the yeast protein kinase c cell integrity pathway mediates ...a set of genes from the protein kinase...

EUKARYOTIC CELL, Dec. 2003, p. 1200–1210 Vol. 2, No. 61535-9778/03/$08.00�0 DOI: 10.1128/EC.2.6.1200–1210.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Yeast Protein Kinase C Cell Integrity Pathway Mediates Tolerance tothe Antifungal Drug Caspofungin through Activation of Slt2p

Mitogen-Activated Protein Kinase Signaling†Cristina Reinoso-Martín, Christoph Schuller,* Manuela Schuetzer-Muehlbauer, and Karl Kuchler*

Department of Medical Biochemistry, Division of Molecular Genetics, Max F. Perutz Laboratories,University and Biocenter of Vienna, A-1030 Vienna, Austria

Received 11 August 2003/Accepted 2 October 2003

The echinocandin caspofungin is a new antifungal drug that blocks cell wall synthesis through inhibition of�-(1-3)-glucan synthesis. Saccharomyces cerevisiae cells are able to tolerate rather high caspofungin concen-trations, displaying high viability at low caspofungin doses. To identify yeast genes implicated in caspofungintolerance, we performed a genome-wide microarray analysis. Strikingly, caspofungin treatment rapidly inducesa set of genes from the protein kinase C (PKC) cell integrity signaling pathway, as well as those required forcell wall maintenance and architecture. The mitogen-activated protein kinase Slt2p is rapidly activated byphosphorylation, triggering signaling through the PKC pathway. Cells lacking genes such as SLT2, BCK1, andPKC1, as well as the caspofungin target gene, FKS1, display pronounced hypersensitivity, demonstrating thatthe PKC pathway is required for caspofungin tolerance. Notably, the cell surface integrity sensor Wsc1p, butnot the sensors Wsc2-4p and Mid2p, is required for sensing caspofungin perturbations. The expressionmodulation of PKC target genes requires the transcription factor Rlm1p, which controls expression of severalcell wall synthesis and maintenance genes. Thus, caspofungin-induced cell wall damage requires Wsc1p as adedicated sensor to launch a protective response through the activated salvage pathway for de novo cell wallsynthesis. Our results establish caspofungin as a specific activator of Slt2p stress signaling in baker’s yeast.

The fungal cell wall is the essential cellular boundary, con-trolling many transport processes, cellular metabolism, as wellas all communication with the extracellular world. Because ofits mechanical strength, it allows cells to withstand turgor pres-sure and consequently prevents cell lysis. Proper cell wall ar-chitecture requires cell wall components such as �-1,3-glucan,chitin, and mannoproteins, all of which form a large complex(21, 22, 25). Their coordinated synthesis represents an essen-tial step for the assembly of a functional cell wall to ensure cellintegrity (10). Certain antifungal drugs, such as caspofungin, asemisynthetic derivative of the secondary metabolite pneumo-candin Bo from the fungus Glarea lozoyensis (1), specificallyblock cell wall synthesis. Caspofungin acts fungicidal, since it isa noncompetitive inhibitor of the 1,3-glucan synthases Fks1pand Fks2p (30), both of which are believed to catalyze thepolymerization of UDP-glucose into �-1,3-glucan during cellwall biogenesis (39). When caspofungin is combined with otherantifungal drugs, such as fluconazole or amphotericin B, syn-ergistic or additive effects against a variety of clinically impor-tant fungal pathogens have been demonstrated in vitro and invivo (56). Cells lacking Fks1p display increased chitin content,elevated levels of the second 1,3-�-glucan synthase, Fks2p (42),as well as altered expression of glycosylphosphatidylinositol

(GPI)-anchored cell surface proteins (57). These changes mayreflect a compensatory response to maintain cell wall integrity.

The intracellular protein kinase C (PKC) signal transductionpathway is essential for sensing cell integrity under a variety ofenvironmental conditions or morphogenetic events. The PKCresponse regulates cell wall and actin cytoskeleton dynamics(13), and it is activated during polarized growth such as bud-ding and mating (64). In addition, PKC signaling is activated byenvironmental conditions that jeopardize cell wall stability,including high temperature (19), hypotonic shock (8), or im-paired cell wall synthesis (24). Accordingly, the absence ofPKC signaling causes cell lysis when yeast is exposed to any ofthese inducing conditions. Osmotic stabilization can preventcell lysis, which also indicates defective maintenance of a func-tional cell wall (34, 58).

Sensing of cell wall perturbations requires dedicated surfacesensors. Genetic studies place the WSC (for cell wall integrityand stress response component) genes upstream of the mito-gen-activated protein (MAP) kinase cascade. The WSC familycomprises four genes: WSC1/HCS77/SLG1, WSC2, WSC3, andWSC4/YFW1 (12, 17, 37, 59, 63). The Wsc1-4p proteins arehighly O glycosylated plasma membrane proteins that containa extracellular domain with a cysteine motif, and an S/T-richdomain that carries glycosylation sites (36, 49, 59). Additionalcell wall stress sensors are the partially redundant Mid2p andMtl1p cell surface proteins. These proteins act as mechanosen-sors of cell wall stress during budding or pheromone-inducedmorphogenesis, high temperature, or other cell wall distur-bances (12, 24, 49, 59).

The activation of the PKC pathway proceeds through thesmall G protein Rho1p, via Pkc1p (35), and a downstreamMAP kinase cascade. Although the molecular mechanisms by

* Corresponding authors. Mailing address: Department of MedicalBiochemistry, Division of Molecular Genetics, Max F. Perutz Univer-sity Laboratories, Campus Vienna BioCenter, Dr. Bohr-Gasse 9/2,A-1030 Vienna, Austria. Phone: 43-1-4277-61807. Fax: 43-1-4277-9618. E-mail for K. Kuchler: [email protected]. E-mail for C.Schuller: [email protected].

† The supplemental material for this article may be found at http://ec.asm.org/.

1200

on February 29, 2020 by guest

http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

which sensors transmit the signal to downstream effectors re-main ill defined, the Rho1-GDP/GTP exchange factor Rom2pmay mediate Rho1p activation (3). Rho1p is a small GTPaseupregulated by the GDP/GTP exchange factors Rom1p andRom2p (46, 48) and downregulated by the GTPase-activatingproteins Sac7p and Bem2p (47, 52). Among other functions,Rho1p binds and activates Pkc1p (20, 45), which in turn acti-vates the MAP kinase kinase kinase Bck1p/Slk1p (6, 33), thefunctionally redundant MAP kinase kinase kinases Mkk1p andMkk2p (15), and the MAP kinase Slt2p/Mpk1p (32, 58). PKCsignaling is constantly guarding cell integrity, and the expres-sion of many cell wall biosynthesis genes requires PKC (14, 65).Nevertheless, a parallel cell integrity signaling mechanism in-volves the Ykr2p and Ypk1p kinases, since the absence of bothof these kinases also leads to cell lysis at elevated temperatures(50).

A previous genome-wide survey of genes whose expressionwas altered in response to Mpk1p/Slt2p activation indicatedthat about 20 genes were upregulated (18). This set containedfive genes encoding GPI-anchored proteins, at least four ofwhich (Ylr194c, Crh1p, Pst1p, and Cwp1p) are also inducedupon loss of Fks1p (57). The PKC pathway is also importantfor other fungal pathogens, including human commensalpathogens such as Candida albicans or Cryptococcus neofor-mans. Mkc1p is the C. albicans homologue of Slt2p, andmkc1�/mkc1� mutant strains display cell surface alterations,an increase in O-glycosylated mannoproteins, hypersensitivityto antifungal agents that inhibit �-1,3-glucan and chitin syn-thesis (43, 44), as well as reduced virulence in vivo (11). Like-wise, PKC signaling mediates response to caspofungin-im-posed cell wall perturbations and high temperature inCryptococcus neoformans (28).

During our efforts to characterize the molecular mechanismsof caspofungin resistance in fungi (54), we noticed that baker’syeast displays much higher tolerance to this new antifungaldrug than did the fungal pathogen C. albicans. Hence, weinvestigated the global response of baker’s yeast to caspofun-gin, exploiting a genome-wide microarray analysis to identifygenes or pathways implicated in caspofungin susceptibility. In-terestingly, our data show that caspofungin rapidly and specif-ically activates Slt2p, leading to PKC pathway activation. Wealso identify the Wsc1p cell surface protein as the dedicatedsensor for caspofungin stress and show that the appropriateresponse to caspofungin-induced cell wall damage requires afunctional PKC pathway.

