Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Unidad de Bioquímica y Genética de Levaduras, 28029 Madrid, Spain1
Botanisches Institut, Universität Basel, Hebelstr 1, CH-4056 Basel, Switzerland2
Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, 48080 Bilbao, Spain3
Author for correspondence: Carlos Gancedo. Tel: +34 91 5854620. Fax: +34 91 5854587. e-mail: cgancedo{at}iib.uam.es
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ABSTRACT |
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Keywords: cell wall, aggregation, stress, antifungals
Abbreviations: T6P, trehalose 6-phosphate
The EMBL accession number for the sequence reported in this paper is AJ242990.
a Present address: Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
b Present address: Département de Biochimie Médicale, Centre Médical Universitaire, 1211 Geneva 4, Switzerland.
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INTRODUCTION |
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METHODS |
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DNA sequencing and sequence analyses.
Sequences were obtained from double-stranded plasmid DNA by the use of the cycle sequencing method and the ABI PRISM 310 capillary sequencer (Perkin Elmer Applied Biosystems). Sequences were analysed by using standard programs.
Isolation of the C. albicans TPS2 gene.
The temperature-sensitive Sac. cerevisiae tps2 strain YSH 6.106-8C was transformed with a C. albicans genomic DNA library in YEp322 (a gift of C. Nombela and J. Pla, Madrid, Spain). About 106 transformants were plated on glucose synthetic medium and incubated at 38·6 °C. Plasmids isolated from positive colonies were tested by PCR using two degenerate oligonucleotides: 5'-GATTACGATGG[CT]AC[CT][CT]T[AGT]AC[CT]CC[AG]AT[CT]GT-3' and 5'-GGACGAACTTC[AG]A[GT][AG]TT[ACGT]GC[CT]TT[ACGT]CC-3', corresponding to highly conserved sequences from known genes encoding T6P phosphatases. A PCR reaction using these primers and DNA from the genomic library mentioned above yielded a fragment of 0·45 kb with sequence similarity to T6P-phosphatase-encoding genes; the same fragment was obtained with DNA of the isolated plasmids. Plasmid YEp352-CaTPS2-1 carried a DNA segment of 3822 bp containing the entire CaTPS2 gene, including 700 bp upstream of the putative initiation codon and 380 bp after the stop codon. The sequence of this fragment is available at the EMBL database under accession no. AJ242990.
Chromosomal disruption of the CaTPS2 gene.
The CaTPS2 gene was disrupted with CaURA3 and CaHIS1 using two disruption cassettes (Fig. 1a). The one with CaURA3 was constructed as follows: a 4 kb BamHIBglII fragment from pCUB6K1 (C. San-José, J. Pla & C. Nombela, unpublished), a derivative of pCUB6 (Fonzi & Irwin, 1993
) containing the hisG-CaURA3-hisG blaster cassette (Fonzi & Irwin, 1993
), was inserted into the unique BamHI site from plasmid YEp352-TPS2-1 to give pOZ11-3. To construct the disruption cassette with CaHIS1, a 1·5 kb BamHIBamHI fragment from p34HHIS1 (given by J. Pla) was ligated into the unique BamHI site from plasmid YEp352-TPS2-1 to produce pOZ11-4. To interrupt the corresponding genomic loci in C. albicans the plasmids were digested with HindIII and the digestion products introduced into the yeast by electroporation. Introduction of the digestion products of pOZ11-3 into C. albicans strain RM1000 produced strain LOZ184, introduction of those of plasmid pOZ11-4 into strain LOZ184 gave strain LOZ200 and introduction of the same products into strain RM1000 provided strain LOZ183. Correct insertion of the disruption cassette was checked by PCR and Southern analysis (Fig. 1b
). C. albicans LOZ250 was obtained by pop-out of the URA3 gene from strain LOZ200 after selection on YNB glucose plates with 20 µg uridine ml-1 and 0·5 mg 5-fluoroorotic acid ml-1.
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Determination of trehalose and T6P.
