School of Life Sciences, Jawaharlal Nehru University, New Delhi-110 067, India
Correspondence
Asis Datta
asisdatta{at}hotmail.com
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ABSTRACT |
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The EMBL accession number for the sequence reported in this paper is AF467941.
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INTRODUCTION |
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This morphological plasticity reflects the interplay of various signalling pathways which control morphogenesis in vivo. In C. albicans, Ras1p is an important regulator of hyphal development and likely functions upstream of the cAMP-dependent protein kinase A (PKA) pathway (Feng et al., 1999). In this pathway, two catalytic subunits or isoforms of PKA, Tpk1p and Tpk2p, have differential effects on hyphal morphogenesis under different hyphal-inducing conditions (Bockmuhl et al., 2001
). Efg1p, a basic helixloophelix (bHLH) protein, plays a major role in hyphal morphogenesis (Leng et al., 2001
; Stoldt et al., 1997
). TPK2 overexpression cannot suppress the efg1/efg1 defect in hyphal development, whereas overexpression of EFG1 can suppress the filamentation defect in tpk2/tpk2, which implies that the function of EFG1 is downstream of TPK2 (Bockmuhl et al., 2001
; Singh et al., 2001
; Sonneborn et al., 2000
). Like in Saccharomyces cerevisiae, Cph1p/Acpr1p, a homologue of Ste12p (Liu et al., 1994
; Singh et al., 1994
, 1997
), and a MAP kinase cascade that includes Cst20p (p21-activated kinase; PAK) (Leberer et al., 1996
, 1997
), Hst7p (MAP kinase kinase; MEK) (Leberer et al., 1996
) and Cek1p (MAPK) (Csank et al., 1998
) are also involved in filamentation in C. albicans. Most importantly, GlcNAc has a dual role to play in that it not only induces the synthesis of its catabolic enzymes, a kinase (Hxk1p), a deacetylase (Dac1p) and a deaminase (Nag1p) (Kumar et al., 2000
), but also regulates GlcNAc-induced transition from a yeast to hyphal form (Singh et al., 2001
). Filamentation regulated by the Nag regulon (HXK1/DAC1/NAG1) is independent of Tpk2p and the Cph1p/Acpr1p-regulated MAP kinase pathway but is dependent on the morphological regulator Efg1p (S. Ghosh and others, unpublished data).
In order to identify and characterize the genes that could be involved in the regulation of morphogenesis and virulence induced by GlcNAc, we performed differential screening of a C. albicans genomic library to identify the genes that are regulated specifically by GlcNAc. Here we report the identification and characterization of the GlcNAc-inducible gene CaGAP1, which is homologous to GAP1, which encodes a general amino acid permease of S. cerevisiae. In yeast, Gap1p is a low-affinity permease with low specificity, which is highly regulated in response to the available nitrogen source (Sophianopoulon & Diallionas, 1995). In the presence of ammonia or glutamine, the amino acid uptake is low, whereas in media containing a poor nitrogen source, e.g. proline, the amino acid uptake is high (Blinder et al., 1996
; Courchesne et al., 1983
). In S. cerevisiae, at least five proteins (Ure2p, Dal80p, Gln3p, Nil1p and Nil2p) function co-ordinately to control the transcription of GAP1 (Blinder et al., 1996
; Cunningham et al., 1993
; Rowen et al., 1997
; Stanbrough et al., 1995
). The nitrogen-dependent regulation of GAP1 is complex, occurring not only at the level of GAP1 transcription but also through Gap1p sorting and degradation by ubiquitin-triggered internalization (Springael et al., 1998
).
In this report, complementation studies by expressing CaGAP1 in a gap1 mutant of S. cerevisiae showed the functional similarity of CaGap1p with the general amino acid permease (Gap1p) of S. cerevisiae. We observed certain differential expression of CaGAP1 in various nitrogen sources as well as in mutants defective in morphogenesis and virulence. We also report some conditions where filamentation and morphogenesis were altered in heterozygous and homozygous disruptants of CaGAP1.
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METHODS |
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Construction of a CaGAP1 expression vector plasmid in S. cerevisiae.