MATERIALS AND METHODS

Yeast strains, growth conditions, and growth inhibition assays. The Saccha-romyces cerevisiae strains used in the present study were isogenic derivatives ofBY4741 (MATa ura3-�0 his3-�1 leu2-�0 met15-�0), all of which were kanamycincassette deletions from the EUROSCARF knockout collection (http://www.uni-frankfurt.de/fb15/mikro/euroscarf/). Strain DL376 (MATa pkc1�::LEU2), a de-rivative of EG123, was kindly provided by David Levin (18). Unless otherwiseindicated, yeast strains were grown routinely at 30°C in YPD medium (1% yeastextract, 2% peptone, 2% glucose). In the case of pkc1�, the media were supple-mented with D-sorbitol at a final concentration of 1 M. Caspofungin (Merck &Co., Whitehouse Station, N.J.), Congo Red (CR; Sigma, St. Louis, Mo.), andcaffeine (Merck & Co., Darmstadt, Germany) were prepared as stock solutionsin sterile water and added to the medium at the desired concentrations. Sensi-tivity phenotypes were assayed with cells grown to the exponential growth phaseand diluted to an optical density at 600 nm (OD600) of 0.2. Identical volumes ofcultures, as well as 1:10, 1:100, and 1:1,000 serial dilutions, were spotted onto

agar plates containing various concentrations of drugs (29). Colony growth wasinspected and recorded after a 48-h incubation at 30°C. To record growth curvesin liquid culture, overnight cultures of wild-type S. cerevisiae cells (BY4741) werediluted to an OD600 of 0.2 and then grown to an OD600 of 1 in YPD medium at30°C for additional 2 h to allow cells to adapt to the medium, followed by theaddition of caspofungin. OD600 values were recorded in a multilabel counter(Wallac 1420; Perkin-Elmer, Turku, Finland).

RNA isolation, radiolabeling, and Northern analysis. Total yeast RNA wasisolated exactly as described previously (53). About 20 �g of glyoxal-treated totalRNA (51) were separated in a 1% agarose gel and transferred to nylon mem-branes (Amersham Biosciences, Little Chalfont, Buckinghamshire, England).Northern blots were hybridized with PCR-amplified probes, which were 32P-labeled dCTP radiolabeled by using a MegaPrime labeling kit (Amersham)under the conditions recommended by the manufacturer. Methylene blue stain-ing of rRNA on nylon membranes was used to control for equal RNA loading(29).

Nylon membranes were prehybridized in 10 ml of 10� Denhardt buffer (1 g ofFicoll 400, 1 g of polyvinylpyrrolidone, 1% [wt/vol] bovine serum albumin frac-tion V), 2� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1%sodium dodecyl sulfate (SDS), and 20 �g of salmon sperm DNA/ml for 3 to 6 hat 65°C. The radiolabeled probes were directly added to the prehybridizationsolution after purification on a NICK column (Amersham) and subsequent heatdenaturation. After an overnight incubation at 65°C, membranes were washed at65°C three times in 2� SSC–1% SDS and three times in 1� SSC–1% SDS andthen exposed to X-ray films at �70°C or analyzed with a PhosphorImager (Storm1840; Molecular Dynamics, Sunnyvale, Calif.).

Fragments of yeast genes used as probes for Northern blots were amplified byPCR with genomic DNA as a template (29). The following PCR program wasused for fragment amplification: denaturation at 94°C for 2 min, followed by 35cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min, with a final extensionat 72°C for 10 min. The primers used in the present study included SLT2s(5�-CAT GGA GCA TAC GGC ATA GT-3�), SLT2as (5�-GCT TGT GAA TTGGCA TTT GG-3�), HSP150s (5�-CTA AGA CTA CCG CTG CTG CT-3�),HSP150as (5�-AGC TGG TGC CAT CTA CGC TG-3�), CWP1s (5�-GTC TGTCGC TTT ATT CGC CT-3�), and CWP1as (5�-GGG CCA TTT CAT ATT ACATTA CGC-3�).

Preparation of yeast extracts and immunoblotting. Yeast cells were grownovernight in YPD medium to the mid-logarithmic growth phase at 30°C. Thecultures were next diluted to an OD600 of 0.2 and then grown to an OD600 of 1,at which point caspofungin was added to the cultures. Aliquots were harvestedafter the time intervals indicated in the figures, and cells were collected bycentrifugation. Cells were lysed in 250 �l of cold YEX lysis buffer (1.85 MNaOH, 7.5% �-mercaptoethanol), and proteins were precipitated with cold 50%(wt/wt) trichloroacetic acid. Cell extracts corresponding to ca. 5 � 106 cells wereseparated in SDS–10% polyacrylamide gels and then transferred to nitrocellu-lose membranes (Protran; Schleicher & Schuell, Dassel, Germany). Phosphory-lated Slt2p was detected by using an anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Cell Signaling, Beverley, Mass.) at a 1:2,000 dilution to detectdually phosphorylated Slt2p. Total Slt2p was detected with anti-GST–Slt2p an-tibodies (a generous gift from Humberto Martín, Madrid, Spain) (40) at a 1:1,000dilution. Polyclonal antibodies to Swi6p (a generous gift from Kim Nasmyth,Vienna, Austria) were used as a loading control. Immunoblots were developedwith the Amersham enhanced chemiluminescence detection system under theconditions recommended by the manufacturer.

DNA microarray experiments. A 20-ml culture of cells in YPD medium wasinoculated from a colony and grown overnight at 30°C and 200 rpm. The culturewas diluted until it reached an OD600 of 0.1, and then it was allowed to recoverfrom the stationary phase for 4 h. The culture was split into two halves; oneremained untreated, and the other was treated with caspofungin by adding thedrug at a concentration of 10 ng/ml. Samples were taken at different time pointsafter drug treatment, followed by preparation of RNA for fluorescence labelingas described below. We used different combinations of cells, such as wild-typeuntreated and treated cells, at an OD600 of 1 (Fig. 1B, arrows A2 and B4) or afterone (arrows A1 and B1), two (arrows A2 and B2), and 3 h (arrows A3 and B3)caspofungin challenges. Cells were pelleted at 3,000 rpm for 5 min and washedwith 1 ml of water, followed by RNA preparation by a routine procedure (53).RNA was quantified by spectrophotometry at 260 nm in Tris-EDTA buffer.

Aliquots containing about 20 �g of total RNA from treated and untreated cellswere used for cDNA synthesis with 200 U of Superscript II reverse transcriptase(Invitrogen, Carlsbad, Calif.). Reactions included either the Cy3-dCTP or theCy5-dCTP kit (Amersham). Labeled cDNAs were pooled, and RNA was de-stroyed by hydrolysis the samples for 20 min in 50 mM NaOH at 65°C, followedby neutralization with acetic acid, and then cDNA was precipitated with isopro-

VOL. 2, 2003 Slt2p MAP KINASE SIGNALING ACTIVATION BY CASPOFUNGIN 1201


http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

panol. Hybridization to whole-genome cDNA microarrays (Ontario Cancer In-stitute, Toronto, Ontario, Canada) was done in DigEasyHyb (Roche, Mannheim,Germany) solution at 37°C overnight with 70 �g of salmon sperm DNA/ml as acarrier. Microarrays were washed three times in 1� SSC–0.1% SDS at 50°C,followed by a 1-min wash in 1� SSC at room temperature. Glass slides were spunfor 5 min at 500 rpm in a tabletop centrifuge to remove liquid, scanned on anAxon 4000B scanner (Axon Instruments, Inc., Union City, N.J.), and analyzed byusing Gene Pix Pro4.1 software (Axon). DNA microarrays and protocols wereobtained from the Ontario Cancer Institute. All microarray experiments werecarried out with independent RNA preparations. Microarray data were collectedand normalized with GenePix Pro4.1, and data filtering was done by usingconventional spreadsheets. The Saccharomyces Genome Database at StanfordUniversity (http://www.yeastgenome.org/) was used to retrieve functional anno-tations of yeast genes. The complete microarray data set is also available assupplementary material.