Trehalose was assayed after hydrolysis with trehalase as described by Blázquez et al. (1994a ), and T6P by measuring the specific inhibition of Yarrowia lipolytica hexokinase as described by Blázquez et al. (1994b
). The Y. lipolytica hexokinase needed was obtained from Sac. cerevisiae CJM291 (Petit et al., 1996
), a strain lacking all glucose-phosphorylating enzymes carrying plasmid pDB20-YlcHXK1 (Petit & Gancedo, 1999
). In some cases, T6P was assayed by HPLC as described by De Virgilio et al. (1993)
.
Analysis of proteins in culture medium.
Cultures were centrifuged and the supernatants collected. Trichloroacetic acid was added at a final concentration of 10% and the mixture was kept at 4 °C for 15 min, centrifuged at 13000 r.p.m. for 15 min in an Eppendorf microcentrifuge and the precipitate resuspended in Laemmli reagent (Laemmli, 1970 ). The samples were applied to an SDS-PAGE (10%) gel and stained with Coomassie blue. To detect glycosylated proteins, PAS staining was performed as described by Kapitany & Zebrowsky (1973)
.
For analysis of intracellular proteins, cell-free extracts were obtained as described by Blázquez et al. (1993) and proteins were assayed using the commercial Pierce reagent.
Propidium iodide staining.
A 25 µl sample of a culture was centrifuged and the cells were resuspended in 200 µl PBS (Sambrook et al., 1989 ) to which 13 µl propidium iodide (50 µg ml-1) was added. The cells were then examined under a fluorescence microscope.
Preparation of samples for electron microscopy.
For scanning electron micrographs, samples were prepared as described by Herrero et al. (1999) and for transmission electron micrographs as described by Wright et al. (1988)
.
Virulence in mice.
Yeasts were grown on Sabouraud Dextrose agar (Difco) at 37 °C for 24 h, washed with 0·85% NaCl and resuspended in the same solution. The number of cells to be inoculated in the mice was determined by counting the cells in an haemocytometer and the viability (c.f.u.) was checked by plating the suspension onto Sabouraud Dextrose Agar plates. In these conditions no appreciable aggregation was observed and a good agreement between c.f.u. data and haemocytometer counting was observed. Groups of 10 female mice (BALB/c, 68 weeks old, 2530 g wt) were inoculated in the lateral caudal vein with 1·5x106 viable C. albicans cells of each strain and observed for 1 month. To determine the c.f.u. in the mice, each organ was removed, weighed and homogenized in 0·85% NaCl. Dilutions were plated on Sabouraud Dextrose at 30 °C and colonies were counted after 48 h. Differences in c.f.u. were analysed by the statistical MannWhitney U test and considered statistically significant at a P<0·05.
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RESULTS |
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Effects of the disruption of the CaTPS2 gene
The Catps2/Catps2 disrupted strain did not accumulate trehalose after a heat shock at 42 °C. Throughout growth, the concentration of T6P in the mutant was 1 nmol (mg dry wt)-1 during the exponential phase and 3 nmol (mg dry wt)-1 in the stationary phase, while in the wild-type it was below our detection level (0·5 nmol). These results strongly indicate that there is only one gene encoding T6P phosphatase in C. albicans.
The Catps2/Catps2 mutant grew at 37 °C and even at 42 °C in YP-glucose (YPD), although slower than the wild-type. Generation times at 37 °C were 60 min for the wild-type and 80 min for the mutant, and 80 min for the wild-type and 130 min for the mutant at 42 °C. In contrast to the wild-type, the mutant cells formed aggregates in stationary phase (Fig. 2). These aggregates were not dispersed by washing with 0·25 M EDTA, a treatment that disperses flocks in Sac. cerevisiae (Stratford, 1989
), but they were dispersed by a mild sonication or by a 10 min treatment with 0·2 mg Zymolyase ml-1 that did not affect cell integrity. Reintroduction of an intact CaTPS2 caused the disappearance of the aggregation phenotype, showing that the effects observed were due to the absence of a CaTPS2 gene (Fig. 2
). Electron microscopic examination of the mutant cells did not show marked morphological abnormalities in the aggregated cells, but a material surrounding them was observed (Fig. 3
). This material was not seen in preparations from exponential-phase cells when aggregation was not observed (results not shown).
