A transcription factor regulatory cascade controls secreted aspartic protease expression in Candida albicans

Authors


*E-mail joachim.morschhaeuser@mail.uni-wuerzburg.de; Tel. (+49) 931 31 21 52; Fax (+49) 931 31 25 78.

Summary

Secreted aspartic proteases (Saps) contribute to the virulence of Candida albicans, a major fungal pathogen of humans. One function of the Saps, which is specifically mediated by the Sap2p isoenzyme, is the degradation of proteins for use as a nitrogen source. The utilization of alternative nitrogen sources in fungi is controlled by GATA transcription factors and we found that C. albicans mutants lacking the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing bovine serum albumin (BSA) as the sole nitrogen source. The growth defect was mainly caused by the inability of gln3Δgat1Δ mutants to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p, and forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. SAP2 is repressed when preferred nitrogen sources are available and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 in the presence of high concentrations of ammonium, glutamine or urea. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that the control of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, secreted aspartic protease expression, a long-known virulence attribute of C. albicans, is controlled by a regulatory cascade in which the general regulators Gln3p and Gat1p control the expression of the transcription factor Stp1p, which in turn mediates SAP2 expression.

Introduction

The yeast Candida albicans is a commensal microorganism on mucosal surfaces in most healthy people but, in immunocompromised patients, it can also cause serious infections of many different organs (Odds, 1988). One of the first recognized virulence attributes of C. albicans was the secretion of aspartic proteases (Staib, 1965; 1969; Remold et al., 1968). These enzymes may help the fungus to gain access to and establish itself in different host niches by degrading tissue barriers (Colina et al., 1996; Morschhäuser et al., 1997), contributing to adherence (Watts et al., 1998), digesting host proteins for nutrient supply (Staib, 1965) and destroying host defence molecules like immunoglobulins and complement proteins (Rüchel, 1981; Kaminishi et al., 1995). C. albicans possesses a family of 10 genes encoding secreted aspartic proteases (Saps), which are differentially expressed and differ in their enzymatic characteristics and substrate specificities and may therefore have specific roles during an infection (Staib et al., 2000; Naglik et al., 2003). The Sap2p isoenzyme is the major in vitro secreted protease (Hube et al., 1994; White and Agabian, 1995). Its expression is induced in media containing proteins as the sole nitrogen source and repressed when preferred nitrogen sources, like ammonium or amino acids, are available (Ross et al., 1990; Banerjee et al., 1991). Sap2p degrades proteins mainly to oligopeptides, which are then taken up into the cell by oligopeptide transporters encoded by the OPT gene family (Reuß and Morschhäuser, 2006). Deletion of SAP2 or several members of the OPT gene family renders C. albicans unable to grow in YCB–BSA medium, which contains bovine serum albumin as the sole nitrogen source (Hube et al., 1997; Staib et al., 2002; 2008; Reuß and Morschhäuser, 2006). Mutants lacking SAP2 also exhibit reduced virulence in animal models of candidiasis (Hube et al., 1997; De Bernardis et al., 1999; Staib et al., 2002), supporting the idea that the ability to utilize proteins as a nitrogen source is important for growth of C. albicans in certain host niches.

Initial work suggested that peptides that are produced from proteins by basal extracellular proteolytic activity serve as the inducers of SAP2 expression (Lerner and Goldman, 1993; Hube et al., 1994). However, it was recently shown that micromolar concentrations of amino acids, which may also be produced during the degradation of extracellular proteins, are the actual inducers of SAP2 (Martinez and Ljungdahl, 2005). Extracellular amino acids are sensed at the cell surface by the SPS sensor, which then proteolytically activates the transcription factors Stp1p and Stp2p. The activated transcription factors are targeted to the nucleus, where Stp1p induces expression of SAP2 and the oligopeptide transporters OPT1 and OPT3 (Martinez and Ljungdahl, 2005).

The use of alternative nitrogen sources in fungi is controlled by GATA transcription factors (Marzluf, 1997). In the yeast Saccharomyces cerevisiae, the two GATA factors Gln3p and Gat1p (Nil1p) activate the expression of catabolic enzymes and uptake systems for secondary nitrogen sources when preferred nitrogen sources are absent or present at insufficient levels for normal growth (Magasanik and Kaiser, 2002). These zinc-finger transcription factors bind to GATA sites in the regulatory region of their target genes to promote transcription, the relative contribution of Gln3p and Gat1p depending on the individual gene and the growth conditions. In the presence of sufficient amounts of a preferred nitrogen source, the GATA factors are retained in the cytoplasm and cannot activate their target genes. This regulation of nitrogen-sensitive genes is known as nitrogen catabolite repression. In addition to the control by the global regulators Gln3p and Gat1p, many nitrogen-regulated genes are also controlled by additional, pathway-specific transcription factors (Marzluf, 1997).

C. albicans possesses homologues of GLN3 and GAT1 which, like in S. cerevisiae, control the expression of nitrogen-regulated genes and are required for optimal growth on a variety of nitrogen sources (Limjindaporn et al., 2003; Dabas and Morschhäuser, 2007; Liao et al., 2008). GLN3, but not GAT1, is also required for filamentous growth, another virulence-associated characteristic of C. albicans, in response to nitrogen starvation (Dabas and Morschhäuser, 2007; Liao et al., 2008). Mutants lacking either GAT1 or GLN3 are attenuated in their virulence, indicating that the ability to properly control the expression of nitrogen-regulated genes is important for the pathogenicity of C. albicans (Limjindaporn et al., 2003; Liao et al., 2008).

Proteins can be considered as an alternative or secondary nitrogen source for C. albicans, as the expression of genes required for their utilization, i.e. SAP2 and the oligopeptide transporters OPT1 and OPT3, is induced in the presence of proteins and SAP2 is repressed even in the presence of proteins when preferred nitrogen sources, like ammonium or amino acids, are available. We therefore investigated if Gln3p and Gat1p are required for the utilization of proteins as a nitrogen source and if and how these transcription factors contribute to the regulation of SAP2 expression.

Results

GAT1 and GLN3 are required for growth of C. albicans on proteins

As GATA transcription factors are known to regulate the expression of genes required for the utilization of alternative nitrogen sources, we tested the ability of our previously constructed gln3Δ, gatΔ and gln3Δgat1Δ mutants to grow in YCB–BSA medium, in which a protein (BSA) is the only available nitrogen source. Growth of the gln3Δ mutants was only slightly reduced as compared with the wild-type parental strain SC5314 (Fig. 1A). In contrast, the gat1Δ mutants had a severe growth defect (Fig. 1B) and growth of the gln3Δgat1Δ double mutants was virtually abolished (Fig. 1C). Re-introduction of a functional copy of GLN3 or GAT1 into the respective single mutants restored wild-type growth (Fig. 1A and B) and the double mutants that were complemented with GLN3 behaved like gat1Δ single mutants (compare Fig. 1B and C). Re-introduction of GAT1 into the double mutants also restored growth (Fig. 1C). However, we noted a GAT1 copy number effect in the absence of GLN3, because the double mutants into which one GAT1 allele was re-introduced grew less well than the gln3Δ single mutants, which contained both GAT1 alleles (compare Fig. 1A and C). The same effect was observed in the gln3Δ mutants in which one of the GAT1 wild-type alleles had been deleted (data not shown). These results demonstrated that both GATA factors, but especially GAT1, are required to efficiently utilize proteins as a nitrogen source.