The CaGAP1 coding region was PCR-amplified from genomic DNA of C. albicans SC5314 using the oligonucleotides 5'-TGATCCTTTAATCTTGGAGAAGG-3' and 5'-TGTTCAACCTGGTCAAAGTCC-3' as primers. The 2·2 kb PCR fragment was cloned into the pGEMT-Easy vector followed by transformation into E. coli strain DH5, as per the manufacturer's instructions (Promega), generating pGPORF. A 2224 bp gel-purified NotI fragment containing the CaGAP1 ORF and downstream portion of the ORF was subcloned into pFL61, a yeast expression vector, under the PGK promoter, generating pFLGP31.
Complementation study of CaGAP1 in S. cerevisiae.
Transformation of S. cerevisiae was carried out by the lithium acetate method as described by Gietz et al. (1992). Five micrograms of plasmid pFLPF31 along with 50 µg denatured calf thymus DNA was transformed into the S. cerevisiae gap1 strain MS143. The transformation mix was plated on SD-URA medium using URA3 as a selection marker. The MS143 (
gap1) strain was plated as a control. The transformants were replica-plated on SD medium containing a suitable amount of supplements without uridine. Ura-positive transformants (
gap1 : CaGAP1) were tested on minimal proline plates containing 20 µg uridine ml-1 and 75 µg mimosine ml-1.
Assay of amino acid uptake.
S. cerevisiae and C. albicans strains to be assayed were cultured in SD medium to OD600 2·0. Cells were collected by filtration on 0·45 µm nitrocellulose filters (Sartorious) and resuspended in SPD medium. [14C]Citrulline was added to exponentially growing cultures. Samples of 0·5 ml were removed periodically for 2·5 min, rapidly collected by filtration through a glass fibre filter (Whatman) and washed with chilled water. Filters were dried under a heat lamp and placed in 5 ml toluene-based liquid scintillation cocktail. The counts were taken in a Wallac DSA-based liquid scintillation counter. The specific activity of [14C]citrulline used was 2·1 GBq mmol-1. Labelled citrulline was obtained from Perkin Elmer Life Sciences.
GlcNAc induction studies of CaGAP1.
C. albicans SC5314 cells were precultured in GPK medium and resuspended in 100x volume of fresh GPK. Cultures were grown to OD600 2·0. Harvested cells were washed twice with 0·3 % KH2PO4, resuspended in an equal volume of NPK, and incubated at 30 °C. The treated cells were harvested at different time points of growth as described in Results and frozen at -20 °C until use. Control cells were resuspended in GPK instead of NPK.
To see the effect of GlcNAc induction in different nitrogen sources, strain SC5314 was precultured in SC, washed once with water, resuspended in SN, SEN, SPN, SAN, SUN and SGN with 2 % GlcNAc and grown for 2 h at 30 °C. The treated cells were harvested and frozen at -20 °C until use. Control cells were resuspended in different media, SD, SED, SPD, SAD, SUD and SGD, with 2 % glucose as a carbon source. For studying the effect of GlcNAc induction in different mutants of C. albicans strains, N-2-1-6, N-2-1-6+p33, A-11-1-1-4, CAN52, AS1 and HLC67 were grown similarly in SN medium with 2 % GlcNAc for 2 h at 30 °C and control cells were cultured in SC with 2 % glucose.
RNA extraction and Northern analysis.
Total RNA was extracted from frozen cells (Ausubel et al., 1994). Then 1·5 % formaldehyde agarose gel electrophoresis was carried out with 40 µg RNA per lane, and subsequent Northern blot analysis was performed as described by Ausubel et al. (1994)
with a 32P-labelled 938 bp EcoRV fragment of CaGAP1 (see Fig. 5a
), excised from pCAGAP1.
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Southern analysis.
For screening of mutants and revertant strains, 5 µg genomic DNA from each transformant and parent strain was digested with AatII and SacI, electrophoresed and transferred (Sambrook et al., 1989) to Genescreen Plus membrane (NEN Research Products). The blots were hybridized with a 32P-labelled 3·5 kb NotINotI fragment from pCaGAP1 (Fig. 1c
).
Induction of filamentation by serum and GlcNAc.
Candida cells were grown to the exponential growth phase in YPD, washed twice with sterile water and shaken for 10 h in water at 30 °C and 100 r.p.m. (Sonneborn et al., 2000). Cells (OD600 0·5) were then induced for germ tube formation with 2·5 mM GlcNAc in salt base containing 0·45 % NaCl and 0·335 % YNB without amino acids at 37 °C for 4 h or with bovine calf serum (Sigma) in YPD at 37 °C for 2 h.
Morphogenesis studies on solid media.
