The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation

Amparo Galán1,{dagger}, Manuel Casanova1,{dagger}, Amelia Murgui2, Donna M. MacCallum3, Frank C. Odds3, Neil A. R. Gow3 and José P. Martínez1

1 Departamento de Microbiologia y Ecologia, Facultad de Farmacia, Universitat de València, Spain
2 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universitat de València, Spain
3 Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK

Correspondence
José P. Martínez
jose.pedro.martinez{at}uv.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14·5 %)/glutamic acid (16·7 %) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4·0, and was regulated by RIM101. A {Delta}ker1/{Delta}ker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAb anti-Ker1p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from {Delta}ker1/{Delta}ker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with {beta}-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the {Delta}ker1/{Delta}ker1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host–fungus interactions.


Abbreviations: CSH, cell-surface hydrophobicity; Con A, concanavalin A

The GenBank accession number for the sequence determined in this work is AF337555. KER1 has the ORF number 6.8869 in the Stanford database and corresponds to IPF 2795 in the Candida database.

{dagger}These two authors contributed equally.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many of the genes required for virulence in bacterial pathogens are regulated in response to environmental signals (pH, temperature, osmotic pressure, and iron and calcium ion concentrations) indigenous to the host niche (Brown & Gow, 1999; Mekalanos, 1992). In the dimorphic, opportunistic fungal pathogen Candida albicans, the ability to respond to ambient pH appears to play a critical role in growth and virulence (for a review see Peñalva & Arst, 2002). It is known that external pH and temperature influence, at least in vitro, the yeast-to-mycelium transition of C. albicans, which is one of the virulence traits of this organism (Buffo et al., 1984; Odds, 1988).

An acidic pH generally encourages growth in the yeast form, whereas a neutral pH promotes hyphal development. The pH response in C. albicans involves the differential expression of at least three genes, PHR1, PHR2 and PRA1 (Mühlschlegel & Fonzi, 1997; Saporito-Irwin et al., 1995; Sentandreu et al., 1998). PHR1 is expressed at a pH above 5·5 and is required for normal morphology at these pH levels. The gene product plays a key role in systematic infections. PHR2 is expressed at an acidic pH, is required for normal morphology at these pH values, and contributes to virulence in the more acidic vaginal environment (De Bernardis et al., 1998; Mühlschlegel & Fonzi, 1997). PHR1 and its functional homologue PHR2 encode glycosidases that are localized at the plasma membrane, and are involved in cross-linking cell-wall glucans, a process required for the maintenance of cell shape and morphology (Fonzi, 1999). PRA1 encodes a protein that was found to be homologous to surface antigens of Aspergillus spp., but whose importance in C. albicans remains to be elucidated (Sentandreu et al., 1998). Overall, these observations indicate that gene expression patterns, cell morphology and virulence are coordinated by pH-responsive signalling pathways in C. albicans.

The pathway controlling pH-responsive gene expression has been most extensively dissected in the ascomycete Aspergillus nidulans (Espeso et al., 1997; Peñalva & Arst, 2002), where it has been demonstrated that the pH response depends on the pH activation of a transcription factor (PacC) encoded by pacC (Tilburn et al., 1995). The transcription factor of A. nidulans homologous to PacC in Saccharomyces cerevisiae and C. albicans is Rim101p, the product of the RIM101 gene (also designated PRR2 in C. albicans) (Davis et al., 2000; Porta et al., 1999; Ramón et al., 1999). Genes known to be under RIM101 regulation in C. albicans include PHR1, PHR2 and PRA1 (Ramón et al., 1999; Sentandreu et al., 1998).

In this paper, a cDNA clone that reacted with polyclonal antibodies towards glycoprotein cell-wall components of C. albicans (Casanova et al., 1989) was isolated and the role of the gene product in morphogenesis examined. This gene encodes a novel lysine/glutamic acid-rich protein (for this reason designated KER1) with no significant homology to known sequences and which is absent from the S. cerevisiae genome. A {Delta}ker1 null mutant was constructed and phenotypic analysis and virulence tests were subsequently conducted. Experimental evidence reported here on the subcellular location and potential functions of KER1 gene suggest that it may be involved in the integrated pH-response pathway, cell-wall biogenesis, and virulence.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
Micro-organisms and growth conditions.
The C. albicans strains used in this study are listed in Table 1. Cells were routinely grown in YPD or SD media at 28 °C with shaking. The effects of acid or alkaline growth conditions were tested in YPD, modified Lee's medium (Lee et al., 1975) containing 0·5 g proline per litre but lacking other amino acids, and medium 199 (Gibco) and adjusted to pH 4 or 7·5 with 155 mM HEPES. Different conditions were used to induce germ-tube formation: (i) addition of 10 % (v/v) fetal calf serum to YPD medium (Gow & Gooday, 1982) or to water, (ii) starvation-stimulated dimorphism as described elsewhere (Casanova et al., 1989), or (iii) changes in pH and temperature (pH 4 to 7·5 and 25 to 37 °C) in modified Lee's medium (Porta et al., 1999). Media were supplemented with 25 µg uridine ml–1 when appropriate. For sensitivity assays, plates containing solid (1·5 % agar) YPD, Spider and modified Lee's media were supplemented with Congo red (50, 100 and 200 µg ml–1), Calcofluor white (5, 20 and 25 µg ml–1) and SDS (0·01 %, 0·025 % and 0·05 %) and incubated at 30 or 37 °C.


