The MAP kinase Mkc1p is activated under different stress conditions in Candida albicans

Federico Navarro-García{dagger}, Blanca Eisman{ddagger}, Sonia M. Fiuza§, César Nombela and Jesús Pla

Dept Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n. E-28040 Madrid, Spain

Correspondence
Federico Navarro-García
fnavarro{at}farm.ucm.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans is an opportunistic pathogen that has adapted to live and grow in the human body as its natural environment. Under these conditions, this fungus faces numerous challenges, including oxidative, osmotic and enzymic processes that may damage external and internal structures. In view of the key role of MAP kinase signalling pathways in the physiology of C. albicans, the effect of agents mimicking in vivo environmental conditions on the activation of the p42-44 MAP kinases has been analysed. It has been found that Mkc1p is phosphorylated in the presence of oxidative stress, changes in osmotic pressure, cell wall damage and a decrease in the growth temperature. This phosphorylation is dependent on Pkc1p, indicating that both proteins operate in the same signalling pathway in C. albicans. Under some stimuli, the phosphorylation of Mkc1p required the presence of Hog1p, the MAP kinase of the high osmolarity glycerol (HOG) pathway. This suggests the existence of a new regulatory role, at least under some conditions, for these MAP kinase pathways in yeast.


Abbreviations: GSNO, S-nitrosoglutathione; HOG, high osmolarity glycerol; SIN-1, 3-morpholinosydnonimine hydrochloride

{dagger}F. Navarro-García dedicates this work to his wife, Marta Muñoz-Fernanz.

{ddagger}Present address: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, E-28049 Madrid, Spain.

§Present address: Unidade I&D ‘Química-Física Molecular’, Faculdade de Ciências e Tecnologia, Univ. Coimbra, 3000 Coimbra, Portugal.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans is a commensal fungus whose natural habitat is the mucous membranes of warm-blooded animals, where it can produce both superficial and difficult-to-treat deep infections (Odds, 1988). In this habitat, the fungus faces numerous challenges involving oxidative and enzymic processes that may damage external and internal structures, and therefore must be able to respond to avoid being killed by the non-specific and specific defences of the human host. To survive, C. albicans needs to continuously monitor the changes taking place in the surrounding environment. In eukaryotes, these external stimuli are usually processed through MAP kinase pathways, in which proteins are sequentially phosphorylated to respond appropriately to external changes.

An in silico analysis of the C. albicans genome (http://genolist.pasteur.fr/CandidaDB/) using the BLAST algorithm and the TEYVATRWYRAPE motif, which is characteristic of the VIII subdomain of the p42-44 MAP kinase family of protein kinases, yields six hits. These hits match the sequence exactly in three cases (Mkc1p, 59 kDa; Cek1p, 48·7 kDa; and Cek2p, 43·3 kDa), and for this reason they can be considered p42-44 homologues. Three additional proteins differ in the TEY signature, namely Hog1p (43 kDa, TGYVSTRYYRAPE) which is considered a p38-type MAP kinase, Smk1p (33 kDa, TNYVATRWYRAPE) and Ipf14035p (59 kDa, TAYVSTRWYRSPE) (bold type indicates the different amino acids). The characterization of Ipf14035p and Smk1p has not been performed, but some studies on the phenotypes of the corresponding mutants have been carried out for the rest of the aforementioned proteins. However, we are currently far from having a complete understanding of the real functions and relationships of these proteins and the pathways in which they operate in C. albicans.

Probably one of the most-studied MAP kinase pathways in C. albicans is the high osmolarity glycerol (HOG) pathway. C. albicans HOG1 has been cloned in budding yeast and complements the lack of growth of a Saccharomyces cerevisiae hog1 mutant in a high-osmolarity medium. Likewise, the C. albicans hog1 mutant has been found to be unable to grow in the presence of high concentrations of extracellular solutes (NaCl, KCl, sorbitol) and also to accumulate internal glycerol in response to an osmotic challenge (San José et al., 1996). It was later found that Hog1p is implicated in morphogenesis, since hog1 mutants form filaments even under non-inducing conditions. Hog1p has a significant influence on virulence, as hog1 mutants are almost completely avirulent (Alonso-Monge et al., 1999). Recent findings show that the HOG signalling pathway is responsible for responding to oxidants in C. albicans by phosphorylating Hog1p (Alonso-Monge et al., 2003). There are some other cloned elements of the putative HOG pathway in C. albicans that are homologous to those of S. cerevisiae, such as HK1, NIK1 and SLN1 (encoding two-component protein-kinase membrane receptors) (Calera et al., 1998; Nagahashi et al., 1998; Srikantha et al., 1998), YPD1 and SSK1 (encoding two-component protein kinases) (Calera et al., 2000; Calera & Calderone, 1999), and the MAP kinase kinase PBS2 (Arana et al., 2005). Nevertheless, the relationship of Hog1p to some of these proteins has only recently been substantiated through the analysis of Hog1p phosphorylation in certain mutants in the presence of different stimuli (Chauhan et al., 2003; Arana et al., 2005). Hog1p is phosphorylated in the presence of NaCl, H2O2 (Alonso-Monge et al., 2003) and a range of other substances, such as farnesol and staurosporine (Smith et al., 2004).