Candida strains were grown in SD at 30 °C, counted using a haemocytometer, and plated at a concentration of 80100 cells per Spider (1 % nutrient broth, 1 % mannitol, 0·2 % K2HPO4, 2 % Bacto agar) or SLAD (0·17 % YNB without amino acids and ammonium sulphate, 2 % glucose, 50 µM ammonium sulphate, 2 % Bacto agar) plate. Plates were incubated at both 30 and 37 °C for 710 days.
Determination of virulence.
Female Swiss mice, 56 weeks old, were intravenously injected with 106 cells of wild-type (SC5314), heterozygous Cagap1 mutant (GP5), homozygous mutant (GP573) and revertant (GP57315) strains of C. albicans. The number of surviving mice was scored.
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RESULTS |
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Effect of nitrogen source on the amino acid analogue resistant phenotypes
Amino acids are transported into S. cerevisiae by both specific and non-specific transport systems. The general amino acid permease system is strongly repressed when growth medium contains (NH4)2SO4 and glutamate (Springael & Andre, 1998). To investigate such an effect on the regulation of CaGap1p we did growth kinetics as well as replica plating of wild-type strain MS138, the mutant MS143 (
gap1) and transformants (
gap1 : CaGAP1) of S. cerevisiae on media containing ammonia (SAD) and glutamate (SED) as nitrogen source in the presence of mimosine. Interestingly, the wild-type strain and the transformants were found to be resistant to mimosine in SAD (Fig. 4b, d
) and SED (data not shown). These results suggested that in ammonia- and glutamate-containing medium, mimosine uptake is lowered due to the inactive general amino acid permease system. Moreover, we observed a similar effect when we did growth kinetics as well as 2 days incubation on solid plates of the C. albicans wild-type strain SC5314 and the null mutant GP573 in ammonia- and glutamate-containing media using both glucose and GlcNAc as a carbon source (data not shown).
Effect of different nitrogen sources on GlcNAc induction of CaGAP1
The CaGAP1 gene was isolated as a result of its differential expression in glucose- and GlcNAc-grown cells. Northern analysis was used to investigate the expression of CaGAP1 in glucose-grown and GlcNAc-grown cultures at various intervals. A significant induction was observed in GlcNAc-grown cells at 2 h growth (Fig. 5b).
Northern blot analysis was also used to investigate the effect of different nitrogen sources upon GlcNAc induction of CaGAP1 (Fig. 5c). The intensity of the individual bands was quantified by densitometry of the autoradiogram, and the fold induction has been represented graphically in Fig. 5(d)
. It was observed that in SEN (glutamate), SPN (proline), SUN (urea) or SGN (glutamine) media, the level of CaGAP1 mRNA was about 1·4-fold higher than that of control cells grown in only GlcNAc-containing medium (SN), whereas the CaGAP1 mRNA level was very low in ammonium-containing SAN medium. There was no change in the level of expression in histidine-containing SHN medium (data not shown). The same experiment was carried out using SED, SPD, SAD, SUD, SGD and SHD media where glucose was supplied as carbon source, but no induction or repression was observed (data not shown).
Expression of CaGAP1 is regulated by Cph1p-mediated Ras1p signalling but is independent of Efg1p
To investigate the effect of different mutations on the expression of GlcNAc-inducible CaGAP1, strains N-2-1-6 (dac1
nag1
hxk1/
dac1
nag1
hxk1), N-2-1-6+p33 (
dac1
nag1
hxk1/DAC1NAG1
hxk1), A-11-1-1-4 (
acpr1/
acpr1), CAN52 (
ras1/
ras1), HLC67 (
efg1/
efg1) and AS1 (
tpk2/
tpk2) were used. Northern blots showed that transcript levels of CaGAP1 mRNA declined in the case of the
ras/
ras and
acpr1/
acpr1 null mutants and remained unaffected in the N-2-1-6, N-2-1-6+p33, HLC67 and AS1 strains (Fig. 5e
). This implies that Acpr1p/Cph1p-mediated Ras1p signalling regulates CaGAP1 whereas the cAMP-dependent protein kinase A and Efg1p-mediated Ras1p signalling pathway is not involved in CaGAP1 expression. Although DAC1, NAG1 and HXK1 are induced by GlcNAc, these GlcNAc catabolic pathway genes are not involved in CaGAP1 expression.