Figure 1.

The GATA transcription factors Gln3p and Gat1p are required for utilization of proteins as a nitrogen source. Overnight cultures of the strains in YPD medium were diluted 10−2 in YCB–BSA medium and incubated at 30°C. Growth was monitored by measuring the optical density of the cultures at the indicated times. All strains were tested in parallel, but the results for the gln3Δ mutants, gat1Δ mutants and gln3Δgat1Δ double mutants are shown separately in A–C for better illustration, together with the results for the corresponding complemented strains. The results for the wild-type strain SC5314 are shown in all three panels. The two independently constructed series of mutants and complemented strains behaved identically and only one of them is shown for better clarity.

Functional analysis of the GAT1 gene

In assembly 19 of the C. albicans genome sequence, the open reading frame (ORF) defined as GAT1 (orf19.1275) is 2067 bp in length and encodes a predicted protein of 688 amino acids. However, Limjindaporn et al. (2003) obtained a plasmid clone from a genomic library of strain SC5314, the strain used for genome sequencing, in which an upstream stop codon was changed to a sense codon, extending the N-terminal part of the GAT1 ORF by 201 bp to encode a predicted protein of 755 amino acids. When we amplified GAT1 from strain SC5314 for re-introduction into the gatΔ single and gln3Δgat1Δ double mutants, we noted that the cloned GAT1 gene in the resulting plasmid pGAT1K1 (see Fig. 2A) contained the stop codon reported in the genome sequence. As the GAT1 alleles of strain SC5314 can be distinguished by a downstream BglII restriction site polymorphism, we amplified the remaining intact GAT1 alleles from the heterozygous GAT1/gat1Δ mutants GAT1M2A and GAT1M2B, in which either of the two alleles was inactivated (see Fig. 2B, lanes 2 and 3). Sequencing of the resulting clones confirmed that strain SC5314 contained two polymorphic GAT1 alleles. The allele located on the smaller BglII fragment, which we had arbitrarily designated as allele 1, was identical to the one reported by Limjindaporn et al. (2003), whereas allele 2 was identical to orf19.1275 of the C. albicans genome sequence. As both heterozygous gat1 mutants grew as well as the wild-type strain in YCB–BSA medium and the growth defect of the homozygous gat1Δ mutants was fully complemented by the GAT1-2 allele containing the upstream stop codon (Fig. 1B), both GAT1 alleles must be functional. To complicate matters, a different start codon located 63 bp further downstream has recently been assigned to orf19.1275 in assemblies 20 and 21 of the C. albicans genome sequence, so that the GAT1 ORF would be only 2004 bp and encode a predicted protein of 667 amino acids. We performed 5′ RACE analysis and found that the GAT1 mRNA starts at an adenine that is located 60 nucleotides upstream of the first start codon and therefore includes all three potential GAT1 ORFs. To investigate if all three potential GAT1 ORFs, which we refer to here as GAT12268, GAT12067 and GAT12004 according to their length, encode functional proteins, we expressed the different ORFs from a tetracycline-inducible promoter in the gat1Δ mutants (see Experimental procedures and Fig. 3A) and tested growth of the corresponding transformants in YCB–BSA. As shown in Fig. 3B, all three GAT1 ORFs fully restored growth of the gat1Δ mutants. Therefore, even the short GAT12004 allele encodes a functional protein, although this finding does not exclude the possibility that translation of Gat1p in the wild-type strain starts further upstream and the N-terminal part of the protein is dispensable.

Figure 2.

Re-introduction of a functional GAT1 copy into gat1Δ mutants.
A. Structure of the DNA fragment from pGAT1K1 (top), which was used for re-integration of an intact GAT1 copy into one of the disrupted gat1Δ::FRT alleles (bottom) in the gat1Δ single and gln3Δgat1Δ double mutants. The GAT1 coding region is represented by the white arrow and the upstream and downstream regions by the solid lines. Details of the SAT1 flipper [grey rectangle bordered by FRT sites (black arrows)] have been presented elsewhere (Reußet al., 2004). The 34 bp FRT sites are not drawn to scale. The probes used for Southern hybridization analysis of the mutants are indicated by the black bars. Only relevant restriction sites are given: A, ApaI; Bg, BglII; H, HindIII; ScI, SacI; ScII, SacII; Xh, XhoI. The BglII site marked in italics is present only in the GAT1-1 allele.
B. Southern hybridization of HindIII/BglII-digested genomic DNA of the wild-type strain SC5314, gat1Δ mutants and complemented strains with the GAT1-specific probe 2. The sizes of the hybridizing fragments (in kb) are given on the left side of the blot and their identities are indicated on the right. Insertion of the SAT1 flipper into either of the two GAT1 alleles of the parental strain SC5314 (lane 1) and subsequent FLP-mediated excision of the cassette produced the heterozygous mutants GAT1M2A and B (lanes 2 and 3). Insertion of the SAT1 flipper into the remaining wild-type GAT1 alleles, followed by recycling of the SAT1 flipper cassette, gave rise to the homozygous gat1Δ mutants GAT1M4A and B (lanes 4 and 5). An intact GAT1 copy was re-introduced into the gat1Δ mutants with the help of the SAT1 flipper, which was subsequently excised again to produce the complemented strains GAT1MK2A and B (lanes 6 and 7). Note that the fragments containing the re-inserted GAT1 copy are larger than the original wild-type fragments as a result of the duplication of part of the GAT1 downstream region.

Figure 3.

Complementation of the growth defect of gat1Δ mutants by different GAT1 alleles.
A. Structure of the DNA cassettes that were used for expression of the GAT12268, GAT12067 and GAT12004 alleles under control of a tetracycline-inducible promoter (Ptet) after integration into the ADH1 locus of the gat1Δ mutants. Bent arrows symbolize promoters (P), the filled circles represent TACT1, which serves for proper transcription termination of the Candida-adapted, reverse tetracycline-dependent transactivator (cartTA) and the target genes in this cassette (Park and Morschhäuser, 2005). Only relevant restriction sites explaining plasmid construction or used to excise the whole cassette from the vector backbone are shown: A, ApaI; B, BamHI; Bg, BglII; S, SmaI; Sl, SalI; ScII, SacII.
B. Growth of the parental strain SC5314 (wild type), gat1Δ mutants and transformants containing the indicated Ptet-GAT1 fusions in YCB–BSA medium in the absence (–) or presence (+) of 50 μg ml−1 doxycycline. Photographs of the cultures were taken after 20 h of growth and their optical densities are given below the tubes. Note that doxycycline slightly reduced growth of the wild-type strain under these conditions.