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Table 1. Strains of C. albicans

 
Escherichia coli strain Y1090 was used for phage amplification. Strains JM109 and DH5{alpha} were used for plasmid propagation. E. coli was routinely grown in LB medium supplemented with 100 µg ampicillin ml–1 or 0·2 mM maltose and 100 mM MgSO4 when necessary.

Screening of libraries and cloning of KER1 gene.
A cDNA library of C. albicans (strain ATCC 26555) germ-tube-specific mRNA in the expression vector {lambda}gt11 (Maneu et al., 1996) was used for immunoscreening with a germ-tube-specific polyclonal antibody (pAb anti-gt), previously obtained by our group (Casanova et al., 1989) at a 1 : 1000 dilution by standard methods (Ausubel et al., 1992). cDNAs from immunoreactive clones were amplified and sequenced and blasted at the C. albicans sequencing project of the Stanford Genome Technology Center (http://www-sequence.stanford.edu/group/candida/search.html) and the University of Minnesota (http://alces.med.umn.edu/gbsearch/ybc.html). The isolated clone described here contained an incomplete ORF showing the high-scoring segment pairs (99 % identity) with the Contig 4-3030 (Contig 6-2517; last release).

For plasmid construction, a DNA genomic fragment of 5400 bps, containing the whole ORF (3591 bps) and 1500 bps and 500 bps from the 5' and 3' flanking regions respectively, was generated by PCR using Expand Long Template PCR System (Roche). The reaction was carried out using as template genomic DNA from C. albicans CAI4 strain and PFC1 and PRC1 primers (Table 2). PCR products were digested, ligated to pUC19 vector (Yanisch-Perron et al., 1985) and used to transform E. coli following standard procedures (Ausubel et al., 1992). Positive transformants were checked by plasmid purification, enzyme digestion and PCR, and sequenced with an Applied Biosystems model 370A automated sequencer.


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Table 2. Primers

 
Nucleic acid manipulation and analysis.
Genomic DNA preparation from C. albicans (Rose et al., 1990), digestion with restriction enzymes, and Southern blotting to nylon filters were carried out by standard protocols (Ausubel et al., 1992). The digoxigenin detection kit was used for DNA hybridization and probe labelling (Roche). Total RNA from C. albicans was prepared basically according to the procedure described by Hube et al. (1994). mRNA from total RNA was isolated using the Dynabeads mRNA Purification Kit (Dynal Biotech ASA). For cDNA synthesis (RT), mRNA was annealed with Oligo d(T)15 primer (Gibco-BRL) at 70 °C for 10 min and cooled on ice for 1 min. The final reaction volume was made up to 40 µl with first-strand buffer, 0·5 mM dNTPs, 80 U RNaseOUT (Gibco-BRL) and 400 U Superscript II RNase H Reverse Transcriptase (Gibco-BRL). cDNA synthesis was carried out at 42 °C for 1 h and the reaction stopped by heating at 70 °C for 15 min.

For RNA expression analysis, semiquantitative RT-PCR was carried out. Appropriate primer pairs to generate unique cDNA amplifications were made using information from the literature, gene databases or sequences for the KER1 and EFB1 genes (Table 2). PCR reaction mixtures were carried out with 1 µg total nucleic acids. Reactions were run for one cycle of 10 s at 94 °C, 28 cycles of 1 min at 94 °C, 1 min at 58 °C and 1 min at 72 °C, and the content in tubes was analysed at cycle number 20 for comparative expression determinations. Differential expression levels were analysed by Molecular Analyst-Bio-Rad software (Bio-Rad). Semiquantitive RT-PCR assays were performed in triplicate to assess its reliability.