Physiological effect of disruption of the CaGAP1 gene
To determine the role of CaGAP1 in the physiology of C. albicans, we disrupted both chromosomal copies of the gene sequentially by the URA-blaster technique (Fonzi & Irwin, 1993). Growth rates of the wild-type (SC5314), heterozygous mutant (GP5), homozygous mutant (GP573) and heterozygous revertant (GP57315) were similar at 30 °C in glucose-containing media (data not shown). In a murine mouse model, no change in virulence was observed with the mutant strains (data not shown). When GlcNAc was used as a carbon source, the growth rate was higher but no striking difference was found among the wild-type and mutant strains. All the strains used for growth kinetics and morphological studies were Ura+. C. albicans can shift from a yeast to a hyphal form when it is cultured at 37 °C in the presence of serum and GlcNAc. This transition was not impaired or affected in a Cagap1/Cagap1 mutant (GP573) in both serum (Fig. 6a, b, c, d
) and GlcNAc (Fig. 6e, f, g, h
) induction media. However, we observed less hyphal clump formation by GlcNAc in the Cagap1/Cagap1 mutant in a shake flask (Fig. 6g
). No difference was found in a heterozygous mutant and a heterozygous revertant with respect to this behaviour (Fig. 6f, h
). We then assessed the filamentous growth from mature colony borders on solid Spider agar in which mannitol, but not glucose, is used as a carbon source at 30 °C. Only the Cagap1 null mutant (GP573) showed less hyphal formation and altered colony morphology which was different from the wild-type strain and the heterozygous mutant (Fig. 6i, j, k
). This phenotype was reversed by reconstituting a single functional copy of the gene (Fig. 6l
). An interesting feature of our analysis was the finding that both the heterozygous and homozygous mutants had an obvious defect in filamentation and drastic abnormal colony morphology on nitrogen-starvation solid SLAD plates at 37 °C (Fig. 6n, o
). Furthermore, the defect in filamentation and colony morphology is not fully suppressed by introduction of a single copy of a functional gene (Fig. 6p
). However, Cagap1/Cagap1 homozygous disruptants were more homogeneous than the heterozygous strain and showed a greater reduction in peripheral hyphal growth, indicating that gene dosage is important for morphogenesis of C. albicans under certain conditions.
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DISCUSSION |
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CaGAP1 (AF467941) is not only homologous to GAP1 (CAA82113) of S. cerevisiae but also shows similarity to other yeast permease genes such as HIP1, TAT2, AGP1 and GPN1 (Jauniaux & Grenson, 1990). The deduced gene product is highly hydrophobic with 1012 transmembrane regions. CaGAP1 was induced by GlcNAc at 2 h growth but was expressed only at a basal level in glucose-containing complete medium. The GlcNAc induction of CaGAP1 was enhanced in synthetic minimal media supplemented with a single amino acid such as glutamate, proline, glutamine or urea but was inhibited by ammonia. The regulation of CaGAP1 at the level of transcription is comparable to GAP1 regulation in yeast, where the transcription factors Gln3p (in the presence of glutamate) and Nil1p (in the presence of urea or proline) are activators (Stanbrough et al., 1995
), while Dal80p (Cunningham & Cooper, 1993
) and Nil2p (Lodish, 1988
; Rowen et al., 1997
) are inhibitors. In the presence of ammonium, Ure2p, another transcriptional repressor, sterically hinders Gln3p from activating GAP1 (Blinder et al., 1996
). These factors bind to an upstream regulatory sequence containing a motif surrounding a core GATA sequence (Springael & Andre, 1998
). An obvious similarity between the CaGAP1 promoter and a nitrogen-regulated gene promoter like GAP1, GLN1, GDH2, etc., of S. cerevisiae is the presence of a GAATAG sequence (Cunningham & Cooper, 1993
). Another feature common to the CaGAP1 and GAP1 promoters is the presence of TTGGT or TTGTT, which plays an auxiliary note in activation by Gln3p (Miller & Magasanik, 1991
). Five GATA-type transcription factors and one gene homologous to URE2 have been reported from the C. albicans Genome Sequencing Project, Stanford. One can therefore presume that the regulation of CaGAP1 might be brought about by all of them.