The GATA transcription factors Gat1p and Gln3p control expression of Sap2p and oligopeptide transporters

Previous work had shown that the secreted aspartic protease Sap2p and oligopeptide transporters are required for the ability of C. albicans to utilize proteins as a nitrogen source and that expression of the SAP2, OPT1 and OPT3 genes is induced in YCB–BSA medium (Hube et al., 1994; Staib et al., 2002; Reuß and Morschhäuser, 2006). To investigate if expression of these genes depends on GLN3 and GAT1, we introduced reporter gene fusions in which GFP was placed under the control of the respective promoters into the gln3Δ, gat1Δ and gln3Δgat1Δ mutants. The strains were grown in YCB–BSA–YE medium, which also induces SAP2 and OPT gene expression but allows growth of sap2Δ and optΔ mutants (Staib et al., 2002; Reuß and Morschhäuser, 2006), and the gln3Δgat1Δ mutants also had no growth defect in this medium. As shown in Fig. 4, the activity of all three tested promoters was reduced in the gln3Δ and gatΔ single mutants, with a stronger effect seen in the gat1Δ mutants. Very low or undetectable promoter activity was observed in mutants lacking both transcription factors. The failure of the gln3Δgat1Δ mutants to express SAP2 was also confirmed by analysing culture supernatants of the wild type and mutants grown in YCB–BSA–YE on SDS gels (Fig. 5). The wild-type strain SC5314 had completely degraded the BSA within 8 h of growth in this medium and Sap2p expression was readily detected by Western immunoblotting with an anti-Sap2p antibody (Fig. 5, lanes 2). BSA degradation was delayed in the gln3Δ (Fig. 5, lanes 3 and 7) and gat1Δ (Fig. 5, lanes 4 and 8) mutants and reduced amounts of Sap2p were detected in the culture supernatant of the latter. However, the strong differences in SAP2 promoter activity in the wild type and gln3Δ and gat1Δ mutants (see Fig. 4) were not reflected in Sap2p levels produced by these strains. This is presumably explained by the fact that in all reporter strains, the promoter activity of the SAP2-1 allele was determined and most of the secreted Sap2p was probably produced from the more strongly expressed SAP2-2 allele, which is essential for growth in YCB–BSA (Staib et al., 2002). In contrast, only very limited BSA degradation was detected in the culture supernatants of the gln3Δgat1Δ double mutants and Sap2p was not detectably expressed in these strains, which behaved similarly to a sap2Δ mutant (compare lanes 5 and 9 with lanes 6 in Fig. 5). In summary, these results demonstrate that the GATA transcription factors Gat1p and Gln3p control the expression of genes that are known to be required for growth of C. albicans on proteins.

Figure 4.

GLN3 and GAT1 control expression of the secreted aspartic protease 2 (SAP2) and the oligopeptide transporters OPT1 and OPT3. YPD overnight cultures of strains expressing GFP under control of the indicated promoter in a wild-type, gln3Δ, gat1Δ or gln3Δgat1Δ background were diluted 10−2 in YCB–BSA–YE medium, grown for 8 h (left panels) or 15 h (right panels) at 30°C, and analysed by flow cytometry. The mean fluorescence of each cell population is given (arbitrary units). The first columns show the results obtained with the A series and the second columns show the results obtained with the B series of the reporter strains (see Table 1). The parental strain SC5314, which does not contain GFP, was used as a negative control.

Figure 5.

Sap2p expression and BSA degradation by the wild-type strain SC5314 (lane 2) and the two independently constructed A and B series of gln3Δ (lanes 3 and 7), gat1Δ (lanes 4 and 8) and gln3Δgat1Δ mutants (lanes 5 and 9). Strains were grown for 8 h (left) or 15 h (right) in YCB–BSA–YE medium at 30°C and the culture supernatants were analysed by SDS-PAGE (top) and by Western immunoblotting with an anti-Sap2p antibody (bottom). A sap2Δ mutant (lane 6) as well as uninoculated medium (lane 1) were included as controls. M, molecular size marker.

Forced expression of SAP2 overcomes the growth defect of gln3Δ gat1Δ mutants

To investigate if the growth defect of mutants lacking the GATA transcription factors Gln3p and Gat1p was caused by their inability to adequately express SAP2 and oligopeptide transporters, we expressed SAP2 and OPT1 from the strong ADH1 promoter in the gln3Δgat1Δ mutants. Expression of OPT1 from the ADH1 promoter has previously been shown to complement the growth defect in YCB–BSA of mutants lacking multiple oligopeptide transporters (Reuß and Morschhäuser, 2006). As can be seen in Fig. 6, forced expression of SAP2 from the ADH1 promoter restored growth of the double mutants, althoug not to wild-type levels. In contrast, expression of OPT1 alone had no effect. These results indicate that the failure to induce SAP2 expression is the main cause of the inability of the gln3Δgat1Δ mutants to utilize proteins as a nitrogen source.

Figure 6.

Forced SAP2 expression restores growth of gln3Δgat1Δ double mutants in YCB–BSA medium. YPD overnight cultures of the wild-type strain SC5314, gln3Δgat1Δ double mutants and transformants expressing OPT1 or SAP2 from the ADH1 promoter were diluted 10−2 in YCB–BSA and incubated at 30°C. Growth was monitored by measuring the optical density of the cultures at the indicated times. The two independently constructed series of mutants and transformants behaved identically and only one of them is shown.

Forced expression of the transcription factor STP1 bypasses the requirement of GATA transcription factors for growth on proteins

It was recently shown that the transcription factor Stp1p regulates expression of SAP2 and OPT1 in response to the presence of micromolar concentrations of extracellular amino acids (Martinez and Ljungdahl, 2005). Under these conditions, Stp1p is proteolytically processed to its activated form and localizes to the nucleus to induce expression of its target genes. Mutants lacking STP1 do not express SAP2 and OPT1 and cannot utilize proteins as a nitrogen source. To study the relationship between Stp1p and the GATA transcription factors Gln3p and Gat1p, we tested whether forced expression of an N-terminally truncated, constitutively active Stp1p would overcome the growth defect of the gln3Δgat1Δ mutants in YCB–BSA medium. For this purpose, a STP1ΔN61 allele was integrated into the genome of the gln3Δgat1Δ mutants under control of the Tet promoter (see Experimental procedures). As can be seen in Fig. 7A, doxycycline-induced expression of the STP1ΔN61 allele fully restored growth of the mutants. In fact, the transformants expressing this allele started to grow even earlier than the wild-type strain SC5314 after transfer from YPD to YCB–BSA medium, presumably because expression of the activated transcription factor allowed a faster adaptation of the cells to the switch of the nitrogen source even in the absence of the GATA transcription factors. This result suggested that Gln3p and Gat1p might be required for STP1 expression or Stp1p activation. To distinguish between these possibilities, we expressed wild-type STP1 from the Tet promoter. Figure 7A shows that doxycycline-induced expression of wild-type STP1 also rescued the growth defect of the gln3Δgat1Δ mutants, although growth of the strains was somewhat delayed in comparison with that of the wild-type strain SC5314. Therefore, forced expression of STP1 bypasses the requirement of GLN3 and GAT1 for growth of C. albicans on proteins.

Figure 7.

A. Forced expression of the transcription factor STP1 overcomes the growth defect of gln3Δgat1Δ double mutants. YPD overnight cultures of the wild-type strain SC5314 and transformants of the gln3Δgat1Δ double mutants expressing full-length STP1 or the constitutively active STP1ΔN61 allele from the Tet promoter were diluted 10−2 in YCB–BSA in the absence or presence of 50 μg ml−1 doxycycline and incubated at 30°C. Growth was monitored by measuring the optical density of the cultures at the indicated times. The two independently constructed series of transformants behaved identically and only one of them is shown.
B. Forced expression of GLN3 or GAT1 does not rescue the growth defect of stp1Δ mutants in YCB–BSA medium. YPD overnight cultures of the wild-type strain SC5314, two independently generated stp1Δ mutants and transformants expressing STP1, GLN3 or the indicated GAT1 ORFs from the Tet promoter were diluted 10−2 in YCB–BSA containing 50 μg ml−1 doxycycline. The optical density of the cultures was measured after 20 h of growth at 30°C.