Plasmid and strain construction for disruption of the KER1 gene.
The technique of Fonzi & Irwin (1993) to disrupt genes in C. albicans was used with minor modifications. DNA sequences flanking the gene were obtained by PCR using as template plasmid DNA obtained from KER1 cloning, and primers F1C1F, F1C1R amplifying for 360 bp (F1C1), and F2C1F and F2C1R amplifying for 732 bp (F2C1) (see Table 2). These fragments were cut with the enzymes matching their respective ends and cloned into pBB510 (Braun & Johnson, 2000) to get the pACA construction. pACA was cut with HindIII and Asp718 to linearize the 5 kb fragment, including F2C1, the hisG–ura3–hisG cassette and F2C1, and transformed into C. albicans CAI4 strain basically according to the lithium acetate procedure described by Gietz et al. (1995). Transformed cells were selected as URA+ on SD medium. One spontaneous URA derivate from a URA+-independent clone was selected on SD medium containing 5'-fluoroorotic acid (5'-FOA) and uridine, and used to delete the second allele of KER1. The disruption transformation was repeated to generate a null mutant. Both strains were verified by PCR and Southern blot analysis. For further phenotypic analysis, CAI4 and C1N7 strains were transformed with CIp10 integrating vector using the RP10 locus for the URA3 gene integration (Murad et al., 2000) to obtain the URA+ CAI4-URA3 and CAC1 strains.

Subcellular fractionation and plasma membrane isolation.
Cells grown in Lee's liquid medium at 30 °C overnight were collected by centrifugation (4000 g, 10 min) and washed twice with chilled 1 mM PMSF in 10 mM Tris buffer (pH 7·2) (buffer A) and broken by shaking glass beads (425–600 µm, Sigma). The cell walls were sedimented from the cell-free homogenate, washed four times with buffer A, boiled for 5 min with 2 % SDS to remove non-covalently bound proteins, and finally washed four more times with buffer A. The purified cell walls were digested in buffer A containing 0·5 mg Zymolyase 20T ml–1 (ICN Biomedicals) for 3 h at 28 °C. After treatment, the wall residue was removed by centrifugation and the solubilized material was concentrated by freeze-drying. The supernatant fluid obtained subsequently to cell breakage was centrifuged at 40 000 g for 40 min to obtain a mixed membrane fraction (P40), and the resulting supernatant was then centrifuged at 100 000 g for 1 h to obtain a microsomal fraction (P100). Plasma-membrane fraction was isolated according to the procedure described by Serrano (1988), slightly modified. The cell homogenate was centrifuged for 10 min at 700 g to remove large debris and the supernatant was further centrifuged for 40 min at 20 000 g. This second pellet, enriched in plasma membranes, was resuspended with about 14 ml 20 % (v/v) glycerol and 0·1 ml 100 mM PMSF and applied to a discontinuous gradient made of 8 ml 53 % (w/w) and 16 ml 43 % (w/w) sucrose solutions in distilled water. Purified plasma membranes were recovered at the interface between the two sucrose solutions after centrifugation for 6 h at 25 000 r.p.m. in a Beckman SW28 rotor. The interface band was diluted in water and pelleted by centrifugation for 20 min at 25 000 r.p.m. The purified membranes were resuspended in 20 % glycerol. The total sugar content in the cell-wall digests was determined by the method of Dubois et al. (1956); whereas the protein content in the other samples (P40 and P100) was determined by the Lowry method.

Antibodies.
An antibody recognizing Ker1p protein was prepared by Sigma-Genosys, by using a synthetic peptide selected from the deduced amino acid sequence from KER1 gene. pAb anti-Ker1p was raised against a 15-mer residue (HIKVPVKFSYHPTLE) derived from the N-terminal domain of the protein. The polyclonal antibody germ-tube-specific (pAb anti-gt) against purified walls from mycelial cells of C. albicans was obtained as described previously (Casanova et al., 1989).

SDS-PAGE and Western blotting.
SDS-PAGE under denaturing conditions was performed basically as described by Laemmli (1970) using slab gradient (4–20 % or 4–10 %) gels. Electrophoretic transfer to nitrocellulose paper was carried out as described previously (Casanova et al., 1989). Blotted proteins were immunodetected by using the primary specific antibodies (pAb anti-gt and pAb anti-Ker1p; see above) diluted (1 : 1000 and 1 : 500 respectively) in 0·01 M Tris/HCl buffer (pH 7·4), containing 0·9 % NaCl, 0·05 % Tween 20 and 3 % bovine serum albumin as a blocking agent. Peroxidase-conjugated secondary antibodies (Bio-Rad) were used at 1 : 2000 dilution, with 4-chloro-1-naphthol as the chromogenic reagent. Concanavalin A (Con A) staining of nitrocellulose blots was conducted as described elsewhere (Casanova et al., 1989).