RESULTS

S. cerevisiae displays higher caspofungin tolerance than C.albicans. Caspofungin is a new antifungal drug, which actsfungicidal, since it blocks cell wall synthesis through inhibition

of fungal �-1,3-glucan synthases. During our attempts to char-acterize fungal caspofungin resistance mechanisms (54), wenoticed that baker’s yeast displayed a much higher caspofungintolerance than the fungal pathogen C. albicans (54). At suble-thal concentrations, yeast cells showed 100% viability on agarplates when treated with 10 ng of caspofungin/ml (Fig. 1A),whereas viability of C. albicans cells was drastically reduced toca. 12% of untreated control cells under the same conditions(data not shown). Hence, we pursued a global microarray anal-ysis to identify yeast genes implicated in caspofungin tolerance.To pinpoint the optimal conditions for RNA isolation, wild-type yeast cells were treated with increasing drug concentra-tions until reduced growth was obvious (Fig. 1B). Notably, cellgrowth was severely impaired in liquid culture at 10 ng ofcaspofungin/ml (Fig. 1B), whereas the viability appeared to beunaffected, as judged from the ability of cells to form colonies(Fig. 1A).

Caspofungin modulates genes implicated in cell wall func-tion and signal transduction. Since 10 ng of caspofungin/mlcaused a strongly reduced growth of baker’s yeast, we chosethis concentration to extract RNA for global microarray anal-ysis. RNA was then isolated from treated and untreated con-trols cells at a similar OD600 or from cells in the same growthphases (Fig. 1B). Several independent experiments were car-ried out to compare transcriptomes of cells after drug treat-ment for 1 h (A1 and B1), 2 h (A2 and B2), and 3 h (A3 andB3), as well as at a similar OD600 of 1 (A2 and B4). Total RNAwas purified and labeled by incorporation of Cy3- and Cy5-dCTP and then hybridized to whole-genome cDNA microar-rays of S. cerevisiae. The representative results from thesemicroarray profiling experiments are represented in Table 1and Table 2 and in the supplementary material. Based onfunctional annotation (http://www.yeastgenome.org/), thegenes whose expression was modulated by caspofungin wereclassified into several categories.

Caspofungin enhanced transcription of genes required forcell wall biogenesis or maintenance of its architecture. At leastone of the three chitin synthase genes, CHS1 (4), as well as fivegenes encoding GPI-anchored proteins (CWP1, SED1, CRH1,YLR194c, and PST1) were induced. Furthermore, PIR2/HSP150, one out of a total of five PIR family members (Pir1p,Pir2p/Hsp150p, Pir3p, Cis3p, and YJ160p) was also induced(23). A similar induction was observed for YGR189c encodinga homolog of bacterial �-glucanases and eukaryotic endotrans-glycosidases. Most strikingly, the MAP kinase SLT2/MPK1 andthe related gene MLP1 (YKL161c), both of which appear to beinduced by active cell integrity signaling (18), were also up-regulated after caspofungin challenge. Remarkably, however,none of the genes involved in general stress response throughMsn2p was induced (Table 1).

Further, caspofungin also caused repression of certain genesinvolved in cell wall organization and biogenesis (Table 2; seealso Supplementary Material), including the putative WSC4stress sensor, the SIT4 phosphatase, and calcineurin CNA1, aswell as the �-1,6-mannosyltransferase OCH1 and the manno-syltransferase KTR4 genes. Taken together, the results of themRNA expression profiling demonstrate the activation of tar-get genes of PKC cell integrity pathway (18). Despite the rapiddeleterious effect of caspofungin on cell proliferation and cell

FIG. 1. Viability and growth of S. cerevisiae in the presence ofcaspofungin. (A) S. cerevisiae (BY4741) cells growing in the exponen-tial growth phase at an OD600 of 1 were treated with 10 ng of caspo-fungin/ml for 1 h. Viability was determined by plating appropriateserial dilutions of treated and control cells on YPD plates. (B) Cellswere from an overnight culture were grown to the early exponentialgrowth phase (OD600 1), diluted into fresh YPD medium, and grownfor another 2 h. The culture was split, and different concentrations ofcaspofungin (10 and 50 ng/ml) were added. The OD of the cultures wasmonitored by spectrophotometry. At the indicated time points (labeledA and B), RNA was extracted and corresponding pairs of labeled RNAsamples from treated and untreated cells (A2 and B4, A1 and B1, A3and B3, and A2 and B2) were used for microarray analysis.

1202 REINOSO-MARTIN ET AL. EUKARYOT. CELL


http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

wall synthesis, other stress-related genes remained unaffectedin their expression profile.

Caspofungin induces Slt2p phosphorylation and activatesthe PKC signaling pathway. The microarray data indicatedthat members of the PKC pathway increased expression uponcaspofungin treatment. Thus, we used Northern blotting to testwhether SLT2 expression is controlled by caspofungin in orderto verify the microarray data. Indeed, induction of SLT2mRNA was observed in cells treated with 10 or 30 ng ofcaspofungin/ml after 30 or 60 min (Fig. 2A).

The activation of the PKC pathway also leads to phosphor-ylation of Slt2p on Thr190/Tyr192 residues (32). Therefore, wealso tested whether caspofungin increased the amount of du-ally phosphorylated Slt2p, which would indicate active cellintegrity signaling. We used a phospho-specific antibody raisedagainst dually phosphorylated p44/42 MAP kinase and a spe-cific anti-Slt2p antibody as described in Material and Methods.Caspofungin triggered a rapid and transient Slt2p phosphory-lation and thus activation of Slt2p within 5 min after drugtreatment (Fig. 2B). The amount of activated phospho-Slt2pkinase decreased after about 30 min, which is consistent withstress adaptation. We used CR, a cell wall-damaging drugknown to induce Slt2p phosphorylation (10, 24, 41), as a pos-itive control. As expected, no Slt2p was detectable in cellslacking SLT2 (Fig. 2B). Interestingly, although the microarraydata indicated slightly upregulated mRNA levels, correspond-ing total Slt2p protein levels did not significantly change under

activating conditions (19), a result perhaps due to autoregula-tory mechanisms (18).

Slt2p is required for caspofungin tolerance. The data indi-cate that caspofungin specifically triggers PKC pathway signal-ing, since it induced Slt2p phosphorylation and thus its activa-tion. We therefore tested whether Slt2p is also required forcaspofungin tolerance. Wild-type and isogenic slt2� cells werediluted and spotted onto plates containing various amounts ofcaspofungin. Indeed, cells lacking Slt2p displayed a severegrowth retardation at low caspofungin doses and were unableto grow at higher concentrations compared to the isogenicwild-type control (Fig. 3A). CR and caffeine, which are drugsknown to inhibit the growth of cells lacking a functional PKCpathway (6, 7), were used as positive controls. Finally, we alsotested the growth of slt2� cells in liquid culture in the presenceof caspofungin (Fig. 3B). Within the first 6 h after caspofunginaddition, no growth differences were detectable between thewild-type and the slt2� cells. However, after prolonged treat-ment, slt2� cells showed dramatic growth defects in liquidculture compared to wild-type cells (Fig. 3B). Taken together,these data demonstrate that the MAP kinase Slt2p is necessaryfor caspofungin tolerance.

The Wsc1p cell surface sensor mediates caspofungin-inducedPKC pathway activation. Because Slt2p mediates caspofungintolerance, we investigated whether other upstream and down-stream components of the PKC pathway (Fig. 4) are implicatedin caspofungin tolerance. We also asked the question which

TABLE 1. S. cerevisiae genes upregulated by caspofungin

Gene ORF IDc FunctionFold repressiona RMb

Avg SD A2-B2 A2-B4 A1-B1 A3-B3

CWP1 YKL096w Structural constituent of cell wall 3.43 1.26 4.40 4.77 2.37 2.18SLT2 YHR030c MAP kinase 2.83 0.67 3.43 2.89 1.53 2.83

YLR194c Structural constituent of cell wall 2.70 1.01 1.57 2.70 3.83SED1 YDR077w Structural constituent of cell wall 2.55 0.48 2.71 3.20 2.13 2.16YPS4 YLR121c Aspartic-type endopeptidase 2.39 0.94 1.36 2.42 3.38CHS1 YNL192w Chitin synthase 2.06 0.56 2.10 1.42 2.66PRB1 YEL060c Endopeptidase 1.98 0.54 1.05 1.93 1.96 2.51NCE103 YNL036w Function unknown 1.91 0.50 1.78 1.28 2.36CRH1 YGR189c Structural constituent of cell wall 1.85 0.57 2.42 2.31 1.12 1.56

YPR146c Function unknown 1.77 0.98 0.59 1.97 2.75PST1 YDR055w Structural constituent of cell wall 1.77 0.69 1.65 2.54 0.82 2.06DOP1 YDR141c Function unknown 1.76 0.35 2.22 1.56 1.51

YLR414c Function unknown 1.76 0.37 1.28 1.86 2.27 1.87MLP1 YKL161c Protein kinase 1.64 0.50 1.20 2.39 1.46 1.53