To investigate if, vice versa, forced expression of GLN3 or GAT1 would also allow cells lacking Stp1p to grow on proteins, we deleted STP1 in the wild-type strain SC5314 and expressed GLN3 and the GAT12268, GAT12067 and GAT12004 alleles from the Tet promoter in the stp1Δ mutants (see Experimental procedures and Table 1 for strain constructions). In agreement with a previous report (Martinez and Ljungdahl, 2005), the stp1Δ mutants exhibited a growth defect in YCB–BSA medium, which was complemented by expression of a STP1 copy from the Tet promoter (Fig. 7B). In contrast, tetracycline-induced expression of GLN3 and GAT1 did not rescue the growth defect of the stp1Δ mutants, indicating that the GATA transcription factors cannot efficiently induce SAP2 expression in the absence of Stp1p.

Table 1. C. albicans strains used in this study.
StrainParentRelevant genotype or characteristicsReference
  • a. 

    SAT1-FLIP denotes the SAT1 flipper cassette.

 SC5314 Wild-type strainGillum et al. (1984)
 SAP2MS4BSC5314sap2Δ::FRT/sap2Δ::FRTStaib et al. (2008)
gln3Δ, gat1Δ and gln3Δgat1Δ mutants and complemented strains
 GLN3M2ASC5314gln3-1Δ::FRT/GLN3-2Dabas and Morschhäuser (2007)
 GLN3M2BSC5314GLN3-1/gln3-2Δ::FRTDabas and Morschhäuser (2007)
 GLN3M4AGLN3M2Agln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
 GLN3M4BGLN3M2Bgln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
 GLN3MK2AGLN3M4AGLN3-FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
 GLN3MK2BGLN3M4Bgln3-1Δ::FRT/GLN3-FRTDabas and Morschhäuser (2007)
 GAT1M2ASC5314gat1-1Δ::FRT/GAT1-2Dabas and Morschhäuser (2007)
 GAT1M2BSC5314GAT1-1/gat1-2Δ::FRTDabas and Morschhäuser (2007)
 GAT1M4AGAT1M2Agat1-1Δ::FRT/gat1-2Δ::FRTDabas and Morschhäuser (2007)
 GAT1M4BGAT1M2Bgat1-1Δ::FRT/gat1-2Δ::FRTDabas and Morschhäuser (2007)
 GAT1MK1AGAT1M4AGAT1-2-SAT1-FLIPa/gat1-2Δ::FRTThis study
 GAT1MK1BGAT1M4Bgat1-1Δ::FRT/GAT1-2-SAT1-FLIPThis study
 GAT1MK2AGAT1MK1AGAT1-2-FRT/gat1-2Δ::FRTThis study
 GAT1MK2BGAT1MK1Bgat1-1Δ::FRT/GAT1-2-FRTThis study
 Δgln3GAT1M2AGLN3M4Agln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
gat1-1Δ::FRT/GAT1-2 
 Δgln3GAT1M2BGLN3M4Bgln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
GAT1-1/gat1-2Δ::FRT 
 Δgln3GAT1M4AΔgln3GAT1M2Agln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
gat1-1Δ::FRT/gat1-2Δ::FRT 
 Δgln3GAT1M4BΔgln3GAT1M2Bgln3-1Δ::FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
gat1-1Δ::FRT/gat1-2Δ::FRT 
 Δgln3GAT1MK2AΔgln3GAT1M4AGLN3-FRT/gln3-2Δ::FRTDabas and Morschhäuser (2007)
gat1-1Δ::FRT/gat1-2Δ::FRT 
 Δgln3GAT1MK2BΔgln3GAT1M4Bgln3-1Δ::FRT/GLN3-FRTDabas and Morschhäuser (2007)
gat1-1Δ::FRT/gat1-2Δ::FRT 
 Δgln3GAT1MK3AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
GAT1-2-SAT1-FLIP/gat1-2Δ::FRT 
 Δgln3GAT1MK3BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/GAT1-2-SAT1-FLIP 
 Δgln3GAT1MK4AΔgln3GAT1MK3Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
GAT1-2-FRT/gat1-2Δ::FRT 
 Δgln3GAT1MK4BΔgln3GAT1MK3Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/GAT1-2-FRT 
gat1Δ mutants expressing different GAT1 versions from a tetracycline-inducible promoter
 Δgat1TET1-GAT1-1AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12268 
 Δgat1TET1-GAT1-1BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12268 
 Δgat1TET1-GAT1-2AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12067 
 Δgat1TET1-GAT1-2BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12067 
 Δgat1TET1-GAT1-3AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12004 
 Δgat1TET1-GAT1-3BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
ADH1/adh1::Ptet-GAT12004 
Strains expressing PSAP2–GFP, POPT1–GFP, POPT3–GFP and PSTP1–GFP reporter gene fusions
 SCSAP2G1A and -BSC5314sap2-1::PSAP2-1–GFP/SAP2-2Reuß and Morschhäuser (2006)
 SCOPT1G22ASC5314OPT1-1/opt1-2::POPT1–GFPReuß and Morschhäuser (2006)
 SCOPT1G22BSC5314opt1-1::POPT1–GFP/OPT1-2Reuß and Morschhäuser (2006)
 SCOPT3G22ASC5314opt3-1::POPT3–GFP/OPT3-2Reuß and Morschhäuser (2006)
 SCOPT3G22BSC5314OPT3-1/opt3-2::POPT3–GFPReuß and Morschhäuser (2006)
 SCSTP1G1 A and -BSC5314STP1/stp1::PSTP1–GFPThis study
 Δgln3SAP2G1AGLN3M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
sap2-1::PSAP2–GFP/SAP2-2 
 Δgln3SAP2G1BGLN3M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
sap2-1::PSAP2–GFP/SAP2-2 
 Δgln3OPT1G22AGLN3M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
OPT1-1/opt1-2::POPT1–GFP 
 Δgln3OPT1G22BGLN3M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
OPT1-1/opt1-2::POPT1–GFP 
 Δgln3OPT3G22AGLN3M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
opt3-1::POPT1–GFP/OPT3-2 
 Δgln3OPT3G22BGLN3M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
OPT3-1/opt3-2::POPT1–GFP 
 Δgln3STP1G1AGLN3M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
STP1/stp1::PSTP1–GFP 
 Δgln3STP1G1BGLN3M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
STP1/stp1::PSTP1–GFP 
 Δgat1SAP2G1AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
sap2-1::PSAP2–GFP/SAP2-2 
 Δgat1SAP2G1BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
sap2-1::PSAP2–GFP/SAP2-2 
 Δgat1OPT1G22AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
OPT1-1/opt1-2::POPT1–GFP 
 Δgat1OPT1G22BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
opt1-1::POPT1–GFP/OPT1-2 
 Δgat1OPT3G22AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
OPT3-1/opt3-2::POPT1–GFP 
 Δgat1OPT3G22BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
opt3-1::POPT1–GFP/OPT3-2 
 Δgat1STP1G1AGAT1M4Agat1-1Δ::FRT/gat1-2Δ::FRTThis study
STP1/stp1::PSTP1–GFP 
 Δgat1STP1G1BGAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
STP1/stp1::PSTP1–GFP 
 Δgln3Δgat1SAP2G1AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
sap2-1::PSAP2–GFP/SAP2-2 
 Δgln3Δgat1SAP2G1BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
sap2-1::PSAP2–GFP/SAP2-2 
 Δgln3Δgat1OPT1G22AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
OPT1-1/opt1-2::POPT1–GFP 
 Δgln3Δgat1OPT1G22BΔgln3GAT1M4Bgat1-1Δ::FRT/gat1-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
opt1-1::POPT1–GFP/OPT1-2 
 Δgln3Δgat1OPT3G22AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
opt3-1::POPT1–GFP/OPT3-2 
 Δgln3Δgat1OPT3G22BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
OPT3-1/opt3-2::POPT1–GFP 
 Δgln3Δgat1STP1G1AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
STP1/stp1::PSTP1–GFP 
 Δgln3Δgat1STP1G1BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
STP1/stp1::PSTP1–GFP 
gln3Δgat1Δ double mutants expressing SAP2 or OPT1 from the ADH1 promoter
 Δgln3Δgat1SAP2ex7AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::PADH1 -SAP2 
 Δgln3Δgat1SAP2ex7BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::PADH1 -SAP2 
 Δgln3Δgat1OPT1E1AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::PADH1 -OPT1 
 Δgln3Δgat1OPT1E1BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::PADH1-OPT1 
gln3Δgat1Δ double mutants expressing STP1 or STP1ΔN61 from a tetracycline-inducible promoter
 Δgln3Δgat1TET1-STP1AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::Ptet-STP1 
 Δgln3Δgat1TET1-STP1BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::Ptet-STP1 
 Δgln3Δgat1TET1-STP1ΔN61 AΔgln3GAT1M4Agln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::Ptet-STP1ΔN61 
 Δgln3Δgat1TET1-STP1ΔN61 BΔgln3GAT1M4Bgln3-1Δ::FRT/gln3-2Δ::FRTThis study
gat1-1Δ::FRT/gat1-2Δ::FRT 
ADH1/adh1::Ptet-STP1ΔN61 
stp1Δ mutants and derivatives expressing STP1, GLN3 or GAT1 from a tetracycline-inducible promoter
 STP1M1A and -BSC5314STP1/stp1Δ::SAT1-FLIPThis study
 STP1M2ASTP1M1ASTP1/stp1Δ::FRTThis study
 STP1M2BSTP1M1BSTP1/stp1Δ::FRTThis study
 STP1M3ASTP1M2Astp1Δ::FRT/stp1Δ::SAT1-FLIPThis study
 STP1M3BSTP1M2Bstp1Δ::FRT/stp1Δ::SAT1-FLIPThis study
 STP1M4ASTP1M3Astp1Δ::FRT/stp1Δ::FRTThis study
 STP1M4BSTP1M3Bstp1Δ::FRT/stp1Δ::FRTThis study
 Δstp1TET1-STP1ASTP1M4Astp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-STP1 
 Δstp1TET1-STP1BSTP1M4Bstp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-STP1 
 Δstp1TET1-GLN3ASTP1M4Astp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GLN3 
 Δstp1TET1-GLN3BSTP1M4Bstp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GLN3 
 Δstp1TET1-GAT1-1ASTP1M4Astp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12268 
 Δstp1TET1-GAT1-1BSTP1M4Bstp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12268 
 Δstp1TET1-GAT1-2ASTP1M4Astp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12067 
 Δstp1TET1-GAT1-2BSTP1M4Bstp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12067 
 Δstp1TET1-GAT1-3ASTP1M4Astp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12004 
 Δstp1TET1-GAT1-3BSTP1M4Bstp1Δ::FRT/stp1Δ::FRTThis study
ADH1/adh1::Ptet-GAT12004 