Flow cytometry analysis.
For flow cytometric determination of cell aggregation, liquid cultures of each strain were filtered through a 30 µm diameter nylon mesh and analysed immediately in an EPICS XL-MCL flow cytometer (Beckman-Coulter). Cell aggregation was estimated from the measurement of forward-angle light scatter (FS Log), an indicator of particle size, and 90 ° side light scatter (SS Log), an indicator of particle complexity (Hewitt & Nebe-von-Caron, 2001) in 10 000 individual cells.

Cell-surface hydrophobicity (CSH).
CSH of individual cells was determined by light microscopy observations, following attachment of latex-polystyrene microspheres (0·760 µm diameter; Sigma), according to the method described by López-Ribot et al. (1991). According to the criterion of Hazen & Hazen (1987), cells with three or more attached microspheres were considered to be positively hydrophobic. CSH of cell populations was determined by an aqueous-hydrocarbon biphasic hydrophobicity assay by mixing 1·2 ml cell samples (OD600 of 0·100) with 0·3 ml of cyclohexane and vigorous vortexing for 3 min. The phases were then allowed to separate, and the percentage change in OD600 of the aqueous phase was considered the hydrophobicity value of the cell population (Hazen & Hazen, 1987).

Virulence tests.
Strains CAF2 and CAC1 (Table 1) were grown in SDGY medium (4 % glucose, 1 % neopeptone, 0·1 % yeast extract, 10 % glycerol; pH 3·5) overnight in a shaking incubator at 30 °C. Harvested cells were washed twice in water, and resuspended in saline solution to give an inoculum of approximately 500 c.f.u. (g mouse body weight)–1 in a final volume of 100 µl. Five DBA/2 mice (Harlan Laboratories) were inoculated intravenously with the CAF2 strain and six DBA/2 mice with the CAC1 strain. Mice weighed approximately 20 g. Survival was monitored twice daily. Animals that became seriously ill, showing hunched posture, ruffled fur and reduced mobility, were humanely terminated and their deaths recorded as occurring on the following day. For viable cell counting, the left kidney and brain of dead mice were removed aseptically post-mortem, weighed and homogenized with an UltraTurrax apparatus in 0·5 ml sterile distilled water. Dilutions of the organ homogenates were plated on Sabouraud agar, containing 5 g chloramphenicol l–1 and 2 g gentamicin sulphate l–1 to determine tissue burdens of C. albicans in each organ.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Isolation, cloning and characterization of the KER1 gene
Immunoscreening of an expression library with a germ-tube-specific polyclonal antibody led to the isolation of a cDNA clone that when blasted against the sequence databases showed a novel incomplete ORF contained in the contig 4-3030. A genomic clone of the gene was obtained by PCR using two primers deduced from the contig sequence, and engineered with the appropriate restriction sites (Table 2) for cloning into pUC19. The resulting construction was confirmed by sequencing. The ORF encodes a putative polypeptide of 1197 amino acids with a deduced molecular mass of 134 027 Da and a pI of 5·11, which is rich in lysine (14·5 %) and glutamic acid (16·7 %). For this reason it was called Ker1p (for lysine/glutamic-acid-rich).

BLAST or FASTA comparisons of the translated amino acid sequence of Ker1p with the translated GenBank databases revealed homology with several proteins known or predicted to encode coiled-coil domains. These include conventional and non-conventional myosins, laminin, caldesmon and intermediate filaments. Best matches were found with a 200 kDa diagnostic antigen of Babesia bigemina (Tebele et al., 2000) and with the liver-stage antigen (LSA-1) of Plasmodium falciparum (Kun et al., 1999). In general, the homologous regions were found throughout the central segment of Ker1p (amino acids 407–830) with unique sequences in the N- and C-terminal regions flanking the large central region containing the predicted coiled-coil domains.

A search for sequence similarities in the S. cerevisiae protein databases (SGD and MIPS) revealed that there was no obvious homologue of Ker1p in this organism. Best alignments were found with Uso1p (Nakajima et al., 1991), Mlp1p (Strambio-de-Castillia et al., 1999a), Slk19p (Strambio-de-Castillia et al., 1999b) and Imh1p (Kjer-Nielsen et al., 1999) proteins with amino acid identities of approximately 20 %, distributed randomly over the sequences. All these proteins have in common a high content in K and E amino acids and predominantly {alpha}-helical structure over the entire length of the protein, which is indicative of structural analogy rather than true homology. Similar rates of identity were observed with ScMnn4p, the product of the MNN4 gene, which regulates mannosyl phosphorylation in S. cerevisiae (Jigami & Odani, 1999). The amino acid sequence analysis of ScMnn4p revealed a striking lysine/glutamic acid repeat region and also predicted the presence of {alpha}-helical conformation. Secondary-structure analysis of Ker1p (Geourjeon & Deleage, 1995), coiled-coil structures (Lupas et al., 1991) and transmembrane domains (Hofmann & Stoffel, 1992) prediction according to Expasy analysis (see Table 3) predicted a transmembrane localization.