YJL171c Structural constituent of cell wall 1.63 0.36 1.37 2.05 1.47 1.64HSP150 YJL159w Structural constituent of cell wall 1.63 0.32 1.90 1.85 1.17 1.59CCW14 YLR391w-a Structural constituent of cell wall 1.61 0.51 1.97 2.18 1.26 1.05SUR1 YPL057c Glycosyl group transferase 1.59 0.54 0.93 1.83 2.01BRE1 YDL074c Ubiquitin-protein ligase 1.59 0.43 1.60 2.12 1.42 1.11

YLR327c Function unknown 1.56 0.19 1.48 1.60 1.61TFB3 YDR460w General RNA polymerase II

transcription1.55 0.66 2.11 1.75 0.87

ORC1 YML065w Adenosinetriphosphatase 1.53 0.56 2.31 1.08 1.60YOR161c Function unknown 1.48 0.50 1.03 1.38 2.03YPL260c Function unknown 1.44 0.48 1.50 1.65 0.98YMR254c Function unknown 1.44 0.83 2.85 1.27 0.91

BGL2 YGR282c Endo-�-1,3-glucanase 1.44 0.45 1.48 2.10 1.10 1.08NUP2 YLR335w mRNA-nucleus export 1.41 0.61 1.92 1.51 0.79

YMR191w Function unknown 1.40 0.57 1.93 1.73 0.82

a That is, the average of all four experiments.b RM, ratio of medians.c ORF ID, open reading frame name according to the Stanford genome database.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

cell surface sensor mediates sensing caspofungin-induced cellwall damage. Several gene products are known to feed into thePKC pathway by transducing surface signals. Mid2p is requiredfor the induction of Slt2p tyrosine phosphorylation after expo-sure to high temperature, mating pheromones or CalcofluorWhite (24, 41). In contrast, Wsc1p/Slg1p/Hcs77p has only beenimplicated in the sensing of thermal stress (12, 59). Hence, weanalyzed levels of phospho-Slt2p in wild-type cells and in iso-genic wsc1�, wsc2�, wsc3�, wsc4�, and mid2� strains, as wellas in an lre1� strain (data not shown), and we also tested theircaspofungin sensitivities. Interestingly, a caspofungin-inducedincrease in Slt2p phosphorylation, and thus activation was de-tected in all mutants tested except for the wsc1� mutant (Fig.5A). All other putative sensor deletion strains tested showednormal caspofungin-induced activation of Slt2p (Fig. 5A). No-tably, levels of phospho-Slt2p were generally lower in wsc4�cells, but the caspofungin-mediated activation was still detect-able. Conversely, wsc2�, wsc3�, and rom2� mutants, as well asthe lre1� mutant (data not shown), displayed higher levels ofdually phosphorylated Slt2p than did the wild-type strain. Im-munoblotting with anti-Slt2p antibodies showed that the Slt2pand Swi6p levels did not change under these conditions, thelatter serving as a loading control (Fig. 5A).

Next, we tested caspofungin susceptibilities of wild-type cellsand isogenic strains carrying deletions of the PKC pathwaygenes, including the deletions mid2�, rho2�, mid2�, mkk2�,

bck1�, wsc1�, wsc2�, wsc3�, wsc4�, lre1�, and slt2�. Strainswere grown in YPD medium, and identical volumes, as well as10-fold serial dilutions, were spotted onto agar plates contain-ing the indicated caspofungin concentrations. The growth as-says demonstrated that all mutants lacking genes of the PKCpathway were also caspofungin hypersensitive (Fig. 5B).Among the most sensitive mutants were bck1� and slt2� cells,as well as pkc1� cells (Fig. 5B). Interestingly, the GDP/GTPexchange factor Rom2p showed higher levels of phospho-Slt2pbut was only slightly sensitive to caspofungin in the plate assay(Fig. 5B). These results indicate that an active PKC pathway isnecessary but not sufficient for full caspofungin tolerance.These data establish the importance of the PKC pathway andsuggest that Wsc1p senses caspofungin-induced cell wall dam-age (Fig. 5B).

The 1,3-�-glucan synthase Fks1p is not required for caspo-fungin-induced Slt2p activation. Caspofungin is a noncompet-itive inhibitor of 1,3-�-glucan synthases encoded by FKS1 andFKS2 (30). The FKS1 gene is expressed during vegetativegrowth, whereas FKS2 is mainly expressed during sporulation(30). We therefore investigated whether cells lacking Fks1pdisplay altered caspofungin sensing. fks1� mutant cells showedhigh levels of phospho-Slt2p even in uninduced cells, suggest-ing compensatory activity of the PKC pathway. However, in thepresence of caspofungin, the level of phospho-Slt2p still in-creased, suggesting FKS1-independent sensing. Likewise,

TABLE 2. S. cerevisiae genes downregulated by caspofungin

Gene ORF IDc FunctionFold repressiona RMb

Avg SD A2-B2 A2-B4 A1-B1 A3-B3

CNE1 YAL058w ER-associated protein catabolism 3.82 3.14 7.41 1.55 2.5BTT1 YDR252w Chaperone 3.42 1.59 3.28 1.9 5.08GFD2 YCL036w Function unknown 3.3 2.25 2.27 1.76 5.88APG14 YBR128c Function unknown 3.27 2.44 6.06 2.26 1.49PHM6 YDR281c Function unknown 2.96 1.64 1.59 1.79 5.13 3.33NCA3 YJL116c Function unknown 2.93 1.85 1.81 1.92 5.06BDH1 YAL060w (R,R)-Butanediol dehydrogenase 2.83 0.96 3.91 2.7 3.1 1.61

YDR114c Function unknown 2.62 1.54 4.85 1.32 2.33 2.0ATO3 YDR384c Transporter activity 2.59 1.11 4.02 1.52 1.92 2.88PTC4 YBR125c Protein phosphatase type 2C 2.55 1.3 1.91 2.3 4.44 1.54MSI1 YBR195c Function unknown 2.53 0.78 3.15 1.65 2.79DUT1 YBR252w dUTP pyrophosphatase 2.51 0.96 3.86 1.64 2.1 2.42DTR1 YBR180w Amine/polyamine transporter 2.36 0.32 2.48 1.94 2.7 2.33GDH3 YAL062w Glutamate dehydrogenase

(NADP�)2.35 0.33 1.97 2.6 2.48

MEU1 YLR017w Glutamate biosynthesis 2.34 0.8 2.95 1.42 3.07 1.92YDR444w Function unknown 2.33 1.1 1.81 1.59 3.6

MFA1 YDR461w Pheromone 2.32 0.67 1.87 1.76 2.43 3.23HXT5 YHR096c Glucose transporter 2.28 0.9 2.46 1.3 3.08ERV46 YAL042w ER-to-Golgi transport 2.26 0.72 3.33 1.82 2.03 1.86PHO11 YAR071w Acid phosphatase 2.24 0.57 2.07 1.77 3.07 2.06TGL2 YDR058c Triacylglycerol lipase 2.19 0.31 1.9 2.52 2.15ADH5 YBR145w Alcohol dehydrogenase 2.19 0.25 2.29 1.81 2.38 2.26ILV6 YCL009c Acetolactate synthase 2.18 0.5 2.19 1.58 2.13 2.8LEU1 YGL009c 3-Isopropylmalate dehydratase 2.14 0.53 2.91 2.06 1.86 1.74LYS20 YDL182w Homocitrate synthase 2.13 0.68 1.9 1.56 3.12 1.96HSM3 YBR272c Function unknown 2.09 0.47 2.48 1.57 2.22

YBR014c Function unknown 2.09 0.86 3.01 1.3 1.97RIB7 YBR153w Vitamin B2 biosynthesis 2.09 0.3 2.34 1.76 2.16

YJR024c Function unknown 2.07 0.39 2.45 1.56 2.03 2.26

a See Table 1, footnote a.b See Table 1, footnote b.c See Table 1, footnote c.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

caspofungin-induced Slt2p phosphorylation was similar to thatof the wild type in fks2� cells (Fig. 6A). As expected, mutantslacking FKS1 also showed pronounced caspofungin hypersen-sitivity in agar plate assays because expression levels of Fks2punder these conditions apparently cannot fully compensate forthe loss of Fks1p (Fig. 6B). Notably, chs3� cells were slightlyhypersensitive. These results demonstrate that an activatedPKC pathway, as well as a functional 1,3-�-glucan synthase, isrequired for caspofungin tolerance (Fig. 6B).