Gln3p and Gat1p control STP1 expression

The results presented above suggested that Gln3p and Gat1p are required for the utilization of proteins because they regulate STP1 expression, which in turn induces SAP2. To investigate whether STP1 expression is regulated by the GATA transcription factors, we placed the GFP reporter gene under control of the endogenous STP1 promoter in the wild-type strain SC5314 and the gln3Δ, gat1Δ and gln3Δgat1Δ mutants and compared STP1 promoter activity during growth of the strains in YCB–BSA–YE medium. As can be seen in Fig. 8A, both GATA transcription factors, especially Gat1p, were required for normal STP1 expression levels. However, a basal STP1 expression was observed in the absence of both transcription factors, as the fluorescence of gln3Δgat1Δ double mutants containing the reporter fusion was significantly above the background.

Figure 8.

Expression of the transcription factor STP1 is controlled by the GATA transcription factors Gln3p and Gat1p and regulated by nitrogen catabolite repression.
A. YPD overnight cultures of reporter strains expressing GFP under control of the STP1 promoter in a wild-type, gln3Δ, gat1Δ or gln3Δgat1Δ background were diluted 10−2 in YCB–BSA–YE medium, grown for 8 h at 30°C, and analysed by flow cytometry.
B. YPD overnight cultures of reporter strains containing a PSTP1–GFP (top) or a PSAP2–GFP fusion (bottom) in a wild-type background were diluted 10−2 in SD medium containing 100 μM or 100 mM ammonium and in YCB–BSA–YE medium without or with 100 mM ammonium, glutamine (Gln) or urea, grown for 8 h at 30°C, and analysed by flow cytometry.
The mean fluorescence of each cell population is shown in A and B. The first columns show the results obtained with the A series and the second columns show the results obtained with the B series of the reporter strains (see Table 1). The parental strain SC5314, which does not contain GFP, was used as a negative control. Background fluorescence values of this strain were between 2.6 and 3.8 in the various media in the experiment shown in B.

Nitrogen catabolite repression of SAP2 is mediated by regulation of STP1 expression

The fact that Gln3p and Gat1p control STP1 expression levels indicated that STP1, like SAP2, is regulated by nitrogen catabolite repression. We therefore determined STP1 promoter activity in the wild-type strain SC5314 in minimal SD medium containing high (100 mM) and low (100 μM) concentrations of the preferred nitrogen source ammonium as well as in YCB–BSA–YE medium in the absence and presence of ammonium. The activity of the SAP2 promoter was monitored under the same conditions for comparison. As shown in Fig. 8B, STP1 expression was repressed about twofold by high ammonium concentrations, both in minimal medium and in YCB–BSA–YE, demonstrating that it is indeed under nitrogen catabolite repression. SAP2 promoter activity was detected only in the inducing medium YCB–BSA–YE and reduced to background levels by the addition of ammonium. A similar repression of STP1 and SAP2 expression was observed when high concentrations of an amino acid, glutamine, or urea were added instead of ammonium to YCB–BSA–YE medium. These findings suggested that nitrogen catabolite repression of SAP2 might be mediated at least in part through the regulation of STP1 expression by the GATA transcription factors Gln3p and Gat1p. In this case, forced expression of STP1 should overcome SAP2 repression by preferred nitrogen sources. To investigate this possibility, we observed BSA degradation and Sap2p expression during growth of the wild-type strain SC5314 and strains expressing wild-type or constitutively active forms of STP1 in YCB–BSA–YE in the absence or presence of high ammonium levels. Figure 9 shows that ammonium suppressed Sap2p expression and BSA degradation by the wild-type strain SC5314, regardless of the presence or absence of doxycycline (Fig. 9, lanes 1–4). In contrast, strains expressing STP1 or STP1ΔN61 from the Tet-inducible promoter degraded the BSA and expressed Sap2p even in the presence of ammonium (Fig. 9, lanes 5–8), demonstrating that forced STP1 expression overcomes the repression of protease secretion by ammonium and that STP1 expression levels play a decisive role in nitrogen catabolite repression of SAP2.