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More than 50% of stationary-phase Catps2/Catps2 cells were permeable to propidium iodide, while less than 1% wild-type cells from the same growth phase took up the dye (Fig. 4), indicating that the mutant had a defect in cell integrity. This idea was also supported by the finding of a large amount of proteins in the medium when mutant cells had grown to stationary phase (Fig. 5a
). The pattern of proteins from the medium was similar to that of a cell-free extract (Fig. 5a
), making it unlikely that the proteins found in the medium were a selected set preferentially secreted by the mutant. Leakage of proteins took place, although at a decreased level in media buffered at pH 5 (Fig. 5b
), suggesting that a certain loss of cell integrity already occurred at this pH.
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Cells bearing the double Catps2/Catps2 disruption formed colonies with a smooth border on plates of Spider medium (Fig. 6) and when challenged with serum at 37 °C showed a delay in the formation of hyphae. Both facts indicate that the mutation interferes with the correct filamentation process.
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DISCUSSION |
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The disruption of CaTPS2 did not cause a strong thermosensitive growth phenotype, even at 42 °C, and produced an aggregation of cells in the stationary phase. This is an important difference with the phenotypes reported for the tps2 mutant of Sac. cerevisiae (De Virgilio et al., 1993 ) and of Sch. pombe (Franco et al., 2000
) where no flocks have been observed and indicates that the accumulation of T6P acts differently in these organisms. The fact that aggregation in the Catps2/Catps2 mutant was apparent only in the stationary phase suggests that the increased levels of T6P influence processes taking place at this phase of growth when an important number of not yet well understood changes occur that affect different cell properties (Werner-Washburne et al., 1996
). Reports of flocculation in C. albicans mutants are scarce; among them one concerns a mutant in a two-component histidine kinase, CaHK1 (Calera & Calderone, 1999
), and another one, a mutant affected in the gene BGL2 encoding a glucosyl transferase (Sarthy et al., 1997
). In the first case the aggregation is due to interactions between the hyphal surfaces, while in the second case no information about how the aggregation occurred is available. In the case of the aggregation observed in the Catps2/Catps2 mutant, hyphae were not seen when aggregation took place. Expression of ALS1, a gene that encodes an agglutinin (Hoyer et al., 1995
), was similar in the wild-type and in the mutant (results not shown), suggesting that changes in Als1 levels are not involved in the aggregation. A modification in hydrophobic properties of the cells could also occur in the mutant and determine its aggregation. The material that surrounded the aggregated cells of the Catps2/Catps2 mutant could have adhering properties that contribute to the aggregation of the cells. We do not know the nature of this material, but it is not too far fetched to think that its presence is a consequence of a defective cell wall. This idea is supported by the deaggregation of the flocks by a mild Zymolyase treatment.
The pH influence on aggregation could be explained by a difference in the charge of some proteins whose levels are changed in the Catps2/Catps2 mutant. The accumulated T6P could also have an effect on the expression of some pH-regulated genes or on the activity of their products. It is well known that pH controls expression of different genes whose products are implicated in morphogenesis in C. albicans and that acidic pH favours the yeast form (El Barkani et al., 2000 ; Fonzi, 1999
; Ramon et al., 1999
). It is noteworthy that aggregation of the CaHK1 mutant also occurred at pH 7·5 and not at acidic pH (Calera & Calderone, 1999
). In Sac. cerevisiae it has been found that cell wall organization is altered when the organism is faced with pH changes; at acidic pH values higher levels of certain cell-wall proteins were produced and resistance to lysis by ß-1,3-glucanase increased (Kapteyn et al., 2001
). The cell wall architecture of C. albicans has been shown to be similar to that of Sac. cerevisiae (Kapteyn et al., 2000
), therefore it is not illogical to think that these changes may also occur in C. albicans and that they are altered in the Catps2/Catps2 mutant. That the changes are specifically related to the absence of an intact CaTPS2 and not to a lack of trehalose is shown by the fact that Catps1/Catps1 mutants did not aggregate nor showed changes similar to those shown by the Catps2/Catps2 mutant (Zaragoza et al., 1998
).