The Slt2p-dependent transcription factor Rlm1p is acti-vated by caspofungin. To identify regulators acting down-stream of the PKC pathway, we investigated the Rlm1p andSwi4p transcription factors implicated in the PKC pathway.Rlm1p and Swi4p (SBF) act as downstream effectors of Slt2p/Mpk1p signaling (14, 16, 38, 61). Most Rlm1p-regulated genesencode cell wall proteins or enzymes involved in cell wallbiosynthesis (18). The Swi4p and Swi6p proteins form a het-erodimeric complex known as SBF, which regulates gene ex-pression during the G1/S transition (27). SBF-activated genes

are involved in budding, as well as in membrane and cell wallbiosynthesis. Previous studies have shown that the Rlm1p tran-scription factor, which is activated by Slt2p-dependent phos-phorylation, is also mediating SLT2 mRNA induction in re-sponse to heat shock (18, 61, 62). To examine the role ofRlm1p and Swi4p in the SLT2 activation upon caspofunginstress, we determined the mRNA levels of SLT2, HSP150, andCWP1 by Northern analysis (Fig. 7). SLT2 and CWP1 expres-sion was induced in wild-type and swi4� cells but stronglyimpaired in the rlm1� mutant strain (Fig. 7). Notably, HSP150mRNA levels were slightly induced by caspofungin after 1 h inwild-type cells but increased significantly in the swi4� mutant.However, similar to SLT2 and CWP1, no induction was ob-served for HSP150 in the rlm1� mutant. ACT1 served as acontrol for RNA loading. Finally, we also tested whether theabsence of Rlm1p causes caspofungin hypersensitivity. How-ever, although Slt2p induction requires Rlm1p, rlm1� cellsfailed to display dramatic caspofungin hypersusceptibilities,implying that Slt2p might require the function of other, as-yet-unknown transcription factors to mediate candin tolerancethrough target genes such as FKS1 or FKS2, since candintolerance is apparently also controlled by other factors (31).Likewise, agar plate assays demonstrated that cells lackingSwi4p displayed normal caspofungin susceptibility comparedto the wild-type control (Fig. 7B). Taken together, our dataestablish caspofungin and perhaps other echinocandins as spe-cific activators of cell integrity signaling through the Wsc1psurface sensor and downstream Slt2p kinase.

DISCUSSION

The cell wall is an essential component of the fungal cell andthe prime barrier to the surrounding environment. Modulationof cell wall architecture is required for cell growth, mating, andadaptation to changing environmental conditions. Hence, cellwall composition perhaps undergoes constant dynamic changescontrolled by a network of sensors regulating cell wall-modi-fying enzymes, most of which appear to be controlled by theSlt2p/Mpk1 MAP kinase pathway (26). We show in this studythat blocking �-1,3-glucan synthesis by caspofungin rapidly andselectively activates the yeast PKC pathway and that the integ-rity of this pathway is required for tolerance to caspofungin.

Caspofungin, an echinocandin family member, kills Candidaand Cryptococcus spp., as well as Aspergillus spp., by inhibitionof the enzymes synthesizing �-1,3-glucan (28, 30, 39, 56). Weused microarray profiling to analyze the global response of S.cerevisiae to sublethal but growth-inhibitory doses of caspofun-gin (Fig. 1B). Although transcript profiles were generated fromdistinct growth phases and at different time points, the datasetsof all experiments are remarkably similar (Table 1 and supple-mentary material). Among the genes induced by caspofungin,we found a group of genes recently identified to be under thecontrol of the PKC pathway (18). These genes also overlapwith those identified to be upregulated by lack of Fks1p, themain yeast 1,3-�-glucan synthase (31, 57). Further, about 20genes are upregulated in response to overexpression of anactivated allele of the MAP kinase kinase Mkk1p (S386P),which is acting upstream of Slt2p (18). Caspofungin also in-duces five GPI-anchored proteins (PST1, CRH1, SED1,YLR194c, and CWP1) important for cell wall function, as well

FIG. 2. Expression of SLT2 mRNA is induced in presence ofcaspofungin. (A) Northern analysis of logarithmically growing culturesin the presence of caspofungin. Wild-type cells (BY4741) and isogeniccells lacking Slt2p (slt2�) were grown at 30°C in the presence orabsence of 10 or 30 ng of caspofungin/ml for 30 or 60 min. Total RNAwas isolated and fractionated in 1% agarose gels. The SLT2 probe wasisolated as described in Materials and Methods. Methylene blue stain-ing of 25S rRNA was used as a loading control. (B) Immunoblotanalysis of total and phosphorylated Slt2p. Exponentially growing wild-type BY4741 cells were exposed to caspofungin (�) for various times,starting immediately after caspofungin addition (0 min). Cell extractswere subjected to immunoblotting with phospho-specific antibodies asdescribed in Materials and Methods. Cells lacking Slt2p (�) were usedas negative control after a 20-min exposure, and the wild-type BY4741cells were treated with 10 �g of CR/ml (�) for 2 h (120 min) as apositive control. Decoration of blots with polyclonal anti-GST-Swi6pantibodies served as a loading control to verify equal protein loading.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

as chitin synthase Chs1p. This is in good agreement with thegenes encoding genes upregulated in fks1� mutants (YLR194c,CRH1, PST1 and CWP1) reported earlier (57). Strikingly,caspofungin specifically induces and activates the MAP kinaseSlt2p and its related serine/threonine protein kinase Mlp1p,

both of which are also induced in strains lacking FKS1 (18, 57).Among genes downregulated by caspofungin, we find RHO4(Rho small monomeric GTPase), the sensor WSC4, the �-1,6-mannosyltransferase MNN10, and the serine/threonine phos-phatase SIT4, the last of which is required for the downregu-lation of the PKC pathway, as well as the calcium-dependentprotein phosphatase calcineurin CNA1.

An exhaustive analysis of transcript profiles of several cellwall biosynthesis mutants also indicated the involvement ofstress response pathways regulated by the transcription factorsMsn2/4p, Crz1p, and Hsf1p (31). Interestingly, and in contrastto these studies, our results do not indicate expression modu-lation of any key genes from these stress pathways. Hence, theexperimental conditions we used in terms of sublethal drugdoses and exponentially growing cells is sufficient to elicit themost specific response, namely, PKC-mediated signaling.

Activation of the PKC pathway by caspofungin is apparentfrom the phosphorylation status of the MAP kinase Slt2p. Weused a combination of an antibody recognizing dually phos-phorylated p44/42 MAP kinase (on Thr202/Tyr204) and a spe-cific anti-Slt2 antibody to detect the phosphorylated and activeform of the MAP kinase Slt2p. The intracellular activation ofMAP kinases requires phosphorylation on conserved Thr andTyr residues in subdomain VIII (5). As for Slt2p, these resi-dues correspond to Thr190 and Tyr192. Phosphorylation of bothsites is essential for MAP kinase activation. Therefore, theamount of dually phosphorylated Slt2p is a direct indicator of

FIG. 3. THe slt2� strain is hypersensitive to cell wall perturbations.(A) Identical volumes of 10-fold serial dilutions of exponentially grow-ing wild-type cells and cells lacking Slt2p (slt2�) were spotted ontoYPD plates containing 10 mM caffeine, 100 �g of CR/ml, or 50 and 150ng of caspofungin/ml and then incubated at 30°C. Colony growth wasinspected after a 48-h incubation at 30°C. (B) Wild-type BY4741 cellsand the mutant strain (slt2�) were grown in the presence or absence of10 ng of caspofungin/ml, and cell growth was recorded in a spectro-photometer. The OD600 was recorded every hour until the cells ap-proached stationary phase.

FIG. 4. Diagram depicting the yeast PKC signaling pathway. Thebasic components of the cell integrity signal transduction pathway aredepicted. Cell surface sensors transduce extracellular signals through aMAP kinase cascade to induce transcription via two main downstreamregulators, Rlm1p and Swi4p, so as to respond to a variety of externalcell wall stresses.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

PKC/Slt2p pathway activation (32). Our results suggest a rapidresponse of the PKC pathway to caspofungin-induced damage,since phospho-Slt2p levels peak after only 10 min of caspofun-gin challenge. The levels of phospho-Slt2p reached a levelcomparable to a 2-h treatment with CR (Fig. 2B). The timeframe of Slt2p activation is quite similar to the induction byhypo-osmotic shock that occurs within 1 min after stress expo-sure (8). These fast kinetics suggests that the status of the PKCpathway is tightly and dynamically linked to the activity of theglucan synthase and cell wall integrity or biosynthesis. Further-more, we also demonstrate that the integrity of the PKC path-way is important for sensing cell wall damage, as well as for thedownstream response to caspofungin. Mutants lacking SLT2are hypersensitive to caspofungin both in plate assays and inliquid culture. In addition, we found that bck1� cells lackingthe MAP kinase kinase kinase and rom2� cells lacking theGDP/GTP exchange factor for the PKC activator Rho1p, aswell as pkc1� mutants, display marked caspofungin hypersen-sitivity.