The CEK1 gene has been discovered to be a suppressor of the cell cycle arrest induced by the {alpha} pheromone in S. cerevisiae (Whiteway et al., 1992). CEK1 disruption reduces both hyphae formation in C. albicans, in the presence of serum and different carbon sources, and virulence in different experimental models of infection (Csank et al., 1998; Guhad et al., 1998). This illustrates the importance of this MAP kinase, homologous to the S. cerevisiae MAP kinases Fus3p and Kss1p, in the course of an infection. The deletion of putative upstream and downstream elements of the same MAP kinase pathway, such as HST7 (S. cerevisiae STE7 homologue), CST20 (S. cerevisiae STE20 homologue) and CPH1 (S. cerevisiae STE12 homologue), also affects virulence and hyphal formation (Liu et al., 1994; Köhler & Fink, 1996; Leberer et al., 1996). The relationship between all these elements has been studied through the analysis of the phenotypes of single and double mutants (Csank et al., 1998), but their influence on Cek1p activation has not yet been tested.

Cek2p, together with Cek1p, plays an important role in the mating process. CEK2 is expressed only in MTL{alpha} cells, but not in cells of MTLa/{alpha}, which is the common sexual type of wild-type strains. cek1 cek2 strains are completely defective in mating (Chen et al., 2002). In addition, other C. albicans genes encoding proteins similar to those of the mating pathway of S. cerevisiae work in this pathway (Magee et al., 2002).

The MKC1 gene has been cloned by complementation of the growth defect of S. cerevisiae slt2 mutants at high temperatures (Navarro-García et al., 1995). SLT2/MPK1 encodes the MAP kinase of the cell integrity signalling pathway in S. cerevisiae. C. albicans mkc1{Delta} mutants display a higher susceptibility to lytic enzyme preparations, to high temperatures and to cell wall antifungals than wild-type strains. Furthermore, the cell wall composition of these mutant cells is different from that of the wild-type cells. All these findings point to a role for Mkc1p in the construction of the cell wall (Navarro-García et al., 1995, 1998). In addition, it has been established that Mkc1p is necessary for virulence in a systemic mouse model of infection (Diez-Orejas et al., 1997). In S. cerevisiae, Pkc1p controls the cell integrity signalling pathway, being upstream of the MAP kinase module. In a similar manner to S. cerevisiae, C. albicans pkc1 mutants have been shown to be much more fragile than mkc1{Delta} mutants, as they are unable to survive in the long term without osmotic protection (Paravicini et al., 1996). In contrast to the HOG pathway, little is known about the physiological conditions that might activate Mkc1p, such as stimuli and the epistatic relationship with PKC1. In an effort to shed light on this question, we have determined the phosphorylation state of the p42-44 MAP kinases under certain stress conditions, such as temperature, osmotic pressure, cell wall damage and oxidizing agents. In addition, information regarding the stimuli that activate other MAP kinases is presented.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
Yeast strains are listed in Table 1. For clarity, and unless otherwise stated, mkc1{Delta} indicates the mkc1{Delta}/mkc1{Delta} Ura+ strain (CM1613), hog1 indicates the hog1/hog1 Ura+ strain (CNC13), cek1{Delta} indicates the cek1{Delta}/cek1{Delta} Ura+ strain (CK43B-16), cek2{Delta} indicates the cek2{Delta}/cek2{Delta} Ura+ strain (BEC73) and pkc1{Delta} indicates the pkc1{Delta}/pkc1{Delta} Ura+ strain (AM-2) (Table 1). RM100 was the wild-type strain in control experiments, since it behaved identically to CAF2 or SC5314 strains with respect to MAP kinase activation in control experiments (data not shown).


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Table 1. Strains used in this work

 
Yeast strains were grown in YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose) or SD minimal medium (2 % glucose, 0·67 % yeast nitrogen base without amino acids), with the appropriate additions depending on auxotrophic requirements. Histidine and uridine were routinely added to liquid and solid media (final concentration 50 µg ml–1). The pkc1{Delta} strain was routinely grown on media supplemented with 0·5 or 1 M sorbitol to allow normal growth. Growth was routinely performed at 37 °C (unless otherwise indicated) in water baths. Usually, overnight cultures were refreshed to OD600 0·05, and experiments were performed when cultures reached OD600 1. After oxidative treatments, culture viability was high (>80 %), as indicated by propidium iodide staining and as shown in a previous study (Alonso-Monge et al., 2003).

Susceptibility to H2O2, menadione, diamide, calcofluor white, Congo red and cold-shock was tested using exponentially growing cells in YPD medium at 37 °C. Cells (2·5x107 ml–1) were transferred to 96-well plates and 1/10 dilutions were performed. Samples (5 µl) were spotted onto YPD plates with the appropriate supplements, incubated at 37 °C for 24 h (except for the cold-shock experiment) and examined.

Calcofluor white was a generous gift from the Bayer Corporation. Amphoterycin B, Congo red, diazinedicarboxylic acid bis(N,N-dimethylamide)(diamide), glycerol, H2O2, menadione sodium bisulfite, 3-morpholinosydnonimine hydrochloride (SIN-1), nikkomycin Z, S-nitrosoglutathione (GSNO) and sorbitol were from Sigma-Aldrich. Zymolyase 20-T was from ICN Biochemicals. Antifungals were generous gifts from Lilly (cilofungin), Pfizer (fluconazol) and Merck (pneumocandin B0 L-688786). Solutions of antifungals and other stress-inducing substances were prepared using the smallest possible volume of sterile double-distilled H2O and YPD or, when liquid, were added directly to the media. Substances were added at the same temperature as the culture and in such a way as to avoid alteration of the nutrient medium concentration and/or volume.