Two facts support the conclusion that when Catps2/Catps2 cells enter stationary phase, they suffer an important loss of viability: the leakage to the culture medium of high amounts of proteins, most of them intracellular, and the permeability to propidium iodide. We could not quantify the loss of viability by colony counting because the aggregation produced highly variable counts. Again, lack of trehalose was not the cause of loss of viability as Catps1/Catps1 cells did not take up propidium iodide (unpublished results). The effects of the mutation are likely to be a consequence of defects in the cell wall as they are suppressed by osmotic stabilizers. Sorbitol also suppressed the phenotype of the CaHK1 mutant (Calera & Calderone, 1999 ) and remediated the germination defect of conidia of A. nidulans orlA mutants defective in T6P phosphatase (Borgia et al., 1996
). However, its action in the first case could be related to an inhibition of hyphal development (Alex et al., 1998
), while in the second case it appears to be due to a stabilization of the cell wall weakened by a decreased chitin content. A decrease in chitin does not seem to be the cause of the increased permeability of the Catps2/Catps2 mutant, since a decrease in chitin does not affect cell stability in C. albicans (Bulawa et al., 1995
; Gow et al., 1994
).
Our hypothesis is that the accumulation of T6P in the mutant is responsible for alterations in the architecture of the cell wall, a structure implicated in the maintenance of cell integrity and in interactions with other cells. Up to now the only role demonstrated for T6P outside the trehalose biosynthetic pathway is its inhibition of hexokinase (Blázquez et al., 1993 ); our results suggest other regulatory roles for this metabolite. In Sac. cerevisiae, tps1 mutants are not thermosensitive in permissive sources (unpublished data; Elliott et al., 1996
), showing that a lack of trehalose is not the cause of the thermosensitive phenotype of tps2 mutants. These latter mutants accumulate T6P and it has been shown that a decrease in T6P intracellular accumulation suppresses their thermosensitive phenotype (Elliott et al., 1996
), indicating that a high concentration of this metabolite interferes with normal cell growth as we have shown for the Catps2/Catps2 mutant. The possibility that T6P plays a role in the regulation of a cell integrity pathway cannot be ruled out.
The altered colony morphology found on Spider medium may be due to interference of the accumulated T6P with changes in cell wall composition that occur during this process (Schwartz & Larsh, 1980 ). However, we cannot exclude that the protein Tps2 itself may have a direct effect on components of the pathway(s) that control hyphae formation.
The decreased infectivity of the Catps2/Catps2 mutant is clearly due to the lack of CaTPS2 and not to a secondary effect originated during the construction of the disrupted strain, as shown by the regain of infectivity of that strain when transformed with a plasmid bearing the CaTPS2 gene. Several causes may explain the greatly decreased infectivity of the Catps2/Catps2 mutant: a somewhat slower growth rate, defects in the cell integrity or impaired hyphae formation. Since a Catps1/Catps1 mutant also has a lower infection potential (Zaragoza et al., 1998 ), the possibility that trehalose may be an important factor that protects C. albicans from some host defence mechanisms cannot be discarded. In fact, the Catps1/tps1 mutant appears to be highly sensitive to free radicals (J. C. Argüelles, personal communication), compounds that have been proposed to act as a host defence (Murphy, 1991
).
Although the disruption of CaTPS2 did not cause a thermosensitive phenotype in C. albicans, the properties of the Catps2/Catps2 mutant and the absence of a trehalose biosynthetic pathway in mammals still make inhibitors of T6P phosphatase attractive candidates for antifungal therapy.
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ACKNOWLEDGEMENTS |
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Received 29 November 2001;
revised 15 January 2002;
accepted 28 January 2002.
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