Figure 9.

Nitrogen catabolite repression of SAP2 is mediated by Gln3p- and Gat1p-dependent control of STP1 expression. The wild-type strain SC5314 and transformants of the gln3Δgat1Δ double mutants expressing full-length STP1 or the constitutively active STP1ΔN61 allele from the Tet promoter were grown for 27 h at 30°C in YCB–BSA–YE in the presence (+) or absence (–) of doxycycline (50 μg ml−1) and ammonium (100 mM) as indicated. The culture supernatants were analysed by SDS-PAGE (top) and by Western immunoblotting with an anti-Sap2p antibody (bottom). Uninoculated growth medium was used as a control. M, molecular size marker.

Discussion

The results described in this study demonstrate that the GATA transcription factors Gln3p and Gat1p control expression of the secreted aspartic protease SAP2, which is required for growth of C. albicans on proteins, and are therefore essential for the utilization of this alternative nitrogen source. Limjindaporn et al. (2003) already reported that in preliminary experiments, a gat1Δ mutant was deficient in the utilization of BSA as a nitrogen source. We could confirm and extend these observations by showing that growth of gat1Δ mutants in YCB–BSA medium was strongly delayed, although the mutants eventually reached the same optical density as the wild type after several days of growth, while mutants lacking both GLN3 and GAT1 failed to grow in this medium. This is in line with the fact that the gln3Δgat1Δ double mutants did not detectably express SAP2, while Sap2p production was still observed in either of the single mutants. Therefore, both Gln3p and Gat1p contribute to SAP2 expression, although Gat1p clearly plays a more prominent role than Gln3p.

The induction of SAP2 expression in the presence of proteins and its repression by sufficient amounts of a preferred nitrogen source, like ammonium or amino acids in high concentrations, has been known for many years (Ross et al., 1990; Banerjee et al., 1991; Hube et al., 1994). However, insights into the molecular basis of SAP2 regulation were obtained only recently when Martinez and Ljungdahl (2005) elucidated how SAP2 expression is induced by proteins. Micromolar concentrations of amino acids, which may be produced by basal extracellular proteolytic activity and signal the availability of proteins, are sensed by the SPS sensor, which then induces the proteolytic activation of two latent transcription factors, Stp1p and Stp2p. Each of these transcription factors has a specific subset of target genes. While Stp2p induces the expression of genes involved in amino acid uptake, Stp1p activates genes that are required for the utilization of proteins as a nitrogen source, the secreted aspartic protease SAP2 and the oligopeptide transporters OPT1 and OPT3. Stp1p is essential for SAP2 and OPT1 expression (OPT3 is also induced by Stp2p) and stp1Δ mutants can not grow on protein as the sole nitrogen source. Conversely, cells expressing a truncated, constitutively active STP1 allele do not need an inducer and express SAP2 and OPT1 even in the absence of proteins. In the presence of high (mM) concentrations of amino acids, Stp1p but not Stp2p levels are downregulated, thereby ensuring that amino acid permeases remain expressed while the expression of enzymes and transporters required for the utilization of proteins is shut off. As Stp1p was readily detected in the presence of high concentrations of ammonium, Martinez and Ljungdahl (2005) concluded that steady-state levels of Stp1p are affected by amino acid availability, but not by the overall nitrogen status of the cell. However, they did not compare Stp1p levels in media containing high and low ammonium levels, and our results clearly demonstrate that STP1 expression is downregulated at high concentrations of ammonium or other nitrogen sources, like amino acids and urea, which may at least partially explain the repression of SAP2 expression when sufficient amounts of preferred nitrogen sources are available.

Our work provides a link between the two types of transcription factors that are essential for SAP2 expression and growth of C. albicans on proteins, the general regulators Gln3p and Gat1p and the specific regulator Stp1p, and explain how both positive and negative control of SAP2 expression is achieved (see Fig. 10). When preferred nitrogen sources like ammonium become limiting, Gln3p and Gat1p increase expression of STP1, which seems to be a prerequisite for SAP2 expression. However, nitrogen starvation alone is not sufficient and SAP2 induction still needs a positive signal, which is provided by the presence of low concentrations of amino acids that result in the proteolytic activation of Stp1p. Conversely, even in the presence of proteins, SAP2 expression is repressed when sufficient amounts of preferred nitrogen sources are available, because under these conditions, STP1 expression is downregulated to levels that may not be adequate for SAP2 expression. It is not clear whether the twofold downregulation of STP1, which we observed in our experiments, is sufficient to abolish SAP2 expression, and it is well possible that Gln3p and Gat1p also directly activate SAP2 or control other genes that contribute to SAP2 expression. However, such an additional effect of Gln3p and Gat1p on SAP2 expression would be insufficient, as stp1Δ mutants do not detectably express SAP2 and are unable to grow in YCB–BSA medium. In contrast, forced expression of STP1 from the Tet-inducible promoter relieved SAP2 expression from its dependence on Gln3p and Gat1p, demonstrating that the control of STP1 expression levels is a central aspect in the regulation of SAP2 and in the decision on whether to use available proteins as a nitrogen source.

Figure 10.

Schematic describing the regulation of SAP2 expression by the transcription factors Stp1p, Gln3p and Gat1p. White arrows and spheres symbolize genes and proteins, respectively, in their inactive state, and the activated state is indicated by the grey shading. Activation of a gene or protein is symbolized by the thin black arrows. An increase in Stp1p levels is indicated by two corresponding spheres instead of only one. CM, cytoplasmic membrane.
A. Under nitrogen-replete conditions (in this model represented by high NH4+), the GATA factors Gln3p and Gat1p have only basal activity and STP1 is expressed at low levels. In the absence of proteins, Stp1p is not activated by the SPS sensor and SAP2 is not expressed.
B. Under nitrogen limiting conditions, Gln3p and Gat1p are activated and induce expression of their target genes, including STP1. However, the absence of proteins prevents activation of Stp1p by the SPS sensor and SAP2 is not expressed.
C. When proteins are the only available nitrogen source, Gln3p and Gat1p ensure high STP1 expression levels. Micromolar concentrations of amino acids generated by basal proteolytic activity induce the SPS sensor to activate Stp1p, which in turn induces SAP2 expression.
D. When both proteins and sufficient concentrations of a preferred nitrogen source, like ammonium, are available, Gln3p and Gat1p are not activated and STP1 expression levels remain low. Stp1p may still be activated by the SPS sensor under these conditions, but this is not sufficient to allow SAP2 expression.
Note that additional possible regulatory mechanisms, e.g. direct regulation of SAP2 by Gln3p and Gat1p or the contribution of other regulators, are not depicted in this model.