CEK2 disruption.
Disruption of the C. albicans CEK2 gene was performed using the Ura blaster protocol (Fonzi & Irwin, 1993), replacing the 1116 bp ORF with a 4·03 kb SalI–NotI fragment obtained from plasmid pDCEK2. This plasmid bears a cat-URA3-cat cassette flanked by a 335 bp fragment from the CEK2 5' region and a 399 bp fragment from the CEK2 3' region. The 5' region was obtained by PCR amplification using primers MKC3-1 (5'-CCTCGTCGACATGAAGACATCTACTGGCCCCAC-3') and MKC3-2 (5'-GTTTAAGCTTCATGTATTCCTGAATAAGATA-3'). The 3' fragment was obtained using primers MKC3-3 (5'-CAGCCACTAGTCAACCGATGAAGACTTGCAG-3') and MKC3-4 (5'-CTTAGTGCGGCCGCTTACGACATGACTATTTCGAAAATT-3'). Both fragments were inserted into pD1 to obtain pDCEK2. pD1 was obtained by ligation of a blunt-ended BamHI-opened SkURACf2 plasmid and a copy of the chloramphenicol acetyltransferase (cat) gene obtained as a HindII–EcoRV fragment from SkCf (R. Alonso-Monge, personal communication). SkURACf2 was obtained by introduction of a HindII–HindIII blunt-ended fragment from SkCf into SkURA1. SkURA1 was obtained by ligation of the URA3 gene extracted as a blunt-ended XbaI–ScaI fragment from pJPDM19 (Pla et al., 1995) into the SmaI site of Bluescript.

C. albicans RM1000 was transformed using standard transformation protocols (Köhler et al., 1997). Transformants were selected and analysed for correct integration into the CEK2 locus by PCR and Southern blot. Correct disruptions of the CEK2 locus were confirmed by PCR using primers MKC3c (5'-GGTGACTAATTTGATCCAGTAATATTG-3') and o-URA3CR (5'-GTGTTACGAATCAATGGCACTACAGC-3') for the Ura+ strains or MKC3c and CF1 (5'-CTCACCGTCTTTCATTGCCATA-3') for Ura strains. Southern blot was performed using ClaI-digested genomic DNA and hybridized with a DIG-labelled probe obtained using primers MKC3c and MKC3-2.

MKC1 reintegration.
MKC1 was reintegrated by inserting a 3·48 kb EcoRV fragment from pSN6 (Navarro-García et al., 1995) into an SmaI-opened pRM1, a C. albicans URA3 replicative plasmid (Pla et al., 1995), to generate plasmid pRMKC10. C. albicans CM1613c (mkc1{Delta}/mkc1{Delta} ura3/ura3) was transformed by standard transformation protocols (Köhler et al., 1997) using either circular pRMKC10 or ClaI-digested pRMCK10. In the latter case, transformants were selected and analysed for correct integration into the MKC1 locus by PCR and Southern blot. Both types of transformants behaved in a similar manner to wild-type strains with respect to phenotypes such as calcofluor white and Congo red resistance. Correct integration was checked by PCR using primers locMKC1up (5'-TAACATGAAGAAAAGGTTATTGGG-3') and locMKC1lower (5'-TACGGATCTGCCATAATATATGGG-3'). Southern blot was performed using XbaI-digested genomic DNA and hybridized with a 0·9 kb KpnI–SmaI DIG-labelled probe, as previously described (Navarro-García et al., 1995).

Obtaining anti-Mkc1 antibodies.
Recombinant Mkc1 protein was expressed and used to immunize rabbits. Briefly, the C. albicans MKC1 ORF was amplified and cloned into the pGEX2T expression vector (Amersham Pharmacia Biotech). The GST fusion protein was expressed in E. coli and excised from gels after SDS-PAGE. Two New Zealand White male rabbits were immunized with the recombinant protein emulsified in 0·5 ml of Freund's complete adjuvant, followed by five booster doses with protein emulsified in Freund's incomplete adjuvant. Immunizations were administered intradermally every week for a total of six immunizations. Blood was collected for antibody analyses prior to and 1–7 weeks after challenge.

Protein extracts and immunoblot analysis.
Yeast strains were grown to OD600 0·8–1 at 37 °C in 25 ml YPD cultures. Stress-inducing substances were added to growing cells as required, but as a general rule, and unless otherwise indicated, a ten-minute treatment was used in most of the experiments before samples were collected. Cell extracts were obtained basically as described previously (Martín et al., 2000), but with several modifications. After treatment, whole cultures (or 5 ml volumes for time-lapse experiments) were collected in sterile cold Falcon tubes half-filled with ice, and harvested at 2500 g and 4 °C for 2 min. Supernatants were discarded and cell pellets were transferred to cold Eppendorf tubes and washed with ice-cold water, and then centrifuged twice at 14 000 g and 4 °C for 2 min, discarding the supernatant. Finally, pellets were dry stored at –80 °C, after liquid nitrogen freezing, until further processing.

For protein extraction, cells were resuspended in lysis buffer and broken using a glass bead protocol. Lysis buffer contained 50 mM Tris/HCl, pH 7·5, 10 % glycerol (v/v), 1 % Triton X-100, 0·1 % SDS, 150 mM sodium chloride, 50 mM sodium fluoride, 10 mM sodium orthovanadate, 50 mM {beta}-glycerol phosphate, 50 mM sodium pyrophosphate, 5 mM EDTA, pH 8, 1 mM PMSF, 25 µg ml–1 N-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), 25 µg ml–1 N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), 25 µg pepstatin A ml–1, 25 µg leupeptin ml–1, 25 µg antipain ml–1 and 25 µg aprotinin ml–1. Cell extracts were obtained using glass beads in a fast prep cell breaker (FastPrep FP120, Bio 101 Savant) applying three 30 s rounds at 5·5 speed with intermediate ice coolings. Extracts were centrifuged at 14 000 g and 4 °C for 15 min and the supernatants were collected and stored at –80 °C until further use.

