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
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ABSTRACT |
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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.
These two authors contributed equally.
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INTRODUCTION |
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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
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.
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METHODS |
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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 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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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 hisGura3hisG 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 (425600 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 ml1 (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 (420 % or 410 %) 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 l1 and 2 g gentamicin sulphate l1 to determine tissue burdens of C. albicans in each organ.
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RESULTS |
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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 407830) 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
-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
-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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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 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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DISCUSSION |
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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 ker1 null mutant, as already suggested for other null mutants of C. albicans (Calera & Calderone, 1999
). In addition,
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 hostparasite 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
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
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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Braun, R. B. & Johnson, A. D. (2000). TUP1, CPH1 and EFG1 make independent contributions to filamentation in Candida albicans. Genetics 155, 5767.
Brown, A. J. P. & Gow, N. A. R. (1999). Regulatory networks controlling Candida albicans morphogenesis. Trends Microbiol 7, 333338.[CrossRef][Medline]
Buffo, J., Herman, M. A. & Soll, D. R. (1984). A characterization of pH-regulated dimorphism in Candida albicans. Mycopathology 85, 2130.
Buurman, E. T., Westwater, C., Hube, B., Brown, A. J., Odds, F. C. & Gow, N. A. R. (1998). Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc Natl Acad Sci U S A 95, 76707675.
Calera, J. A. & Calderone, R. A. (1999). Flocculation of hyphae is associated with a deletion in the putative CaHK1 two-component histidine kinase gene from Candida albicans. Microbiology 145, 14311442.[Medline]
Casanova, M., Gil, M. L., Cardeñoso, L., Martínez, J. P. & Sentandreu, R. (1989). Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans. Infect Immun 57, 262271.[Medline]
Chaffin, W. L., López-Ribot, J. L., Casanova, M., Gozalbo, D. & Martínez, J. P. (1998). Cell wall and secreted proteins of Candida albicans: identification, function and expression. Microbiol Mol Biol Rev 62, 130180.
Davis, D., Wilson, R. B. & Mitchell, A. P. (2000). RIM101-dependent and -independent pathways govern pH responses in Candida albicans. Mol Cell Biol 20, 971978.
De Bernardis, F., Mühlschlegel, F. A., Cassone, A. & Fonzi, W. A. (1998). The pH of the host niche controls gene expression in and virulence of Candida albicans. Infect Immun 66, 33173325.
De Groot, P. W. J., Ruiz, C., Vázquez de Aldana, C. R. & 14 other authors (2001). A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genom 2, 124142.[CrossRef]
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350356.
Eroles, P., Sentandreu, M., Elorza, M. V. & Sentandreu, R. (1997). The highly immunogenic enolase and Hsp70 are adventitious Candida albicans cell wall proteins. Microbiology 143, 313320.[Medline]
Espeso, E. A., Tilburn, J., Sánchez-Pulido, L., Brown, C. V., Valencia, A., Arst, H. N., Jr & Peñalva, M. A. (1997). Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC. J Mol Biol 274, 466480.[CrossRef][Medline]
Fonzi, W. A. (1999). PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of -1,3- and
-1,6-glucans. J Bacteriol 181, 70707079.
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
Geourjeon, C. & Deleage, G. (1995). SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 11, 681684.[Abstract]
Gietz, R. D., Schiestl, R. H., Willems, A. R. & Woods, R. A. (1995). Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11, 355360.[Medline]
Gillum, A. M., Tsay, E. Y. & Kirsch, D. R. (1984). Isolation of Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198, 179182.[Medline]
Gow, N. A. R. & Gooday, G. W. (1982). Vacuolation, branch production and linear growth of germ tubes of Candida albicans. J Gen Microbiol 128, 21952198.[Medline]
Hazen, K. C. (1990). Cell surface hydrophobicity of medically important fungi, specially Candida species. In Microbial Cell Surface Hydrophobicity, pp. 249295. Edited by R. J. Doyle & M. Rosenberg. Washington, DC: American Society for Microbiology.
Hazen, K. C. & Hazen, B. W. (1987). A polystyrene microsphere assay for detecting surface hidrophobicity variations within Candida albicans populations. J Microbiol Methods 6, 289299.[CrossRef]
Hewitt, C. J. & Nebe-Von-Caron, G. (2001). An industrial application of multi-parameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44, 179187.[CrossRef][Medline]
Hofmann, K. & Stoffel, W. (1992). PROFILEGRAPH: an interactive graphical tool for protein sequence analysis. Comput Appl Biosci 8, 331337.[Abstract]
Hoyer, L. L., Scherer, S., Shatzman, A. R. & Livi, G. P. (1995). Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol 15, 3954.[Medline]
Hube, B., Monod, M., Schofield, D. A., Brown, A. J. P. & Gow, N. A. R. (1994). Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 14, 8799.[Medline]
Jigami, Y. & Odani, T. (1999). Mannosylphosphate transfer to yeast mannan. Biochim Biophys Acta 1426, 335345.[Medline]
Kjer-Nielsen, L., Teasdale, R. D., Van Vliet, C. & Gleeson, P. A. (1999). A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins. Curr Biol 9, 385388.[CrossRef][Medline]
Kun, J. F. K., Waller, K. L. & Coppel, R. L. (1999). Plasmodium falciparum: structural and functional domains of the mature-parasite-infected erythrocyte surface antigen. Exp Parasitol 91, 258267.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lee, K. L., Buckley, M. R. & Campbel, C. C. (1975). An amino acid liquid synthetic medium for development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148153.[Medline]
López-Ribot, J. L., Casanova, M., Martínez, J. P. & Sentandreu, R. (1991). Characterization of cell wall proteins of yeast and hydrophobic mycelial cells of Candida albicans. Infect Immun 62, 742746.