One of the GATA transcription factors, Gln3p, also controls nitrogen starvation-induced filamentous growth of C. albicans by regulating expression of MEP2, an ammonium permease that stimulates filamentation under these conditions (Biswas and Morschhäuser, 2005). The filamentous growth defect of gln3Δ mutants can be overcome by forced overexpression of MEP2 (Dabas and Morschhäuser, 2007) and the requirement of MEP2 can be bypassed by dominant-active RAS1G13V or GPA2Q354L alleles (Biswas and Morschhäuser, 2005). However, high concentrations of ammonium still suppress filamentous growth in cells expressing hyperactive RAS1 or GPA2 alleles, demonstrating that ammonium can also act downstream of these regulators. In contrast, ammonium could not inhibit SAP2 expression in strains expressing STP1 from a tetracycline-inducible promoter, lending further support to the idea that the regulation of STP1 expression is a decisive factor in the control of SAP2 expression. By using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this long-known virulence attribute.

Experimental procedures

Strains and growth conditions

C. albicans strains used in this study are listed in Table 1. The strains were routinely grown in YPD medium (10 g yeast extract, 20 g peptone, 20 g glucose per litre) at 30°C. To prepare solid media, 1.5% agar was added before autoclaving. For selection of nourseothricin-resistant transformants, 200 μg ml−1 nourseothricin (Werner Bioagents, Jena, Germany) was added to YPD agar plates. To obtain nourseothricin-sensitive (NouS) derivatives in which the SAT1 flipper was excised by FLP-mediated recombination, transformants were grown for 6 h in YPM medium (10 g yeast extract, 20 g peptone, 20 g maltose per litre) without selective pressure to induce the MAL2 promoter. A total of 100–200 cells were then spread on YPD plates containing 20 μg ml−1 nourseothricin and grown for 2 days at 30°C. NouS clones were identified by their small colony size and confirmed by re-streaking on YPD plates containing 100 μg ml−1 nourseothricin as described previously (Reußet al., 2004). To test for growth on BSA as the sole nitrogen source, strains were grown at 30°C in YCB–BSA medium (23.4 g yeast carbon base, 4 g BSA per litre, pH 4.0). Expression of SAP2, OPT1, OPT3 and STP1 was monitored in YCB–BSA–YE medium (YCB–BSA + 0.2% yeast extract), which also induces SAP2 expression (Staib et al., 2002) and allowed normal growth of gat1Δ and gln3Δgat1Δ mutants. Nitrogen catabolite repression was tested by growing strains in SD medium [1.7 g yeast nitrogen base without amino acids (YNB; BIO 101, Vista, Calif.), 20 g glucose per litre] containing low (100 μM) or high (100 mM) ammonium concentrations as well as in YCB–BSA–YE without or with 100 mM ammonium, glutamine or urea.

Plasmid constructions

Plasmid pGAT1K1 (see Fig. 2A) was generated for re-introduction of a functional GAT1 copy into the gat1Δ single and gln3Δgat1Δ double mutants. The GAT1 coding region and flanking sequences were amplified from genomic DNA of strain SC5314 with the primers GAT1 and GAT7 (primer sequences are provided in Table 2) and the ApaI/XhoI-digested PCR product substituted for the GAT1 upstream region in the previously described pGAT1M2 (Dabas and Morschhäuser, 2007). For expression of different versions of the GAT1 gene under control of a tetracycline-inducible promoter, the following plasmids were generated (see Fig. 3A). pTET1-GAT1-1 contains the GAT1 ORF described by Limjindaporn et al. (2003), which is 2268 bp in length and here referred to as GAT12268. It was obtained by amplification from genomic DNA of the heterozygous mutant GAT1M2B, in which the GAT1-1 allele is intact (see Fig. 2B, lane 3), with the primers GAT1-3 and GAT1-2. The PCR product was digested with SalI/SmaI and SmaI/BamHI and the two fragments were ligated together into the SalI/BglII-digested plasmid pNIM1 (Park and Morschhäuser, 2005). pTET1-GAT1-2 contains the GAT1 ORF (orf19.1275) described in assembly 19 of the C. albicans genome sequence, which is 2067 bp in length and here referred to as GAT12067. It was obtained by amplification from genomic DNA of the heterozygous mutant GAT1M2A, in which the GAT1-2 allele is intact (see Fig. 2B, lane 2), with the primers GAT1-1 and GAT1-2. The PCR product was digested with SalI/SmaI and SmaI/BamHI and the two fragments were ligated together into the SalI/BglII-digested plasmid pNIM1. pTET1-GAT1-3 contains the GAT1 ORF (orf19.1275) described in assembly 21 of the C. albicans genome sequence, which is 2004 bp in length and here referred to as GAT12004. It was obtained by amplification from genomic DNA of the heterozygous mutant GAT1M2A, in which the GAT1-2 allele is intact, with the primers GAT1-4 and GAT1-2. The PCR product was digested with SalI/SmaI and SmaI/BamHI and the two fragments were ligated together into the SalI/BglII-digested plasmid pNIM1. To express SAP2 from the ADH1 promoter, the coding region of the SAP2-1 allele was amplified from genomic DNA of strain SC5314 with the primers SAP2ex1 and SAP2ex2. The PCR product was digested with SalI/PstI and PstI/BamHI and the two fragments were ligated together into the XhoI/BglII-digested pOPT4E1 (Reuß and Morschhäuser, 2006) to generate pSAP2ex7. Plasmid pTET1-STP1ΔN61, which contains an N-terminally truncated, constitutively active STP1 allele (Martinez and Ljungdahl, 2005) under control of the tetracycline-inducible promoter, was obtained by amplifying the STP1 ORF lacking codons 2–61 with the primers STP1-1 and STP1-2 and ligating the SalI/BglII-digested PCR product between the same sites in pNIM1. To express the full-length STP1 gene from the tetracycline-inducible promoter, the STP1 ORF was PCR-amplified with the primers STP1-4 and STP1-2, digested at the SalI site introduced before the start codon and at an internal SphI site, and cloned into the SalI/SphI-digested pTET1-STP1ΔN61 to generate pTET1-STP1. For expression of GLN3 from the tetracycline-inducible promoter, the GLN3 coding region was amplified with the primers GLN10 and GLN11, and the PCR product digested at the introduced XhoI and BamHI sites and cloned in pBluescript to generate pGLN3. The XhoI-BamHI fragment from pGLN3 was then ligated between the SalI and BglII sites of pNIM1 to produce pTET1-GLN3.

Table 2.  Primers used in this study.
PrimerSequence
  1. Restriction sites introduced into the primers are underlined; start and stop (reverse sequence) codons are highlighted in bold.