Equal amounts of proteins, usually 150 µg per lane (as assessed by 280 nm measurement and Ponceau red staining of the membranes prior to blocking and detection), were loaded onto 10 % SDS-PAGE, separated and transferred to Hybond membranes (Amersham Pharmacia) using the Bio-Rad II miniprotean system. Blots were probed with anti-phospho-p38 MAP kinase (Thr180/Tyr182) antibody (Anti-p38-P in the figures), anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody (Anti-p42-44-P) (Cell Signalling Technology, Inc.), anti-ScHog1 polyclonal antibody (Anti-HOG1) (Santa Cruz Biotechnology) and anti-GST-Mkc1p antibody (Anti-MKC1) using 1 : 2000 dilutions, and anti-ScKss1 polyclonal antibody (Anti-KSS1) (Santa Cruz Biotechnology) at 1 : 75 dilution. Primary antibodies were detected using a horseradish peroxidase-conjugated anti-rabbit IgG and developed according to the manufacturer's recommendations using the Hybond ECL kit (Amersham Pharmacia Biotech).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mkc1p phosphorylation is triggered by oxidative stress
In order to understand the stimuli that increase the phosphorylation of Mkc1p, the MAP kinase of the cell integrity pathway, we used commercial antibodies raised against the phosphorylated forms of p42-44 MAP kinases (phosphorylated at the threonine and tyrosine residues of the TEY signature of mammalian ERK1 and ERK2). These antibodies recognize the phosphorylated forms of S. cerevisiae Slt2p, Fus3p and Kss1p (p42-44-related MAP kinases) (Bardwell et al., 1996; Martín et al., 2000), Schizosaccharomyces pombe Spm1 (Zaitsevskaya-Carter & Cooper, 1997) and Ustilago maydis Crk1 (Garrido et al., 2004). In C. albicans, these antibodies should also detect the simultaneous phosphorylation at the threonine and tyrosine residues of the TEY signature of the Mkc1p, Cek1p and Cek2p proteins (a motif absent in other C. albicans MAP kinases). First, we checked the effect of H2O2, which has been shown to increase the phosphorylation of C. albicans Hog1p (Alonso-Monge et al., 2003). Cells growing in exponential phase were exposed to a challenge of 10 mM H2O2 and their protein extracts were analysed by Western blot using these antibodies. We detected two bands corresponding to TEY-phosphorylated proteins (Fig. 1A). The mobility of the upper band (61 kDa) corresponded to the expected molecular mass of Mkc1p (59 kDa) (Fig. 1A). Moreover, this band disappeared in an mkc1{Delta} mutant and reappeared in two different MKC1 reintegrant strains under inducing conditions (Fig. 1A). Furthermore, when using a specific polyclonal antibody raised against Mkc1p, a unique band of the same mobility (61 kDa) was detected (Fig. 1A). This band disappeared when extracts of the mkc1{Delta} mutant were used, reappearing when MKC1 was reintroduced in the genome (Fig. 1A). Thus, this band corresponds to Mkc1p which is phosphorylated in the presence of H2O2. The lower band observed in the blots treated with anti-phosphorylated p42-44 antibodies (49 kDa) likely corresponds to the phosphorylated form of Cek1p, since it presented the expected mobility on SDS-PAGE for this protein (48·7 kDa) (Cek2p is 43·3 kDa), and it is absent in a cek1{Delta} mutant (see below, zymolyase treatment, Fig. 5B).



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Fig. 1. Mkc1p is activated in the presence of H2O2, but is not necessary for survival under this condition. (A) Mkc1p is phosphorylated in the presence of H2O2. Western blotting analysis of protein extracts from the indicated strains after a ten-minute treatment with 10 mM H2O2. (B) Sensitivity of the indicated strains to oxidizing agents. Exponentially growing cultures were diluted to obtain cell suspensions of appropriate cell concentrations. Volumes of 5 µl were spotted onto YPD or YPD+0·5 M sorbitol plates containing different oxidants at the concentrations indicated.

 


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Fig. 5. Mkc1p is activated in the presence of caffeine and zymolyase, but not at high temperature. (A) MAP kinase activation in response to caffeine, zymolyase and high temperature. Western blotting of protein extracts of wild-type cells collected after different treatments: Zymolyase 20T (2 units ml–1) for 30 min (Z-20T); 25 mM caffeine for 2 h (Caf) and 42 °C for 10 min (42 °C). (B) MAP kinase activation by Zymolyase 20T in cek1{Delta} and cek2{Delta} strains. Western blotting of protein extracts of cek1{Delta} and cek2{Delta} strains growing in YPD after a thirty-minute treatment with 2 units ml–1 Zymolyase 20T. –, No treatment; +, zymolyase 20T treatment.

 
Since Mkc1p is phosphorylated in the presence of H2O2, Mkc1p could be necessary to survive in the presence of oxidants. In order to test this hypothesis, we analysed the cell viability of mkc1{Delta} mutants, and also hog1 and pkc1{Delta} mutants, in the presence of H2O2, menadione (a superoxide-generating oxidant) and diamide, a thiol-oxidizing agent that causes oxidative stress by multiple mechanisms, including the enlargement of cell wall pores (de Souza & Geibel, 1999) and the depletion of cellular levels of reduced glutathione. As shown in Fig. 1(B), mkc1{Delta} mutants only showed sensitivity to diamide. Sensitivity to oxidants was reduced when sorbitol was included in the plates, except for the pkc1{Delta} mutants in the presence of diamide (Fig. 1B). Therefore, Mkc1p is phosphorylated in response to H2O2, but it is not necessary for cell survival in the presence of H2O2.