The PKC pathway is also important for other fungal species,

FIG. 5. Caspofungin-induced Slt2p phosphorylation is severely im-paired in wsc1� mutant cells. (A) Exponentially growing wild-typeBY4741 and their isogenic derivatives lacking PKC pathway genes orupstream sensors (slt2�, wsc1-4�, rom2�, and mid2�) were treatedwith 10 ng of caspofungin/ml for 20 min at 30°C. Cell extracts wereprepared and subjected to immunoblotting with a polyclonal anti-Slt2pantiserum, as well as phospho-specific anti-Slt2p antibodies, as de-scribed in Materials and Methods. Untreated cells (�) and cells lack-ing Slt2p (slt2�) were used as negative controls. Decoration of blotswith polyclonal anti-GST-Swi6p antibodies served as loading control toverify equal protein loading. (B) Identical volumes of 10-fold serialdilutions of exponentially growing wild-type BY4741 and EG123 cellsand their isogenic derivatives lacking various PKC pathway genes(pkc1� or slt2�, wsc1-4�, rom2�, lre1�, rho2�, mkk2�, bck1�, andmid2�) were spotted onto YPD plates containing various concentra-tions of caspofungin as indicated and then incubated at 30°C. Colonygrowth was inspected after a 48-h incubation at 30°C.

FIG. 6. Slt2p is still activated in cells lacking Fks1p and Fks2p.(A) Exponentially growing wild-type BY4741 cells and cells lacking�-1,3-glucan synthase genes (fks1� and fks2�) were treated with 10 ngof caspofungin/ml for 20 min at 30°C. Cell extracts were prepared andsubjected to immunoblotting with phospho-specific Slt2p antibodiesand anti-Slt2p antibodies as described in Materials and Methods. Ex-tracts from untreated cells (�) were used as a negative control. Dec-oration of blots with polyclonal anti-GST-Swi6p antibodies served asloading control to verify equal protein loading. (B) Identical volumesof 10-fold serial dilutions of exponentially growing wild-type cells andcells lacking cell wall synthesis genes (fks1�, fks2�, and chs3�) or Slt2p(slt2�) were spotted onto YPD plates containing various concentra-tions of caspofungin as indicated and then incubated at 30°C. Colonygrowth was inspected after a 48-h incubation at 30°C.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

such as C. albicans or Cryptococcus neoformans. The C. albi-cans MAP kinase MKC1 complements the lytic phenotype ofthe S. cerevisiae slt2 mutants, thus representing a functionalhomologe of the yeast SLT2 gene. This is further supported bythe fact that homozygous C. albicans mkc1�/mkc1� mutantstrains display cell surface alterations and increased sensitivitytoward antifungals that inhibit �-1,3-glucan and chitin synthe-sis (11, 43, 44). Likewise, PKC signaling is involved in responseto caspofungin-imposed perturbations of cell wall biosynthesisin Cryptococcus neoformans (28). The Cryptococcus neoformansMpk1 homologue of yeast Slt2p controls the cellular responseto high temperature, as well as challenge by cell wall synthesisinhibitors such as caspofungin (28). A C. albicans mkc1�/mkc1� strain displays caspofungin hypersensitivity, eventhough C. albicans cells are already at least 10 times moresensitive to caspofungin than yeast (C. Reinoso-Martin et al.,unpublished data). Thus, it appears likely that the response to

caspofungin is conserved between S. cerevisiae, C. albicans, andCryptococcus neoformans, suggesting that the PKC signalingpathway could harbor several potential drug targets for novelantifungals interfering with cell wall function or architecture.

We also analyzed the role of the transcription factors Rlm1pand Swi4p, which are acting downstream of Slt2p (2, 60). Theactivation of Slt2p by caspofungin also results in Rlm1p-de-pendent transcription activation, whereas a lack of Swi4p doesnot reduce the caspofungin-mediated gene regulation, at leastfor the genes analyzed in the present study. Nevertheless, andsomewhat unexpectedly, cells lacking Rlm1p are not caspofun-gin sensitive (Fig. 7B). However, this is consistent with the factthat the rlm1� mutant cells do not show a cell integrity defectand, more importantly, FKS1 levels do not change upon loss ofRlm1p (18). These findings support our data demonstratingthe activation of the PKC pathway by caspofungin but alsoindicate that certain Rlm1p-dependent target genes are notessential for caspofungin tolerance.

The rapid response of the PKC pathway to caspofungin-induced damage requires a highly active sensing machinery.Among several cell surface factors that could potentially signalthrough the PKC pathway, we show that only wsc1� mutants,but not mutants in other cell wall damage sensor proteins, arehypersensitive to caspofungin. Wsc1p localizes to the plasmamembrane and is a member of the WSC family composed offour genes (WSC1 to WSC4) (17, 59). Notably, deletion ofeither ROM2 or WSC1 leads to a defect of �-1,3-glucan syn-thesis (55), which is consistent with our data. Furthermore,Wsc1p colocalizes with Fks1p (9), the actual target of caspo-fungin. The C-terminal cytoplasmic domains of Wsc1p andMid2p interact with Rom2p, a guanine nucleotide exchangefactor for Rho1p (48). However, Mid2p activates Pkc1p with-out affecting �-1,3-glucan synthesis. Given the role of Wsc1p inthe regulation of Fks1p, the caspofungin sensitivity of thewsc1� mutant comes not entirely unexpected. However, wefind that absence of Wsc1p also prevents phosphorylation andactivation of Slt2p. This result supports the notion of a signal-ing feedback from the cell wall via Wsc1p to monitor theactivity of Fks1p. The situation is probably similar to fks1�cells, which have a reduced glucan content of the cell wall andcompensate for this by increasing the chitin content as well asby the higher levels of the second glucan synthase gene, FKS2(42, 65). We and others (10) have shown that fks1� cellsdemonstrate increased basal levels of phospho-Slt2p, a findingthat further supports the existence of a feedback loop. Ourresults suggest a highly dynamic connection between Fks1pand Wsc1p and offer exciting possibilities to address the tan-talizing question as to how the actual sensing is accomplishedby Wsc1p. To address this possible interplay, we constructed awsc1� fks1� strain. Interestingly, a wsc1� fks1� double mu-tant already exhibits a drastic slow-growth phenotype onYPD medium lacking any drugs (data not shown). Thesedata not only show a genetic interaction between WSC1 andFKS1 but also strongly support the role of Wsc1p in sensingcell wall damage that arises from a loss of glucan synthase.Our data suggest that baker’s yeast can sense the presenceof caspofungin through rapid activation of the PKC path-way, leading to the induction of a salvage response to main-tain cell wall integrity. The results may also explain theinherently higher tolerance of baker’s yeast against this

FIG. 7. Caspofungin-mediated activation of the PKC pathway re-quires Rlm1p. (A) Northern analysis of logarithmically growing cul-tures in the presence of caspofungin. Wild-type cells and isogenic cellslacking Rlm1p (rlm1�) or Swi4p (swi4�) were grown at 30°C in theabsence (�) or presence of 10 ng of caspofungin/ml for 30 or 60 min.Total RNA was isolated and fractionated in 1% agarose gels. Theprobes to detect SLT2, HSP150, and CWP1 mRNAs were as describedin Materials and Methods. ACT1 blotting served as a loading control.(B) Identical volumes of 10-fold serial dilutions of exponentially grow-ing wild-type cells and cells lacking transcriptional regulators (swi4�and rlm1�) or Slt2p (slt2�) were spotted onto YPD plates containingvarious concentrations of caspofungin as indicated and then incubatedat 30°C. Colony growth was inspected after a 48-h incubation at 30°C.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

drug. Recent experimental data demonstrate that caspofun-gin also activates the Mkc1p orthologue of Slt2p in C. albi-cans (data not shown). However, we do not know at thispoint whether and how C. albicans cells can counteractcaspofungin-induced cell wall perturbations.

ACKNOWLEDGMENTS

We thank all laboratory members, and especially Yasmine M. Mam-nun, for critical manuscript reading and helpful discussions. MichaelSchuster is acknowledged for help with computational analysis. Wethank Jeremy Thorner, Kim Nasmyth, Maria Molina, Humberto Mar-tın, David Levin, Javier Arroyo, and Frans Klis for providing strains,plasmids, and antibodies or for stimulating discussions about unpub-lished data.