GAT15′-CCGATAACAATAAGGGCCCTCCCAATCAG-3′
GAT75′-ATAGATGACACTCGAGTTGATGATTGGGTTG-3′
GAT135′-TGTTGAAATCATGGTGCGGTTCAGGTTGG-3′
GAT145′-TTGTGATAATTGGGACATTGTATTAGTGGCAG-3′
GAT155′-AAGTCATACCACCACCTGAACTACCTCTGTC-3′
GAT1-15′-ATATGTCGACAATGTACTACCGTGCTCGTCACTC-3′
GAT1-25′- ATATGGATCCTAATAATTCATGTTTAACCAATC-3′
GAT1-35′-ATATGTCGACAATGACAATGAATTTTAATCAAACA GG-3′
GAT1-45′-ATATGTCGACAATGATGTATATTAACAACAAATCG-3′
GLN105′-TTTTCTCGAGGGACAAATGACTACATCG-3′
GLN115′-ACGTGGATCCTCAAATGTCAAACTTCAACCAAT CC-3′
SAP2ex15′-ACCAGTCGACAATGTTTTTAAAGAATATTTTCAT-3′
SAP2ex25′-ACCCCGGATCCTTAGGTCAAGGCAGAAATACTGG AAGC-3′
STP1-15′-ATATAGTCGACAAAATGCCACCAATACAAAAGATT AAG-3′
STP1-25′-AAACTAGATCTTAATCTAGTAATAGATTGC-3′
STP1-45′-AACCAATGTCGACAACTATGTTGATACTTTCCATA GG-3′
STP1-55′-ATGAAAAGATAAGGGCCCATGGAAAGCC-3′
STP1-65′-AGTTATAGTCGACGTTCTTTAATATG-3′
STP1-75′-ATATATCCTGCAGGTGTAAAGGC-3′
STP1-85′-TAATGAAGAGCTCGAACCTGAACG-3′
STP1-95′-AATGGAAGATCCGCGGCTGTTTCC-3′

To express GFP under control of the STP1 promoter, the STP1 upstream region was amplified with the primers STP1-5 and STP1-6 and the PCR product digested at the introduced ApaI and SalI sites and substituted for the OPT1 upstream region in the ApaI/SalI-digested plasmid pOPT1G22 (Reuß and Morschhäuser, 2006) to produce pSTP1PG1. A STP1 downstream fragment was then amplified with the primers STP1-7 and STP1-8, digested at the introduced PstI and SacI sites, and ligated between the same sites in pSTP1PG1 to generate pSTP1PG2. To obtain a STP1 deletion cassette, the ApaI-SalI STP1 upstream fragment from pSTP1G1 was substituted for the GAT1 upstream fragment in the ApaI/XhoI-digested pGAT1M2 to result in pSTP1M1. A STP1 downstream fragment was then obtained by PCR with the primers STP1-9 and STP1-8, digested at the introduced SacII and SacI sites, and cloned between the same sites in pSTP1M1 to generate pSTP1M2.

Candida albicans transformation

C. albicans strains were transformed by electroporation (Köhler et al., 1997) with the following gel-purified linear DNA fragments: the ApaI-SacI fragment from pGAT1K1 to re-introduce the GAT1 gene at its original locus in the gat1Δ single and gln3Δgat1Δ double mutants (see Fig. 2A), the SacII-ApaI fragments from pTET1-GAT1-1, pTET1-GAT1-2 and pTET1-GAT1-3 to express the GAT12268, GAT12067 and GAT12004 alleles from the Tet promoter in the gat1Δ mutants (see Fig. 3A), an XbaI-HindIII fragment from pSAP2G1 and ApaI-SacI fragments from pOPT1G22, pOPT3G22 (Reuß and Morschhäuser, 2006) and pSTP1PG2 to express the GFP reporter gene from the SAP2, OPT1, OPT3 and STP1 promoters, the ApaI-SacII fragments from pSAP2ex7 and pOPT1E1 (Reuß and Morschhäuser, 2006) to express SAP2 and OPT1 from the ADH1 promoter, the SacII-ApaI fragments from pTET1-STP1 and pTET1-STP1ΔN61 to express wild type and constitutively active STP1 alleles from the Tet promoter in the gln3Δgat1Δ double mutants, the ApaI-SacI fragment from pSTP1M2 to delete the STP1 alleles in strain SC5314 and the SacII-ApaI fragments from pTET1-STP1, pTET1-GLN3, pTET1-GAT1-1, pTET1-GAT1-2 and pTET1-GAT1-3 to express STP1, GLN3 and the GAT12268, GAT12067 and GAT12004 alleles from the Tet promoter in the stp1Δ mutants. Selection of nourseothricin-resistant transformants was performed as described previously (Reußet al., 2004). Single-copy integration of each construct at the desired genomic locus was confirmed by Southern hybridization with specific probes.

Isolation of genomic DNA and Southern hybridization

Genomic DNA from C. albicans strains was isolated as described previously (Millon et al., 1994). Ten micrograms of DNA was digested with appropriate restriction enzymes, separated on a 1% agarose gel and, after ethidium bromide staining, transferred by vacuum blotting onto a nylon membrane and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence-labelled probes was performed with the Amersham ECL Direct Nucleic Acid Labelling and Detection System (GE Healthcare, Braunschweig, Germany) according to the instructions of the manufacturer.

5′-Rapid amplification of cDNA ends

C. albicans strain SC5314 was grown to log-phase in liquid SLAD medium and total RNA was isolated using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). 5′-Rapid amplification of cDNA ends (5′ RACE) analysis was performed using the 5′/3′ RACE kit, 2nd Generation (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's specifications. Briefly, 2 μg of total RNA was reverse-transcribed using the antisense primer GAT13. A homopolymeric dA-tail was added to the purified cDNA using recombinant terminal transferase and dATP provided with the kit. The dA-tailed cDNA was amplified using the 5′ RACE oligo dT-anchor primer and the GAT1-specific antisense primer GAT14. The product was further amplified using the 5′ RACE PCR anchor primer and the GAT1-specific antisense primer GAT15, which was also used for direct sequencing of the PCR product.

Flow cytometry

Cells were grown for the indicated times in the various test media, washed and suspended in phosphate buffered saline to an OD600 of 0.1 and kept on ice. FACS analysis was performed using a FACSCalibur cytometry system equipped with an argon laser emitting at 488 nm (Becton Dickinson, Heidelberg, Germany). Fluorescence was measured on the FL1 fluorescence channel equipped with a 530 nm bandpass filter. Twenty thousand events were counted at low flow rate. Fluorescence data were collected using logarithmic amplifiers. The mean fluorescence values were determined using CellQuest Pro (Becton Dickinson) software.

SDS-PAGE and Western immunoblotting

C. albicans strains were grown in YCB–BSA–YE at 30°C for the indicated times. Fifteen microlitres of the culture supernatants was loaded on two separate SDS 12% polyacrylamide gels. One gel was stained with Colloidal Coomassie dye and the molecular weight of the proteins was determined by use of the Precision Plus Protein Standards all blue size marker (Bio-Rad, München, Germany). The other gel was used for Western immunoblotting. Proteins were transferred onto nitrocellulose membranes using a Semi-Dry-Trans-Blot SD blot apparatus (Bio-Rad). Sap2p was detected with an antibody raised against Sap2p and an anti-rabbit antibody as a second antibody. Signals were detected with ECL detection solutions on ECL films (GE Healthcare).

Acknowledgements

We thank Michel Monod for providing the anti-Sap2p antibody. Sequence data for C. albicans were obtained from the Candida Genome Database (http://www.candidagenome.org/). This study was supported by the Deutsche Forschungsgemeinschaft (DFG Grants MO 846/4 and MO 846/5 and SFB630).

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