In our induction studies we also saw that CaGAP1 is GlcNAc-inducible, but in the GlcNAc catabolic pathway mutants dac1
nag1
hxk1/
dac1
nag1
hxk1 (Nag regulon mutated) and
dac1
nag1
hxk1/DAC1NAG1
hxk1 (hexokinase mutant), which are incapable of utilizing GlcNAc (Singh et al., 2001
), there was no change in induction of CaGAP1 when GlcNAc was added to the media. This fact implies that catabolism of GlcNAc is not required for expression of CaGAP1, but whether GlcNAc directly enhances the expression of CaGAP1 or whether it binds to some surface receptor which transmits the signals via some other intermediate proteins is still unknown. However, GlcNAc induction of the CaGAP1 gene is less in cph1/cph1 and ras1/ras1 null mutants while no striking change of expression was found in efg1/efg1 and tpk2/tpk2 mutant strains. It was also reported that the N-terminal region of Acprp/Cph1p can recognize and bind PREs in vitro like Ste12p of S. cerevisiae (Malathi et al., 1994
). Interestingly, one PRE sequence, TGAAACA, is also present in the CaGAP1 promoter. This clearly showed the role of Cph1p-dependent Ras1p signalling in GlcNAc-induced CaGAP1 expression.
Gap1p of S. cerevisiae is not only regulated transcriptionally but its activity also depends on the external nitrogen source. Addition of ammonium ions (Springael & Andre, 1998; Bernard & Andre, 2001
) or glutamate (Roberg et al., 1997
) inhibits the activity of Gap1p in S. cerevisiae. We found in our study that mimosine inhibited the growth of a wild-type strain and transformants (
gap1 : : CaGAP1) of S. cerevisiae on minimal proline media but was unable to do so in ammonium- or glutamate-containing media. This indicates that CaGap1p is probably not functional in ammonia- or glutamate-grown cells. Similarly in C. albicans, mimosine affected the growth of wild-type strain SC5314 and the revertant strain (GP57315) while a Cagap1 null mutant (GP573) could resist the drug effect in minimal proline medium. In Candida strains, the effect of mimosine persists for a maximum of 1015 h, which may be because of the higher growth rate of this micro-organism.
Yeast possesses many amino acid permeases with overlapping substrate specificities. The general amino acid permease Gap1p, which can transport most amino acids, can be specifically assayed by uptake of [14 C]citrulline (Grenson et al., 1970). To demonstrate the import of amino acids by CaGap1p, a citrulline uptake assay was performed in minimal proline medium. General amino acid activity was increased 2·5-fold when the CaGAP1 gene was expressed in the gap1 mutant strain (
gap1 : : CaGAP1) of S. cerevisiae. On the other hand, the Cagap1 mutant (GP573) of C. albicans showed 50 % less citrulline uptake than the wild-type strain (SC5314) and the permease activity was regained when the CaGAP1 gene was recombined back in the CaGAP1 locus of the Cagap1 mutant strain (GP57315). Therefore, we could not exclude the possibility that the transport pattern of the general amino acid permease is the same in both S. cerevisiae and C. albicans.
We have also shown here that Cagap1/Cagap1 has defects in filamentation on solid Spider and SLAD medium, forming only a few short hyphae instead of the florid filaments that emanate from the wild-type strain. Despite this defect, Cagap1/Cagap1 could not block the induction of filaments by serum response, but we found less hyphal clump formation in GlcNAc inducing conditions. Defective morphology and less filamentation of both the heterozygous and homozygous mutants during nitrogen starvation strongly suggest that the GlcNAc-inducible CaGAP1 is regulated by the external nitrogen source. Thus one interpretation of these data is that GlcNAc-induced hyphal formation is sensitive to the dosage of the CaGAP1 gene under nitrogen source control. Herein lies the importance of GlcNAc, which not only acts as an inducer of hyphal formation (Mattia et al., 1982; Simonitti et al., 1974
) but also regulates the expression of a number of genes within the cell. Through the induction of CaGAP1, GlcNAc might indirectly alter the nutritional status of the cell, by causing an increased uptake of amino acids. Again, depending on the source of nitrogen in the extracellular medium, CaGAP1 is induced or repressed. In a poor nitrogen source like minimal proline medium or under nitrogen starvation conditions, CaGAP1 is induced by GlcNAc through the Cph1p-mediated Ras1p signalling pathway, which leads to a morphological change. This interplay between GlcNAc and different nitrogen sources probably brings about a co-ordinated regulation of CaGAP1 expression and morphogenesis.
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ACKNOWLEDGEMENTS |
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Received 31 December 2002;
revised 10 April 2003;
accepted 24 April 2003.