We next checked the phosphorylation status of MAP kinases in C. albicans using the above-mentioned oxidants and NO generators, such as S-nitrosoglutathione (GSNO), 3-morpholinosydnonimine hydrochloride (SIN-1), which also produces the superoxide anion, and peroxynitrite (ONOO). We detected an increased phosphorylation of Mkc1p in the presence of all these substances, while Hog1p was only phosphorylated in the presence of H2O2 and SIN-1 (Fig. 2A–D). Thus, the cell integrity pathway seems to be involved in the response to a diverse range of oxidants. Furthermore, Mkc1p was not phosphorylated in a pkc1{Delta} mutant in the presence of any of these agents except for diamide (Fig. 2D and data not shown). Strikingly, the phosphorylation of Mkc1p was dependent on the presence of Hog1p (Fig. 2A, C and D), except in those cases where menadione and diamide were present (Fig. 2B, D).



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Fig. 2. Mkc1p is activated in the presence of different oxidants. (A) Mkc1p and Hog1p are phosphorylated in a range of concentrations of H2O2. Western blotting of protein extracts from the indicated strains after a ten-minute treatment with different concentrations of H2O2. (B) Mkc1p is activated in the presence of menadione. Western blotting of protein extracts from the indicated strains after a ten-minute treatment with different concentrations of menadione. (C) Mkc1p, but not Hog1p, is activated in the presence of S-nitrosoglutathione (GSNO). Western blotting of protein extracts from the indicated strains after a ten-minute treatment with different concentrations of GSNO. (D) MAP kinase activation in the presence of diamide and 3-morpholinosydnonimine hydrochloride (SIN-1). Western blotting of protein extracts from the indicated strains after a ten-minute treatment with different concentrations of diamide or SIN-1. D, diamide; S, SIN-1; H, 10 mM H2O2.

 
Mkc1p responds to changes in osmotic pressure
Oxidants could affect the cell wall, and sorbitol could rescue the cells through cell wall stabilization (as shown in Fig. 1B), since it is firmly established that sorbitol protects against cell wall challenges (Torres et al., 1991). In S. cerevisiae, sorbitol prevents the phosphorylation of Slt2p at high temperatures due to the physical stabilization of the cell surface (Martín et al., 2000). Could sorbitol also preclude the H2O2 phosphorylation of Mkc1p (and/or Hog1p) in C. albicans? In order to test this, we analysed the Mkc1p phosphorylation of cells treated with 1 M sorbitol in a ten-minute treatment (S1M), the same treatment plus 10 mM H2O2 for the last 5 min (S1M H) or 20 mM H2O2 plus 1 M sorbitol for the last 5 min (H S1M). Surprisingly, in the absence of any other stimulus, Mkc1p, but not Hog1p, was phosphorylated in the presence of sorbitol (Fig. 3A). The addition of 1 M glycerol mimicked the effect of sorbitol (Fig. 3A). Interestingly, the absence of Hog1p blocked the phosphorylation of Mkc1p in both cases (Fig. 3A). Therefore, the results impeded checking if sorbitol prevents the H2O2-induced phosphorylation of Mkc1p.



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Fig. 3. Mkc1p is phosphorylated due to changes in osmotic pressure. (A) Mkc1p, but not Hog1p, is phosphorylated in the presence of 1 M sorbitol. Western blotting of protein extracts from the indicated strains after 10 min treatments shown as follows. A 10 ml volume of growing culture in YPD was supplemented with: –, 10 ml YPD; S1M, 10 ml YPD+2 M sorbitol; S1M H, 10 ml YPD+2 M sorbitol and 5 min later H2O2 to a final concentration of 10 mM; H S1M, H2O2 to a final concentration of 20 mM and 5 min later 10 ml YPD+2 M sorbitol; 1 M glycerol, 10 ml YPD+2 M glycerol. Sorbitol (2 M) or glycerol was added to the medium to obtain a final concentration of 1 M, since the added volume and the culture volume were equal. (B) A hyposmotic shock, as well as an oxidative shock, activates Mkc1p under conditions of osmotic stabilization. Western blotting of protein extracts from wild-type and pkc1{Delta} strains after 10 min treatments. For oxidative shock, Wt and pkc1{Delta} strains were grown in YPD+S1M medium and H2O2 was added to a final concentration of 10 mM. For hyposmotic shock, wild-type or pkc1{Delta} strains were grown in YPD plus 1 M sorbitol and centrifuged for 10 s at 4000 g and resuspended in either pre-warmed fresh YPD (YPD) or YPD+S1M (S1M) and incubated again for 10 min before collection for protein extraction. (C) Mkc1p is activated in the presence of calcium. Western blotting of protein extracts from the indicated strains after a ten-minute treatment with 100 mM CaCl2. (D) Mkc1p is phosphorylated under different concentrations of calcium and magnesium. Western blotting of protein extracts from a wild-type strain after a ten-minute challenge with different concentrations of CaCl2 and MgCl2.