This study was supported by grants from the Austrian Science Foun-dation (P-15934-B08) and the Austrian National Bank (OeNB-9985)to K.K. C.R.-M. is a recipient of an FP5 Marie Curie postdoctoralfellowship from the European Commission.

REFERENCES

1. Abruzzo, G. K., C. J. Gill, A. M. Flattery, L. Kong, C. Leighton, J. G. Smith,V. B. Pikounis, K. Bartizal, and H. Rosen. 2000. Efficacy of the echinocandincaspofungin against disseminated aspergillosis and candidiasis in cyclophos-phamide-induced immunosuppressed mice. Antimicrob. Agents Chemother.44:2310–2318.

2. Baetz, K., J. Moffat, J. Haynes, M. Chang, and B. Andrews. 2001. Transcrip-tional coregulation by the cell integrity mitogen-activated protein kinase Slt2and the cell cycle regulator Swi4. Mol. Cell. Biol. 21:6515–6528.

3. Bickle, M., P. A. Delley, A. Schmidt, and M. N. Hall. 1998. Cell wall integritymodulates RHO1 activity via the exchange factor. ROM2. EMBO J. 17:2235–2245.

4. Bulawa, C. E. 1993. Genetics and molecular biology of chitin synthesis infungi. Annu. Rev. Microbiol. 47:505–534.

5. Cobb, M. H., and E. J. Goldsmith. 1995. How MAP kinases are regulated.J. Biol. Chem. 270:14843–14846.

6. Costigan, C., S. Gehrung, and M. Snyder. 1992. A synthetic lethal screenidentifies SLK1, a novel protein kinase homolog implicated in yeast cellmorphogenesis and cell growth. Mol. Cell. Biol. 12:1162–1178.

7. Costigan, C., D. Kolodrubetz, and M. Snyder. 1994. NHP6A and NHP6B,which encode HMG1-like proteins, are candidates for downstream compo-nents of the yeast SLT2 mitogen-activated protein kinase pathway. Mol. Cell.Biol. 14:2391–2403.

8. Davenport, K. R., M. Sohaskey, Y. Kamada, D. E. Levin, and M. C. Gustin.1995. A second osmosensing signal transduction pathway in yeast. Hypotonicshock activates the PKC1 protein kinase-regulated cell integrity pathway.J. Biol. Chem. 270:30157–30161.

9. Delley, P. A., and M. N. Hall. 1999. Cell wall stress depolarizes cell growthvia hyperactivation of RHO1. J. Cell Biol. 147:163–174.

10. de Nobel, H., C. Ruiz, H. Martin, W. Morris, S. Brul, M. Molina, and F. M.Klis. 2000. Cell wall perturbation in yeast results in dual phosphorylation ofthe Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2-lacZexpression, glucanase resistance and thermotolerance. Microbiology 146:2121–2132.

11. Diez-Orejas, R., G. Molero, F. Navarro-Garcia, J. Pla, C. Nombela, and M.Sanchez-Perez. 1997. Reduced virulence of Candida albicans MKC1 mu-tants: a role for mitogen-activated protein kinase in pathogenesis. Infect.Immun. 65:833–837.

12. Gray, J. V., J. P. Ogas, Y. Kamada, M. Stone, D. E. Levin, and I. Herskowitz.1997. A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiaein bud emergence and identification of a putative upstream regulator.EMBO J. 16:4924–4937.

13. Heinisch, J. J., A. Lorberg, H. P. Schmitz, and J. J. Jacoby. 1999. The proteinkinase C-mediated MAP kinase pathway involved in the maintenance ofcellular integrity in Saccharomyces cerevisiae. Mol. Microbiol. 32:671–680.

14. Igual, J. C., A. L. Johnson, and L. H. Johnston. 1996. Coordinated regulationof gene expression by the cell cycle transcription factor Swi4 and the proteinkinase C MAP kinase pathway for yeast cell integrity. EMBO J. 15:5001–5013.

15. Irie, K., M. Takase, K. S. Lee, D. E. Levin, H. Araki, K. Matsumoto, and Y.Oshima. 1993. MKK1 and MKK2, which encode Saccharomyces cerevisiaemitogen-activated protein kinase-kinase homologs, function in the pathwaymediated by protein kinase C. Mol. Cell. Biol. 13:3076–3083.

16. Iyer, V. R., C. E. Horak, C. S. Scafe, D. Botstein, M. Snyder, and P. O.Brown. 2001. Genomic binding sites of the yeast cell-cycle transcriptionfactors SBF and MBF. Nature 409:533–538.

17. Jacoby, J. J., S. M. Nilius, and J. J. Heinisch. 1998. A screen for upstreamcomponents of the yeast protein kinase C signal transduction pathway iden-tifies the product of the SLG1 gene. Mol. Gen. Genet. 258:148–155.

18. Jung, U. S., and D. E. Levin. 1999. Genome-wide analysis of gene expressionregulated by the yeast cell wall integrity signalling pathway. Mol. Microbiol.34:1049–1057.

19. Kamada, Y., U. S. Jung, J. Piotrowski, and D. E. Levin. 1995. The proteinkinase C-activated MAP kinase pathway of Saccharomyces cerevisiae medi-ates a novel aspect of the heat shock response. Genes Dev. 9:1559–1571.

20. Kamada, Y., H. Qadota, C. P. Python, Y. Anraku, Y. Ohya, and D. E. Levin.1996. Activation of yeast protein kinase C by Rho1 GTPase. J. Biol. Chem.271:9193–9196.

21. Kapteyn, J. C., R. C. Montijn, E. Vink, J. de la Cruz, A. Llobell, J. E. Douwes,H. Shimoi, P. N. Lipke, and F. M. Klis. 1996. Retention of Saccharomycescerevisiae cell wall proteins through a phosphodiester-linked �-1,3-/�-1,6-glucan heteropolymer. Glycobiology 6:337–345.

22. Kapteyn, J. C., H. Van Den Ende, and F. M. Klis. 1999. The contribution ofcell wall proteins to the organization of the yeast cell wall. Biochim. Biophys.Acta 1426:373–383.

23. Kapteyn, J. C., P. Van Egmond, E. Sievi, H. Van Den Ende, M. Makarow,and F. M. Klis. 1999. The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and�-1,6-glucan-deficient mutants. Mol. Microbiol. 31:1835–1844.

24. Ketela, T., R. Green, and H. Bussey. 1999. Saccharomyces cerevisiae Mid2p isa potential cell wall stress sensor and upstream activator of the PKC1-MPK1cell integrity pathway. J. Bacteriol. 181:3330–3340.

25. Klis, F. M. 1994. Cell wall assembly in yeast. Yeast 10:851–869.26. Klis, F. M., P. Mol, K. Hellingwerf, and S. Brul. 2002. Dynamics of cell wall

structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26:239–256.27. Koch, C., and K. Nasmyth. 1994. Cell cycle regulated transcription in yeast.

Curr. Opin. Cell Biol. 6:451–459.28. Kraus, P. R., D. S. Fox, G. M. Cox, and J. Heitman. 2003. The Cryptococcus

neoformans MAP kinase Mpk1 regulates cell integrity in response to anti-fungal drugs and loss of calcineurin function. Mol. Microbiol. 48:1377–1387.

29. Kren, A., Y. M. Mamnun, B. E. Bauer, C. Schuller, H. Wolfger, K. Hatzix-anthis, M. Mollapour, C. Gregori, P. Piper, and K. Kuchler. 2003. War1p, anovel transcription factor controlling weak acid stress response in yeast. Mol.Cell. Biol. 23:1775–1785.

30. Kurtz, M. B., and C. M. Douglas. 1997. Lipopeptide inhibitors of fungalglucan synthase. J. Med. Vet. Mycol. 35:79–86.

31. Lagorce, A., N. C. Hauser, D. Labourdette, C. Rodriguez, H. Martin-Yken, J.Arroyo, J. D. Hoheisel, and J. Francois. 2003. Genome-wide analysis of theresponse to cell wall mutations in the yeast Saccharomyces cerevisiae. J. Biol.Chem. 278:20345–20357.

32. Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsu-moto, and D. E. Levin. 1993. A yeast mitogen-activated protein kinasehomolog (Mpk1p) mediates signaling by protein kinase C. Mol. Cell. Biol.13:3067–3075.