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Table 3. Secondary-structure analysis (predicted topology)

The predicted {alpha}-helix content is 66 %. The amino acid positions of predicted coiled coils and transmembrane domains are shown.

 
Regulation of KER1 expression
Differential expression of KER1 was assessed by semiquantitative RT-PCR using conditions favouring yeast or hyphal development. As shown in Fig. 1(a), maximum expression was found in modified Lee's medium in both yeast and hyphal cultures. However, KER1 expression was down-regulated during growth in rich culture media. Interestingly, KER1 expression was also down-regulated when cells were grown in Lee's medium at pH 4, suggesting that KER1 expression may also be pH-dependent. To explore this possibility, the effect of media at different pH and incubation temperatures on KER1 expression was assessed (Fig. 1b). The highest transcription levels were observed when cells were grown at pH 7·5 in Lee's or M199 medium regardless of the growth temperature employed. In C. albicans, it has been demonstrated that the RIM101 pathway is required for some alkaline responses (Davis et al., 2000). Therefore, expression of KER1 was analysed in {Delta}rim101/{Delta}rim101 mutants that are deficient in a transcription factor regulating pH responses. Since the ratio KER1/EFB1 is higher at pH 4 than at pH 7·5 (Fig. 1b), Rim101p could be controlling KER1 by repressing its expression at an acidic pH. These results demonstrate that RIM101 is required for the expression of KER1.



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Fig. 1. (a) Expression analysis of KER1 transcript in C. albicans CAI4-URA3 strain by semiquantitative RT-PCR. The KER1/EFB1 expression ratio in each experimental condition was assessed by software analysis, by comparing the results of amplification with the KER1 primers (SC1F-SC1R) (see Table 2) with the EFB1 primers (see Table 2) as internal control. Conditions 1–5 correspond to budding yeast growing conditions in YPD (1), YNB (2), modified Lee's liquid medium (3) and stationary-phase cells in modified Lee's liquid medium (4), all of them incubated at 30 °C, and cells grown in modified Lee's medium at pH 4 and 25 °C (5). Conditions 6–9 correspond to cells grown under different germ-tube-induction conditions: starvation and pH and temperature shifts in modified Lee's medium (6 and 7), in YPD medium supplemented with serum (8), and in water supplemented with serum (9). (b) Analysis of the effects of temperature and pH on KER1 expression. Strain CAI4-URA3 was inoculated in modified Lee's medium (conditions 1 and 2) or M199 medium (3–5), adjusted to pH 4 (1, 3 and 4) or 7·5 (2 and 5) and incubated at 25 °C (1 and 3) or 37 °C (2, 4 and 5). KER1 expression was also analysed in the {Delta}rim101 null mutant (CAR2 strain) grown in Lee's medium at pH 4 (acid conditions; 6) and at pH 7·5 (alkaline conditions; 7). KER1 transcripts were also analysed when CAI4-URA3 cells were grown in YPD at pH 6 (8) and at pH 7·5 (9).

 
Construction of a KER1 null mutant
Disruption of KER1 gene was achieved by the ura-blaster technique (Fonzi & Irwin, 1993). A disruption cassette (see Methods) was used to replace a 3587 kb fragment of KER1 which included the entire ORF (Fig. 2a). The resultant C1H heterozygous and C1N7 null mutant strains (Table 1) were confirmed by PCR and Southern analysis (Fig. 2b and c, respectively). To confirm knockout of both KER1 gene alleles in the null mutant, RT-PCR was performed using specific primers for KER1 (SC1F-SC1R; see Table 2). The intron-containing gene that encodes the elongation factor EF-1{beta} (EFB1) of C. albicans (Maneu et al., 1996) was included as internal control for possible contamination by genomic DNA. Results shown in Fig. 2(d) indicated the absence of KER1 transcripts under both growth conditions.