 
We hypothesized that a prolonged incubation in culture medium supplemented with 1 M sorbitol would lead to the adaptation of the pathway and, therefore, to a low level of phosphorylation of Mkc1p. Then, the effect of an oxidative challenge could be detected in osmotically stabilized cells. For this purpose, mid-log cells grown in YPD supplemented with 1 M sorbitol were treated with 10 mM H2O2 for 10 min. As can be observed in Fig. 3(B), the oxidative stress increased the phosphorylation level of Mkc1p. This result indicates that the osmotic stabilization does not prevent the phosphorylation of Mkc1p upon oxidative challenge, suggesting that the phosphorylation could be attributed to alterations other than cell wall structural damage. In addition, we were able to determine that the phosphorylation of Mkc1p depends on Pkc1p upon either an oxidative (pkc1 H2O2 lane) or a hyperosmotic challenge (pkc1 lane) (Fig. 3B).

Since the opposite change in the medium osmolarity, hypo-osmotic shock, has been shown to activate the cell integrity pathway in S. cerevisiae (Davenport et al., 1995), we tested it in C. albicans. As can be observed in Fig. 3(B), when cells growing in YPD+1 M sorbitol were shifted to YPD, both Mkc1p and Hog1p were phosphorylated (Wt YPD lane). In addition, the hypo-osmotic shock effect on Mkc1p was found to be PKC1-dependent (pkc1 S1M and pkc1 YPD lanes). All these findings suggest that Mkc1p is involved in the response to osmolarity changes in the surrounding medium.

In order to check if the response of Mkc1p to 1 M sorbitol is an example of a general response to osmotic shock, we treated C. albicans cells with ionic extracellular solutes. The presence of 1 M or 2 M KCl or NaCl failed to phosphorylate Mkc1p, contrary to that which occurred with Hog1p (data not shown). Therefore, the Mkc1p pathway seems to respond selectively to certain osmotic stresses.

Since we previously demonstrated that mkc1{Delta} mutants are sensitive to high concentrations of Ca2+ (Navarro-García et al., 1995), we tested the effect of this divalent ion in the activation of Mkc1p by adding CaCl2 to exponentially growing cells in YPD medium. As shown in Fig. 3(C), Mkc1p was phosphorylated in the presence of CaCl2, the effect being dependent on the presence of Hog1p. In fact, concentrations as low as 25 mM were enough to lead to the phosphorylation of Mkc1p (Fig. 3D), suggesting that the effect was non-osmotic and that there was a specific mechanism for the activation by calcium.

Cell wall perturbations increase the phosphorylation of Mkc1p in a PKC1-dependent manner
Following previous results that suggested that the Mkc1p pathway is involved in cell wall construction (Navarro-García et al., 1995, 1998), we hypothesized that the alteration of the cell wall would lead to the activation of this pathway. To test this, we checked the phosphorylation state of Mkc1p after treatment with the cell-wall-disrupting agents calcofluor white and Congo red. After 2 h, an increase in the phosphorylation of Mkc1p was detected (Fig. 4A). Interestingly, the phosphorylation of Mkc1p was not blocked in a hog1 mutant, and was in fact increased compared to a wild-type strain, a result that could explain the augmented resistance of this mutant to both substances [Fig. 1(A) and Alonso-Monge et al. (1999)]. The activation of the Mkc1p-mediated pathway following cell wall alterations is consistent with the increased sensitivity of pkc1{Delta} and mkc1{Delta} mutants to these substances, which can be alleviated by the addition of sorbitol (Fig. 4B).



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Fig. 4. Mkc1p is activated by cell wall challenges. (A) Mkc1p is phosphorylated in the presence of calcofluor white (CW) and Congo red (CR). Western blotting of protein extracts of the indicated strains grown in YPD supplemented with 20 µg ml–1 CW or CR and incubated for 2 h before recovery and processing. (B) Sensitivity of MAP kinase mutants to calcofluor white (CW) and Congo red (CR). Exponentially growing cultures were diluted to obtain cell suspensions of appropriate cell concentrations. A volume of 5 µl was spotted onto YPD or YPD+1 M sorbitol (Sorb.) plates with or without calcofluor white (CW) or Congo red (CR) at the concentrations indicated in the figure. (C) Mkc1p is activated in the presence of antifungal substances. Western blotting of protein extracts of wild-type cells after a ten-minute treatment with 5 µg ml–1 of different fungicides: Amph, amphotericin B; Cilo, cilofungin; Fluco, fluconazol; Nikko, nikkomycin Z; PNB0, pneumocandin B0 (L-688786-000Z041).

 
mkc1{Delta} mutants have also been described to be more sensitive to antifungal agents that affect cell wall construction, such as cilofungin, echinocandin and nikkomycin Z (Navarro-García et al., 1998). Upon treatment with these substances, Mkc1p was phosphorylated, except in the case of cilofungin (Fig. 4C). Moreover, substances interfering with the structure (amphotericin B) or the synthesis (fluconazol) of the cell membrane also increased the phosphorylation of Mkc1p.

mkc1{Delta} mutants have been shown to be more sensitive to caffeine, zymolyase and high temperature (Navarro-García et al., 1995, 1998), conditions that have also been shown to affect the yeast cell surface. Mkc1p was phosphorylated in the presence of caffeine and zymolyase 20T (Fig. 5A), as it was dependent on HOG1 (data not shown). Surprisingly, high-temperature treatments did not lead to Mkc1p phosphorylation (Fig. 5A), even when different high temperatures (42–55 °C) and periods of time (1 min to 4 h) were tested (data not shown). In contrast to the other conditions tested so far, Cek1p was phosphorylated upon treatment with zymolyase 20T (Fig. 5A). Additionally, under this condition, another band was clearly detected with a molecular mass over 36·5 kDa (Fig. 5A). In order to investigate the nature of this lower-molecular-mass band, we subjected cek1{Delta} and cek2{Delta} MAP kinase mutants to this treatment, since the predicted molecular mass of Cek2p is 43·3 kDa. Strikingly, the appearance of this lower-molecular-mass band was dependent on the presence of CEK1 (Fig. 5B). A possibility that we are currently exploring is that this smaller band is a C-terminal fragment of Cek1p produced by protein processing of the full-size version of Cek1p, since no alternative ATG codon exists in CEK1. Consistent with this hypothesis, these two bands were detected using a commercial antibody raised against the C-terminus of the S. cerevisiae MAP kinase Kss1p, which is homologous to Cek1p (Fig. 5B).