33. Lee, K. S., and D. E. Levin. 1992. Dominant mutations in a gene encoding aputative protein kinase (BCK1) bypass the requirement for a Saccharomycescerevisiae protein kinase C homolog. Mol. Cell. Biol. 12:172–182.

34. Levin, D. E., and E. Bartlett-Heubusch. 1992. Mutants in the Saccharomycescerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect.J. Cell Biol. 116:1221–1229.

35. Levin, D. E., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990.A candidate protein kinase C gene, PKC1, is required for the Saccharomycescerevisiae cell cycle. Cell 62:213–224.

36. Lodder, A. L., T. K. Lee, and R. Ballester. 1999. Characterization of theWsc1 protein, a putative receptor in the stress response of Saccharomycescerevisiae. Genetics 152:1487–1499.

37. Lussier, M., A. M. White, J. Sheraton, T. di Paolo, J. Treadwell, S. B.Southard, C. I. Horenstein, J. Chen-Weiner, A. F. Ram, J. C. Kapteyn, T. W.Roemer, D. H. Vo, D. C. Bondoc, J. Hall, W. W. Zhong, A. M. Sdicu, J.Davies, F. M. Klis, P. W. Robbins, and H. Bussey. 1997. Large scale iden-tification of genes involved in cell surface biosynthesis and architecture inSaccharomyces cerevisiae. Genetics 147:435–450.

38. Madden, K., Y. J. Sheu, K. Baetz, B. Andrews, and M. Snyder. 1997. SBF cellcycle regulator as a target of the yeast PKC-MAP kinase pathway. Science275:1781–1784.

39. Marco, F., M. A. Pfaller, S. A. Messer, and R. N. Jones. 1998. Activity ofMK-0991 (L-743,872), a new echinocandin, compared with those ofLY303366 and four other antifungal agents tested against blood streamisolates of Candida spp. Diagn. Microbiol. Infect. Dis. 32:33–37.

40. Martin, H., J. Arroyo, M. Sanchez, M. Molina, and C. Nombela. 1993.Activity of the yeast MAP kinase homologue Slt2 is critically required for cellintegrity at 37 degrees C. Mol. Gen. Genet. 241:177–184.

41. Martin, H., J. M. Rodriguez-Pachon, C. Ruiz, C. Nombela, and M. Molina.2000. Regulatory mechanisms for modulation of signaling through the cellintegrity Slt2-mediated pathway in Saccharomyces cerevisiae. J. Biol. Chem.275:1511–1519.

42. Mazur, P., N. Morin, W. Baginsky, M. el-Sherbeini, J. A. Clemas, J. B.Nielsen, and F. Foor. 1995. Differential expression and function of twohomologous subunits of yeast 1,3-�-D-glucan synthase. Mol. Cell. Biol. 15:5671–5681.

43. Navarro-Garcia, F., R. Alonso-Monge, H. Rico, J. Pla, R. Sentandreu, and C.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

Nombela. 1998. A role for the MAP kinase gene MKC1 in cell wall construc-tion and morphological transitions in Candida albicans. Microbiology 144:411–424.

44. Navarro-Garcia, F., M. Sanchez, J. Pla, and C. Nombela. 1995. Functionalcharacterization of the MKC1 gene of Candida albicans, which encodes amitogen-activated protein kinase homolog related to cell integrity. Mol. Cell.Biol. 15:2197–2206.

45. Nonaka, H., K. Tanaka, H. Hirano, T. Fujiwara, H. Kohno, M. Umikawa, A.Mino, and Y. Takai. 1995. A downstream target of RHO1 small GTP-bindingprotein is PKC1, a homolog of protein kinase C, which leads to activation ofthe MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 14:5931–5938.

46. Ozaki, K., K. Tanaka, H. Imamura, T. Hihara, T. Kameyama, H. Nonaka, H.Hirano, Y. Matsuura, and Y. Takai. 1996. Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein inSaccharomyces cerevisiae. EMBO J. 15:2196–2207.

47. Peterson, J., Y. Zheng, L. Bender, A. Myers, R. Cerione, and A. Bender. 1994.Interactions between the bud emergence proteins Bem1p and Bem2p andRho-type GTPases in yeast. J. Cell Biol. 127:1395–1406.

48. Philip, B., and D. E. Levin. 2001. Wsc1 and Mid2 are cell surface sensors forcell wall integrity signaling that act through Rom2, a guanine nucleotideexchange factor for Rho1. Mol. Cell. Biol. 21:271–280.

49. Rajavel, M., B. Philip, B. M. Buehrer, B. Errede, and D. E. Levin. 1999. Mid2is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae.Mol. Cell. Biol. 19:3969–3976.

50. Roelants, F. M., P. D. Torrance, N. Bezman, and J. Thorner. 2002. Pkh1 andPkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and defineprotein kinase modules required for maintenance of cell wall integrity. Mol.Biol. Cell 13:3005–3028.

51. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorymanual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.

52. Schmidt, A., M. Bickle, T. Beck, and M. N. Hall. 1997. The yeast phospha-tidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the ex-change factor ROM2. Cell 88:531–542.

53. Schmitt, M. E., T. A. Brown, and B. L. Trumpower. 1990. A rapid and simplemethod for preparation of RNA from Saccharomyces cerevisiae. NucleicAcids Res. 18:3091–3092.

54. Schuetzer-Muehlbauer, M., B. Willinger, G. Krapf, S. Enzinger, E. Presterl,and K. Kuchler. 2003. The Candida albicans Cdr2p ATP-binding cassette

(ABC) transporter confers resistance to caspofungin. Mol. Microbiol. 48:225–235.

55. Sekiya-Kawasaki, M., M. Abe, A. Saka, D. Watanabe, K. Kono, M. Mine-mura-Asakawa, S. Ishihara, T. Watanabe, and Y. Ohya. 2002. Dissection ofupstream regulatory components of the Rho1p effector, 1,3-�-glucan syn-thase, in Saccharomyces cerevisiae. Genetics 162:663–676.

56. Stone, E. A., H. B. Fung, and H. L. Kirschenbaum. 2002. Caspofungin: anechinocandin antifungal agent. Clin. Ther. 24:351–377.

57. Terashima, H., N. Yabuki, M. Arisawa, K. Hamada, and K. Kitada. 2000.Upregulation of genes encoding glycosylphosphatidylinositol (GPI)-attachedproteins in response to cell wall damage caused by disruption of FKS1 inSaccharomyces cerevisiae. Mol. Gen. Genet. 264:64–74.

58. Torres, L., H. Martin, M. I. Garcia-Saez, J. Arroyo, M. Molina, M. Sanchez,and C. Nombela. 1991. A protein kinase gene complements the lytic pheno-type of Saccharomyces cerevisiae lyt2 mutants. Mol. Microbiol. 5:2845–2854.

59. Verna, J., A. Lodder, K. Lee, A. Vagts, and R. Ballester. 1997. A family ofgenes required for maintenance of cell wall integrity and for the stressresponse in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94:13804–13809.

60. Watanabe, M., C. Y. Chen, and D. E. Levin. 1994. Saccharomyces cerevisiaePKC1 encodes a protein kinase C (PKC) homolog with a substrate spec-ificity similar to that of mammalian PKC. J. Biol. Chem. 269:16829–16836.

61. Watanabe, Y., K. Irie, and K. Matsumoto. 1995. Yeast RLM1 encodes aserum response factor-like protein that may function downstream of theMpk1 (Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 15:5740–5749.

62. Watanabe, Y., G. Takaesu, M. Hagiwara, K. Irie, and K. Matsumoto. 1997.Characterization of a serum response factor-like protein in Saccharomycescerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1(Slt2) mitogen-activated protein kinase pathway. Mol. Cell. Biol. 17:2615–2623.

63. Wei, Y. F., B. J. Chen, and L. Samson. 1995. Suppression of Escherichia colialkB mutants by Saccharomyces cerevisiae genes. J. Bacteriol. 177:5009–5015.

64. Zarzov, P., C. Mazzoni, and C. Mann. 1996. The SLT2(MPK1) MAP kinaseis activated during periods of polarized cell growth in yeast. EMBO J.15:83–91.

65. Zhao, C., U. S. Jung, P. Garrett-Engele, T. Roe, M. S. Cyert, and D. E. Levin.1998. Temperature-induced expression of yeast FKS2 is under the dualcontrol of protein kinase C and calcineurin. Mol. Cell. Biol. 18:1013–1022.



http://ec.asm.org/

Dow

nloaded from

http://ec.asm.org/

the yeast protein kinase c cell integrity pathway mediates ...a set of genes from the protein kinase...

Documents