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Fig. 2. (a) Schematic representation of the construction, integration and selection of the cassette used to disrupt the KER1 gene. Methionine and stop codons are represented by checkered boxes. Significative primers for the disruption strategy are indicated in bold. (b) Ethidium-bromide-stained agarose (0·8 %) gel of PCR products obtained from C. albicans wild-type strain CAI4 (lanes 1 and 2), heterozygous strain C1H (lanes 3 and 4) and homozygous strain C1N7 (lanes 5 and 6). PCR was performed using whole cells as template and AC1F-SC1R (lanes 1, 3 and 5) and AC1F-HisG1 (lanes 2, 4 and 6) as primers amplifying for 1140 bp and 760 bp respectively. (c) Southern blot analysis of representative {Delta}ker1 mutants obtained during the disruption process. Genomic DNA (10 µg) from C. albicans CAI4 (lanes 1 and 4), C1H (lanes 2 and 5) and C1N7 (lanes 3 and 6) strains was digested with EcoRV (lanes 1–3) and with EcoRV–BamHI combination (lanes 4–6), subjected to electrophoresis, blotted and hybridized with F2C1 probe obtained by PCR and labelled with digoxigenin-dUTP, under high-stringency conditions. F2C1 probe detected a 9·5 kb and a 4·5 kb fragment for each restriction enzyme combination respectively for the wild-type strain, and a 3·3 kb fragment for the null mutant strain. (d) Qualitative analysis of KER1 expression. cDNA from strains CAI4 (lanes 1 and 2) and C1N7 (lanes 3 and 4), incubated under conditions to induce growing as budding yeast (lanes 1 and 3) and as germ tubes (lanes 2 and 4), was amplified by multiplex PCR using specific primers for KER1 (SC1F-SC1R; see panel a and Table 2) and EFB1 as internal control (Table 2).

 
Subcellular localization of Ker1p
Different subcellular fractions (cell-wall extract, P40 and P100) obtained from the wild-type and CAC1 null mutant strains were analysed by SDS-PAGE and Western immunoblotting using pAb anti-gt (Fig. 3a) and pAb anti-Ker1p (Fig. 3b) as probes. An immunoreactive 134 kDa species was detected in the P40 fraction (mixed membranes), whereas no immunoreactive bands were detected in the P40 obtained from the null mutant strain, suggesting a membrane localization for the Ker1p protein. The null mutant also displayed an altered pattern of immunoreactive bands in Zymolyase cell-wall digests with respect to the wild-type strain (compare lanes 3 and 4 in Fig. 3a and b, respectively). Further SDS-PAGE and Western immunoblotting analysis of the purified plasma-membrane fraction revealed the presence of a 134 kDa polypeptide band only in the parent CAI4-URA3 strain (Fig. 3c, arrow).



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Fig. 3. Western immunoblotting analysis of subcellular fractions from C. albicans strains grown at 30 °C in modified Lee's liquid medium. Slab gradient polyacrylamide gels (4–20 % in a, and 4–10 % in b and c) were loaded with P40 fraction (P40) (a and b, lanes 1 and 2; 50 µg of material expressed as total protein content per well), with cell-wall Zymolyase digests (Zy) (lanes 3 and 4; 200 µg of material expressed as total sugar content per well), and the purified plasma-membrane fraction (PM) (c, 50 µg of material expressed as total protein content per well) from strains CAI4-URA3 (lanes 1 and 3) and CAC1 (lanes 2 and 4). After SDS-PAGE, polypeptides were transferred to nitrocellulose sheets and immunodetected with pAb anti-gt in (a) and with pAb anti-Ker1p in (b) and (c).

 
Phenotypic analysis of the {Delta}ker1 mutant
The effect of the KER1 gene deletion on the phenotype of the {Delta}ker1 mutant was studied under conditions that normally promote both yeast and hyphal growth in C. albicans. The CAC1 mutant and the control CAI4-URA3 strains were grown in modified Lee's medium at 25 °C (pH 4) and 37 °C (pH 7·5). Under incubation conditions that promote hyphal growth, cells of the CAC1 strain tended to aggregate. Aggregation was most dramatic at pH 7·5 and 37 °C after 3 h incubation, as assessed by macroscopic and light microscopy observations and flow cytometry analysis (data not shown). Overall, late-stationary-phase cells grown under different incubation conditions (i.e. in Lee's medium at 28 °C or YPD medium at 30 °C) also tended to aggregate (not shown).

Western blotting analysis revealed that Con A-reactive mannoprotein species present in the cell-wall Zymolyase digests from CAC1 mutant strain exhibited different electrophoretic mobilities and a greater polydispersity when compared to their counterparts in the homologous extracts from CAI4-URA3 parental wild-type strain (not shown), thus suggesting that mannosylation of cell-wall glycoproteins could be affected by loss of KER1 function. Besides, Ker1p appears to be involved in cell surface hydrophobicity (CSH), a biological property considered to be an important virulence trait in C. albicans, and that appears to be associated with the glycosylation levels of cell-wall glycoproteins (Masuoka & Hazen, 1997). CSH determined by an aqueous-hydrocarbon biphasic partition assay (see Methods), showed that 92·5±4·2 % of cells in the cultures of CAC1 strain displayed CSH, whereas only 61·3±2·9 % of the cells in the cultures of the parental strain were found to be hydrophobic (values are the mean of three independent experiments carried out in duplicate±standard deviations). Attachment of latex-polystyrene microspheres (see Methods), confirmed the previous results.