Mkc1p is phosphorylated at low temperatures
The absence of phosphorylation in Mkc1p after a shift to high temperatures (Fig. 5A) could suggest that this pathway is not involved in the response to temperature changes. Alternatively, C. albicans may not sense an increase in temperature as a stressful condition. In order to distinguish between these two possibilities, we performed experiments in which C. albicans cultures growing at 37 °C were shifted to lower temperatures. The results shown in Fig. 6(A) favour the second hypothesis, since Mkc1p was phosphorylated upon exposure to these changes. This effect was found to be dependent on PKC1 (data not shown). Nevertheless, mkc1{Delta} mutants were not cold-sensitive, while pkc1{Delta} mutants displayed reduced growth ability at 15 °C, a phenotype that could be suppressed by adding 1 M sorbitol to the medium (Fig. 6B).



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Fig. 6. Lowering the growth temperature promotes Mkc1p phosphorylation. (A) MAP kinase activation at low temperatures. Western blotting of protein extracts of wild-type or mkc1{Delta} cells growing at 37 °C, shifted to water baths at the indicated temperatures and incubated for a further 10 min before cell recovery. (B) pkc1{Delta} mutants are cold-sensitive. Exponentially growing cultures were diluted to obtain suspensions at appropriate cell concentrations. A volume of 5 µl was spotted onto YPD or YPD+1 M sorbitol (Sorb.) plates. Plates were incubated at the indicated temperatures for 4 days.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Although knowledge of different phenotypic aspects of C. albicans MAP kinase mutants is extensive, we lack information about the real functional activity of these proteins in different situations. This aspect is especially marked in the case of p42-44 MAP kinases, in contrast to p38 MAP kinases, the activity of which is better understood (Alonso-Monge et al., 2003; Smith et al., 2004). To understand the physiological roles of the p42-44 MAP kinases, we studied different stimuli that lead to the phosphorylation of these MAP kinases. A common approach to detect the phosphorylation of MAP kinases is the use of antibodies raised against the phosphorylated forms of these kinases. According to previous work with the MAP kinases of different organisms, phosphorylation of the threonine and tyrosine residues in the TEY signature of domain VIII of the p42-44 MAP kinase family is necessary for phosphorylation activity. When both residues are dephosphorylated, p42-44 MAP kinases are unable to phosphorylate their substrates (Anderson et al., 1990; Nishida & Gotoh, 1993; Flury et al., 1997). Accordingly, antibodies against the phosphorylated forms of p42-44 MAP kinases are used in many eukaryotic organisms (fungal, mammalian and plant) as indicators of the activation of these MAP kinases. This is the approach we used to detect and determine the phosphorylation and thereby the activation of different p42-44 MAP kinases, mainly Mkc1p, in C. albicans.

After analysing the results presented here, several main conclusions could be drawn: first, Mkc1p participates in the response to different kinds of stress in the cell, including osmotic, oxidative and cell wall stress; second, Pkc1p, as well as Hog1p, controls the activation of Mkc1p; and third, the activation of Mkc1p triggered by some specific types of stress, frequently used in the laboratory, must be carefully interpreted from an ecological point of view.

Here, we show for the first time that Mkc1p, as well as Hog1p, is phosphorylated in the presence of oxidative stress. In accordance with the putative involvement of the Mkc1p pathway in the maintenance of the cell wall, cell wall damage would be a reasonable trigger for the oxidation-mediated activation of Mkc1p. Nevertheless, this is not the case, because the presence of sorbitol in the medium does not prevent Mkc1p and Hog1p phosphorylation (Fig. 3A, B). What would be the purpose of the Mkc1p activation under these conditions? A very appealing explanation is that C. albicans adopts survival mechanisms similar to those of the host cells. In macrophages, ERK1/2 kinases are activated in the presence of biologically active NO derivatives. This activation would trigger self-protection, since the absence of the activity of these kinases in the macrophage leads to apoptosis in the presence of GSNO (Callsen & Brune, 1999). Since C. albicans undergoes apoptosis in the presence of oxidative stress (Phillips et al., 2003), Mkc1p phosphorylation could avoid apoptosis in the presence of NO radicals generated by macrophages. This activation could contribute to the survival rate of 80 % of C. albicans after macrophage endocytosis (Ibata-Ombetta et al., 2001), a process that increases the expression of ROS detoxifying genes and DNA-damage repair genes (Lorenz et al., 2004). In fact, mkc1{Delta} mutants are more virulent in a NO-deficient mouse strain (DBA/2) than in a NO-competent strain (Diez-Orejas et al., 1997). We are performing experiments to detect the response of Mkc1p and other C. albicans MAP kinases to macrophages and other cells of the immune system. Preliminary experiments indicate that Mkc1p is activated in the presence of activated macrophages thus suggesting the importance of the Mkc1p MAP kinase pathway in C. albicans in producing an infection and overcoming the immune system attack.