Lupas, A., Van Dyke, M. & Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 11621164.[Medline]
Lussier, M., Sdicu, A. M., Shahinian, S. & Bussey, H. (1998). The Candida albicans KRE9 gene is required for cell wall -1,6-glucan synthesis and is essential for growth on glucose. Proc Natl Acad Sci U S A 95, 98259830.
Maneu, V. E., Cervera, A. M., Martínez, J. P. & Gozalbo, D. (1996). Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1. FEMS Microbiol Lett 145, 157162.[CrossRef][Medline]
Martínez, J. P., Gil, M. L., López-Ribot, J. L. & Chaffin, W. L. (1998). Serologic response to cell wall mannoproteins and proteins of Candida albicans. Clin Microbiol Rev 11, 121141.
Masuoka, J. & Hazen, K. C. (1997). Cell wall protein mannosylation determines Candida albicans cell surface hidrophobicity. Microbiology 143, 30153021.[Medline]
Masuoka, J. & Hazen, K. C. (1999). Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells. Glycobiology 9, 12811286.
Mekalanos, J. J. (1992). Environmental signals controlling expresión of virulence determinants in bacteria. J Bacteriol 174, 17.[Medline]
Monteoliva, L., Lopez-Matas, M. L., Gil, C., Nombela, C. & Pla, J. (2002). Large-scale identification of putative exported proteins in Candida albicans by genetic selection. Eukaryot Cell 1, 514525.
Mühlschlegel, F. & Fonzi, W. A. (1997). PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression. Mol Cell Biol 17, 59605967.[Abstract]
Munro, C. A., Winter, K., Buchan, A., Henry, K., Becker, J. N., Brown, A. J., Bulawa, C. E. & Gow, N. A. (2001). Chs1 of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity. Mol Microbiol 39, 14141426.[CrossRef][Medline]
Murad, A. M. A., Lee, P. R., Broadbent, I. D., Barelle, C. J. & Brown, A. J. P. (2000). CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16, 325327.[CrossRef][Medline]
Nakajima, H., Hirata, A., Ogawa, Y., Yonehara, T., Yoda, K. & Yamasaki, M. (1991). A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol 113, 245260.[Abstract]
Odds, F. C. (1988). Candida and Candidosis. A Review and Bibliography, 2nd edn. London: Baillière Tindall.
Peñalva, M. A. & Arst, H. N., Jr (2002). Regulation of gene exprssion by ambient pH in filamentous fungi and yeasts. Microbiol Mol Biol Rev 66, 426446.
Porta, A., Ramón, A. M. & Fonzi, W. A. (1999). PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans. J Bacteriol 181, 75167523.
Ramón, A. M., Porta, A. & Fonzi, W. A. (1999). Effect of environmental pH on morphological development of C. albicans is mediated via the PacC-related transcription factor encoded by PRR2. J Bacteriol 181, 75247530.
Rose, M. D., Winston, F. & Hieter, D. (1990). Methods in Yeast Genetics: a Laboratory Course Manual. Plainview, New York: Cold Spring Laboratory Press.
Saporito-Irwin, S. M., Birse, C. E., Sypherd, P. S. & Fonzi, W. A. (1995). PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis. Mol Cell Biol 15, 601613.[Abstract]
Sentandreu, M., Elorza, M. V., Sentandreu, R. & Fonzi, W. A. (1998). Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans. J Bacteriol 180, 282289.
Serrano, R. (1988). H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: purification and reconstitution. Methods Enzymol 157, 533544.[Medline]
Southard, S. B., Spetch, C. A., Mishra, C., Chen-Weiner, J. & Robbins, P. W. (1999). Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins. J Bacteriol 181, 74397448.
Staab, J. F., Ferrer, C. A. & Sundstrom, P. (1996). Developmental expression of a tandemly repeated, proline- and glutamine-rich amino acid motif on hyphal surfaces of Candida albicans. J Biol Chem 271, 62986305.
Strambio-de-Castillia, C., Blobel, G. & Rout, M. P. (1999a). Proteins connecting the nuclear pore complex with the nuclear interior. J Cell Biol 144, 839855.
Strambio-de-Castillia, C., Blobel, G. & Rout, M. P. (1999b). Slk19p is a centromere protein that functions to stabilize mitotic spindles. J Cell Biol 146, 415425.
Tebele, N., Skilton, R. A., Katende, J., Wells, C. W., Nene, V., McElwain, T., Morzaria, S. P. & Mosoke, A. J. (2000). Cloning, characterization and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina. J Clin Microbiol 38, 22402247.
Tilburn, J., Sarkar, S., Widdick, D. A., Espeso, E. A., Orejas, M., Mungroof, J. M., Peñalva, A. & Arst, H. N., Jr (1995). The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid and alkaline expressed genes by ambient pH. EMBO J 14, 779790.[Abstract]
Timpel, C., Strahl-Bolsinger, S., Ziegelbauer, K. & Ernst, J. F. (1998). Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans. J Biol Chem 273, 2083720846.
Timpel, C., Zink, S., Strahl-Bolsinger, S., Schroppel, K. & Ernst, J. F. (2000). Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen Candida albicans. J Bacteriol 182, 30633071.
Warit, S., Zhang, N., Short, A., Walmsley, R. M., Oliver, S. G. & Stateva, L. I. (2000). Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species. Mol Microbiol 36, 11561166.[CrossRef][Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide references of the M13 mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Received 5 November 2003;
revised 25 May 2004;
accepted 27 May 2004.
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