Finally, the sensitivity of {Delta}ker1 mutants to substances that interferred with cell-wall assembly was examined. No significant differences in sensitivity to Congo red, SDS and Calcofluor white were found between mutant and parental strains when incubated either at 30 °C or at 37 °C in several rich media (data not shown). However, when CAC1 cells were grown in solid Lee's medium at 37 °C, sensitivities to Calcofluor white and Congo red increased with respect to those of the parental strain at alkaline pH but not at acidic pH (Fig. 4). These observations are in agreement with results from the KER1 expression analysis, since the highest level of KER1 expression was observed at pH 7·5 in Lee's medium.



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Fig. 4. Effect of KER1 gene disruption on cell-wall integrity. Cells were grown at 37 °C and pH 4 and pH 7·5 in modified Lee's solid medium supplemented with Calcofluor white (30 µg ml–1; CFW) and Congo red (50 µg ml–1; CR). In each panel, the upper row corresponds to the wild-type strain CAI4-URA3 and the bottom row corresponds to the null mutant strain CAC1. Drops (5 µl) of serial cell suspensions containing from 106 to 102 cells µl–1 (from left to right in each panel) were inoculated onto the surface of culture medium.

 
Effect of KER1 deletion on virulence
The virulence of the Ura+ parental and disrupted strains was studied in a murine model of disseminated C. albicans infection. Prior to animal studies, two factors that might also affect the virulence of the mutant strains, the generation time, which was found to be the same in both strains, and the effect of URA3 gene disruption, were evaluated. To ensure full URA3 expression in the null mutant, strain C1N7 was transformed to strain CAC1 with the CIp10 integrating vector. Mice infected with the parental control strain (CAF2) succumbed to infection within 5 days of challenge, while all the mice infected with CAC1 survived to day 10. Two of the six animals challenged with CAC1 were still alive 28 days after challenge (Fig. 5). As shown in Table 4, tissue burdens of C. albicans recovered from organs post-mortem also showed a lower severity of infection by CAC1 as compared to CAF2. For all CAF2-infected mice, samples from the left kidney and brain were culture-positive, with mean burdens of 6·4±0·2 and 4·9±0·4 log10(c.f.u. g–1), respectively. For the six CAC1-infected mice, one kidney homogenate and two brain homogenates were culture-negative for C. albicans. Mean burdens for positive organs were significantly lower than for CAF2-infected mice at 5·5±0·3 and 3·8±0·5 log10(c.f.u. g–1), respectively (P<0·05, Student's t-test).



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Fig. 5. Virulence of CAC1 ({circ}) and CAF2 ({bullet}) C. albicans strains in immunocompetent mice. See Methods for experimental details.

 

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Table 4. Recovery of C. albicans from infected tissues

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Molecular genetic approaches have identified several genes encoding hypha-specific cell-surface proteins that may contribute to differences in cell-wall structure or function in C. albicans (Hoyer et al., 1995; Staab et al., 1996). In this context, immunoscreening of a cDNA library with a germ-tube-specific polyclonal antibody (Casanova et al., 1989) led to the isolation of a novel gene, designated KER1, encoding a putative polypeptide of 1197 amino acids, rich in lysine (14·5 %) and glutamic acid (16·7 %). A search for sequence similarities in the current protein databases revealed no obvious homologue of Ker1p in other organisms.

Although the polyclonal antiserum used in the immunoscreening was mostly directed towards cell-wall components of C. albicans (Casanova et al., 1989), bioinformatic and Western immunoblotting analysis suggest that KER1 encodes an integral membrane protein rather than a cell-wall protein. These findings are not unusual since it has already been reported that antibodies raised against purified cell-wall preparations may also cross-react with non-wall cell components released during the isolation and purification processes, and that may be present as contaminants in the isolated walls (Eroles et al., 1997).