Using different oxidative, osmotic and cell wall stresses, we have established for the first time that Pkc1p and Mkc1p work in the same pathway in C. albicans, since the absence of PKC1 precludes the activation of Mkc1p. Nevertheless, contrary to S. cerevisiae Slt2p (Vilella et al., 2005), diamide activates Mkc1p even in a pkc1{Delta} mutant, and mkc1{Delta} mutants are more sensitive to this agent. Diamide could directly affect either Mkc1p or elements of the cell integrity pathway other than Pkc1p. This situation has already been detected in S. cerevisiae, in which ‘lateral inputs' can intrude into the cell integrity pathway (Harrison et al., 2004).

Thus, we have confirmed that Mkc1p and Pkc1p are elements of the cell integrity pathway, since they are necessary to appropriately respond to cell wall injuries. In addition, since it is activated following treatment with zymolyase, Cek1p could play a role in a putative STE vegetative growth (SVG) pathway. In S. cerevisiae, both pathways collaborate in the maintenance of cell wall integrity (Lee & Elion, 1999).

Moreover, the dependence of cell integrity MAP kinase activation on the presence of the HOG MAP kinase in response to certain stimuli is a completely new finding in yeasts. A possible explanation could be the alteration of the sensors that activate the Mkc1p pathway. Recently, in S. cerevisiae, it has been shown that the incorrect O-mannosylation of the Wsc1p, Wsc2p and Mid2p receptors in pmt2{Delta} pmt4{Delta} mutants prevents the activation of Slt2p upon exposure to elevated temperature (Lommel et al., 2004). In C. albicans, Hog1p might control the expression of the proteins involved in glycosylation of the receptors that activate the Mkc1p MAP kinase pathway. In fact, in the C. albicans genome there are some genes that encode proteins which display a high degree of homology to MID2 and different members of the WSC family. However, a more direct relationship between these two MAP kinases cannot be ruled out.

A striking observation is the fact that some stimuli activate the Mkc1p MAP kinase but that the corresponding mutant does not display an increased sensitivity under the same conditions. These phenotypic differences could indicate that C. albicans ‘understands' the stress in terms of its normal habitat, the mucous membranes of warm-blooded animals. An interesting example is the activation of Mkc1p in the presence of 1 M sorbitol, rather than of Hog1p, as in S. cerevisiae, although mkc1{Delta} mutants are not more sensitive. C. albicans may interpret a high concentration of non-ionic extracellular solutes as a stimulus that is usually present in its natural environment, but that does not compromise its cell physiology. Our hypothesis is that 1 M sorbitol (or glycerol) is interpreted by C. albicans as an external condition equivalent to contact with host tissue, to which it must respond. However, the Mkc1p pathway is not the only pathway necessary for host-tissue invasion, although its functionality could be required at the beginning of invasion. mkc1{Delta} mutants are unable to form pseudohyphae and to penetrate into Spider medium, while the overexpression of MKC1 in S. cerevisiae CGX69 strains enhances its penetration into the agar (Navarro-García et al., 1998). In addition, the mkc1{Delta} mutants are found in lower counts in the target organs of infected mice, and their hyphae are shorter than those of wild-type strains (Diez-Orejas et al., 1997).

Another example can be found in the cold-shock activation of Mkc1p. Although we did not find cold-sensitivity in mkc1{Delta} mutants, they did die at high temperatures (Navarro-García et al., 1995). Why then is Mkc1p not activated at high temperatures? The ecological/physiological significance could be linked to the normal growth temperature of C. albicans inside the human body: 37 °C. A real challenge to this micro-organism would therefore be a decrease in temperature, since this could indicate that it was close to the outside of its ecological niche, the body. The lack of viability of the mkc1{Delta} mutant could be explained through architectural defects in its cell wall, not because of a defect in signalling.

In conclusion, we have shown that Mkc1p is activated by several stresses that may mimic those produced in essential biological processes. The unexpected phosphorylation of Mkc1p, Cek1p and Hog1p activated by various stimuli reinforces the importance of studying the activation of the C. albicans pathways in order to understand their function. To accomplish this task, it will be necessary to interpret the results from an ecological point of view. C. albicans seems to have developed its own (and different) way to use the same set of tools that it shares with other yeasts to respond and adapt to the challenges in the human body.


   ACKNOWLEDGEMENTS
 
Professor Humberto Martín (Dept Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid) and Dr Miguel Ángel Blázquez (Instituto de Biología Molecular y Celular de Plantas, Univ. Politécnica de València-CSIC) are greatly acknowledged for their help, suggestions and support throughout this work. Esther Martín provided excellent technical assistance for some parts of this work (financed through the FINNOVA programme from Comunidad Autónoma de Madrid). We acknowledge helpful suggestions and privileged communications from Dr María Ángeles de la Torre (Univ. Lleida, Spain). We thank Kevin Wood at the Modern Language Center of Universidad Complutense de Madrid for corrections of the manuscript. B. E. was supported by a predoctoral fellowship from Universidad Complutense de Madrid and by Proyecto Estratégico de la Comunidad Autónoma de Madrid (CPGE 1010/2000). S. F. was sponsored by the Erasmus programme. C. N. is Director of the Special MSD Chair in Genomics and Proteomics. This work is supported by Grant BIO2000-0729 and by Proyecto Estratégico de la Comunidad Autónoma de Madrid (CPGE 1010/2000).


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Received 15 March 2005; revised 24 May 2005; accepted 7 June 2005.



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