration of the -factor pheromone, they initiate and terminate growth of mating projections with regular periodicity (Bucking-Throm et al., 1973). It has been demonstrated that the regulators of Cdc42 activity control the initiation of mating projection formation (Bidlingmaier & Snyder, 2004). Mutation of Cdc24, the guanine nucleotide exchange factor (GEF) for Cdc42, results in a longer mating projection initiation period, and mutation of Bem3, which is a GAP for Cdc42, leads to a shorter mating projection period (Bidlingmaier & Snyder, 2004). Therefore, it is apparent that Cdc42 activation by its GEF, Cdc24, initiates mating projection formation, and this initiation is inhibited by the inactivation of Cdc42 by its GAP, Bem3. As detailed above, ExoS has been shown to ADP-ribosylate Rac1 and Cdc42 in vivo, and ADP-ribosylation of Rac1 by ExoS activates this Rho GTPase. One could therefore speculate that ExoS might ADP-ribosylate and activate Cdc42 in S. cerevisiae, leading to an increase in the initiation of -factor-induced mating projection formation.
Effects of ExoS on Cdc42 may also provide an explanation for the strange bud growth observed after -factor release in yeast expressing ExoS. ExoS expression resulted in many S. cerevisiae cells with large buds and elongated necks. Although we have been unable to discover in the literature any yeast mutants that have a similar phenotype, it is slightly reminiscent of the filamentous growth that results during cell stress (Pruyne & Bretscher, 2000). During filamentous growth, Cdc42 does not redistribute over the bud surface during G2-M, but remains localized at the tip of the bud, and directs further apical growth. Also, constitutive activation of Cdc42, or loss of its GAPs, Bem3 and Rga1, locks Cdc42 into a polarized distribution, and hyperpolarizes growth (Stevenson et al., 1995; Ziman et al., 1991). Therefore, if ExoS activated Cdc42 by ADP-ribosylation, this may lead to the strange bud-growth phenotypes we observed.
When we released S. cerevisiae from -factor arrest, yeast not expressing an active ADPRT domain of ExoS were able to exit G1, and proceed with DNA synthesis in S phase. However, when ExoS with an active ADPRT domain was expressed in yeast, no DNA synthesis was observed. This finding is consistent with what has been observed in mammalian cells, where ExoS inhibits DNA synthesis, and the ADPRT domain was shown to be responsible for this phenotype (Fraylick et al., 2001; Olson et al., 1999). It is not clear which target(s) of the ExoS ADPRT domain is responsible for this inhibition of DNA synthesis. Ras is integral to signal transduction pathways that affect DNA synthesis, so it may be the target (Takai et al., 2001). GTP-bound Ras binds and activates its effector, Raf. Raf is a protein kinase that induces gene expression through the mitogen-activated protein kinase cascade, and this drives the cell cycle. Therefore, inhibition of Ras by ADP-ribosylation may result in the observed inhibition of DNA synthesis. It has been demonstrated that ADP-ribosylation of Ras by ExoS is not required for the cytotoxicity of this protein (Pederson et al., 2002). Therefore, if inhibition of DNA synthesis is the trigger for ExoS-induced cell death, another protein that is involved in inducing DNA synthesis must be inactivated by ExoS ADP-ribosylation.
What of the role of the GAP domain of ExoS? The experiments reported here show that, unlike the Yersinia type III secreted toxin YopE with GAP activity, the ExoS GAP domain produces no discernible effect on yeast actin (Lesser & Miller, 2001). However, the ExoS GAP domain did inhibit yeast growth to a lesser degree than the ADPRT domain, and was expressed (Figs 1 and 4). Thus, although ExoS and YopE have a 54 % amino-acid identity over the N-terminal GAP domain (Frithz-Lindsten et al., 1997), they appear functionally distinct. This is also borne out by studies in mammalian cells. YopE produces disruption of the actin cytoskeleton in a number of cell types (Andor et al., 2001; Von Pawel-Rammingen et al., 2000). The GAP domain of ExoS, however, although producing an expected rounding of cells when introduced by transfection, only gave minimal effects when directly introduced into cells by type III secretion (Fraylick et al., 2001; Pederson & Barbieri, 1998). Thus, although, biochemically, the GAP domain of ExoS has a wide number of targets, when introduced into living cells, its effects seem much more limited. The effects of the GAP domain on yeast growth, although less than the ADPRT domain, clearly show an inhibitory effect (Fig. 1). However, the GAP domain did not prevent DNA synthesis following mating-factor-induced growth arrest (Fig. 6). The mechanism of its growth inhibitory effect remains unclear.
In conclusion, we have developed for the first time a model of ExoS function in the yeast S. cerevisiae. ExoS is a powerful inhibitor of yeast growth, largely due to its ADPRT activity, which also produces an unusual change in actin distribution, suggestive of actin stabilization, and alterations in the numbers and development of mating-factor projections. This suggests that activation of small GTPases might play a more important role in ExoS action than previously thought. The model system described here will be very useful in analysing these changes in more detail.
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REFERENCES |
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