Environmental pH strongly influences morphogenesis in C. albicans. The RIM101-dependent pH signalling pathway plays a central role in the control of pH responses, morphogenesis and niche-specific responses during C. albicans infections (De Bernardis et al., 1998). In the absence of RIM101, KER1 gene was no longer expressed under alkaline conditions, suggesting that KER1 is a component of the same pH response induced at an alkaline pH (Davis et al., 2000). However, ambient pH was not the sole factor influencing its expression, since KER1 mRNA was also detected at low levels in cells grown in YNB, YPD medium supplemented with serum, or YPD buffered at pH 7·5. These results suggest that a RIM101-independent pathway, possibly related to stress and starvation conditions, may also be required for the activation of KER1.

Homozygous mutant cells lacking KER1 grew in the same manner as wild-type cells on a number of different carbon sources, on both rich and minimal culture media, and at various temperatures. The most obvious consequence of KER1 deletion was that null mutant cells flocculated extensively under a variety of conditions, particularly in media that encourage germ-tube formation, which is consistent with the fact that aggregation may occur primarily by interaction of hyphal cell surfaces in the {Delta}ker1 null mutant, as already suggested for other null mutants of C. albicans (Calera & Calderone, 1999). In addition, {Delta}ker1 null mutant cells in stationary phase tended to aggregate under growing conditions that do not promote KER1 expression.

C. albicans mutants defective in N-linked mannosylation, like those strains lacking CaSRB1, the gene encoding GDP-mannose pyrophosphorylase (Warit et al., 2000), and MNN9, encoding the mannosyltransferase (Southard et al., 1999), were also found to flocculate, thus suggesting that aggregation phenotype could be due, at least partly, to an impairment in the N-mannosylation pathway of cell-wall mannoproteins. On the other hand, CSH which is considered to play an important role in host–parasite interaction and virulence of C. albicans (Chaffin et al., 1998; Hazen, 1990) has also been related to N-mannosylation since it has been shown that CSH is increased when the extent of N-linked protein mannosylation is decreased (Masuoka & Hazen, 1997, 1999). As previously stated, cells of {Delta}ker1 null mutant strain described in this work showed an increased CSH and displayed changes in the pattern of species released by Zymolyase digestion of the cell wall and that were reactive towards the polyclonal antibodies used here as probes (pAb anti-gt and pAb anti-Ker1p) and Con A. In addition, growth of {Delta}ker1 null mutant cells was inhibited by the presence of Calcofluor white and Congo red in the culture medium under conditions that enhanced KER1 expression. All these findings suggest that Ker1p could play a role in influencing cell-wall biogenesis.

Although in pathogenic fungi, cell-wall proteins play a key role in the relationship between the fungal cell and the environment through adhesion phenomena and modulation of the immune response (Chaffin et al., 1998; Martínez et al., 1998), membrane proteins also play an essential role in fungal physiology because they are involved in nutrient transport, energy generation, and signal transduction pathways, ultimately leading to growth and host adaptation (Monteoliva et al., 2002). It has been estimated in S. cerevisiae that about 1200 genes may be involved in cell-wall construction as deletion of these genes resulted in an altered cell wall (De Groot et al., 2001). In this context, KER1 could be added to the growing list of Candida genes involved in cell-wall structure that when mutated are uniformly impaired to some degree in morphogenesis. These include the genes required for synthesis or assembly of glucan (e.g. CaKRE9, PHR1 and PHR2) (Fonzi, 1999; Lussier et al., 1998), chitin (e.g. CHS1) (Munro et al., 2001) and mannan (e.g. MNN9, MNT1, SRB1, PMT1 and PMT6) (Buurman et al., 1998; Southard et al., 1999; Timpel et al., 1998, 2000; Warit et al., 2000). Overall, our results indicate that deletion of the KER1 gene clearly resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties that may be essential in the interaction of the fungal cells with the environment.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grant BMC2001-2975 from the Programa Nacional de Promoción General del Conocimiento, Ministerio de Ciencia y Tecnología, Spain (to J. P. M.), grant CTIDIB/2002/3 from the Subsecretaria de la Oficina de Ciencia y Tecnología de la Generalitat Valenciana, Valencia, Spain (to M. C.) and grants 063204 and 056847 (to N. A. R. G. and F. C. O.) from the Wellcome Trust. A. G. is the recipient of a predoctoral fellowship from the Conselleria de Cultura, Educació y Ciència, Generalitat Valenciana, Valencia, Spain. We thank J. E. O'Connor (Departamento de Bioquímica y Biología Molecular, Universitat de València, Valencia, Spain) for his assistance with flow cytometry experiments, Burk Braun for pBB510 vector, Alistair J. P. Brown for CIp10 vector and CAR2 strain, and Gwyneth Bertram for CAI4-URA3 strain. Assistance from the SERBIO and SCSIE (Universitat de València) is acknowledged.


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Received 5 November 2003; revised 25 May 2004; accepted 27 May 2004.



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