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Infection and Immunity, April 2008, p. 1686-1694, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01497-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Combined Inactivation of the Candida albicans GPR1 and TPS2 Genes Results in Avirulence in a Mouse Model for Systemic Infection{triangledown}

Mykola M. Maidan,1,2 Larissa De Rop,1,2 Miguel Relloso,3 Rosalia Diez-Orejas,3 Johan M. Thevelein,1,2 and Patrick Van Dijck1,2*

VIB Department of Molecular Microbiology,1 Laboratory of Molecular Cell Biology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium,2 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain3

Received 9 November 2007/ Returned for modification 19 December 2007/ Accepted 1 February 2008


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ABSTRACT
 
Inhibition of the biosynthesis of trehalose, a well-known stress protectant in pathogens, is an interesting approach for antifungal or antibacterial therapy. Deletion of TPS2, encoding trehalose-6-phosphate (T6P) phosphatase, results in strongly reduced virulence of Candida albicans due to accumulation of T6P instead of trehalose in response to stress. To further aggravate the deregulation in the pathogen, we have additionally deleted the GPR1 gene, encoding the nutrient receptor that activates the cyclic AMP-protein kinase A signaling pathway, which negatively regulates trehalose accumulation in yeasts. A gpr1 mutant is strongly affected in morphogenesis on solid media as well as in vivo in a mouse model but has only a slightly decreased virulence. The gpr1 tps2 double mutant, on the other hand, is completely avirulent in a mouse model for systemic infection. This strain accumulates very high T6P levels under stress conditions and has a growth defect at higher temperatures. We also show that a tps2 mutant is more sensitive to being killed by macrophages than the wild type or the gpr1 mutant. A double mutant has susceptibility similar to that of the single tps2 mutant. For morphogenesis on solid media, on the other hand, the gpr1 tps2 mutant shows a phenotype similar to that of the single gpr1 mutant. Taken together these results show that there is synergism between Gpr1 and Tps2 and that their combined inactivation results in complete avirulence. Combination therapy targeting both proteins may prove highly effective against pathogenic fungi with increased resistance to the currently used antifungal drugs.


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INTRODUCTION
 
Trehalose is known as a stress-protective sugar synthesized under unfavorable growth conditions in a variety of microorganisms (4). It can be synthesized by several pathways (1), but in yeasts it is made only in a two-step process carried out by trehalose-6-phosphate (T6P) synthase (Tps1) and T6P phosphatase (Tps2). Tps1 catalyzes the conversion of UDP-glucose and glucose-6-phosphate into T6P. The second enzyme, Tps2, dephosphorylates T6P to produce trehalose. In Saccharomyces cerevisiae, these enzymes have been studied extensively (4). Apart from a catalytic function in the biosynthesis of trehalose, the ScTps1 enzyme also plays a crucial role in controlling the influx of glucose into glycolysis (5). As a consequence, a yeast tps1{Delta} mutant is not able to grow on glucose because of a very fast deregulation of metabolism due to strong accumulation of sugar phosphates in the first part of glycolysis (31). Deletion of the yeast TPS2 gene results in a thermosensitivity phenotype, which is also caused by deregulation of trehalose metabolism. At higher temperatures (or under any other stress condition), the Sctps2{Delta} mutant accumulates a high level of T6P, resulting in rapid deprivation of free phosphate (9, 28). Because the enzymes of trehalose metabolism are not present in humans and because trehalose is an important stress-protecting molecule in many fungi, these enzymes have been investigated as potential targets for antifungals. Initial work has shown that prevention of trehalose accumulation in Candida albicans by deletion of the TPS1 gene renders the cells less virulent in a mouse model for systemic infection. Interestingly, in this pathogen, the Tps1 enzyme is not essential for growth on glucose (38). Later, it was shown that deletion of TPS2 has an even stronger effect, probably because such a strain not only lacks trehalose but also accumulates high levels of T6P under stress conditions (32, 39). As a result the tps2{Delta} strain is also thermosensitive. It is strongly affected in virulence in a mouse model for systemic infection, despite normal filamentation on various hypha-inducing media. In addition, a tps2 mutant is also affected in the cell integrity pathway (39). For Aspergillus nidulans it was also shown that conidia and hyphae of the orlA1 mutant have a defect in chitin production when incubated at high temperatures. ORLA1 is the ortholog of TPS2 (6).

Despite the deregulation of trehalose metabolism and the deficient cell wall, deletion of TPS2 does not block pathogenicity completely in a mouse model of systemic infection. We have tried to further enhance the accumulation of T6P by increasing the flux into the trehalose biosynthesis pathway. Previously, it was shown that both ScTPS1 expression (26, 37) and ScTps1 activity are under the negative control of the cyclic AMP (cAMP)-protein kinase A (PKA) pathway (14). As a consequence, deletion of the GPR1 gene, encoding a G protein-coupled receptor that activates the cAMP-PKA pathway, results in higher trehalose levels (16, 18, 20). The homologous receptor in C. albicans also functions upstream of the cAMP-PKA pathway, but how it precisely activates the pathway is still unclear (21, 25).

Here we show that also in C. albicans the activity of the trehalose biosynthesis machinery is under the negative control of Gpr1. As a result the double gpr1 tps2 mutant accumulates much more T6P than the single tps2 mutant and it is completely avirulent in a mouse model system for systemic infection. The avirulence phenotype appears to be due to a combination of effects, such as the metabolic imbalance, defective morphogenesis, and enhanced sensitivity to killing by macrophages.


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MATERIALS AND METHODS
 
Yeast strains and growth conditions. All strains used in this study derive from the KAR5 (TPS2/tps2 ura–) and the EL17 (tps2{Delta}/tps2{Delta} ura–) strains (32) and are presented in Table 1. The construction of the different C. albicans gpr1{Delta} strains is described below. The yeast cells were grown with shaking at 28°C in YPD medium (1% yeast extract, 2% peptone, 2% glucose). To study colony morphology, stationary-phase cells were resuspended in fresh YPD medium and diluted to obtain single colonies on plates. After 5 days at 30°C, individual colonies were photographed. The formation of hyphae and colony morphology were tested on different media including medium containing fetal calf serum (Sigma), Lee's medium (17), and SLAD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% glucose, 50 µM ammonium sulfate). Cultivation under embedded-growth conditions was performed as described by Brown et al. (8).


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  		TABLE 1. C. albicans strains used in this studya

Growth curves were determined with a Bioscreen apparatus (Life Sciences). Strains were grown overnight in YPD and diluted to optical density at 600 nm (OD600) of 0.05 in fresh YPD, and 300 µl was added into the wells of the Honeywell plate and incubated at the indicated temperatures with 30 s of shaking every minute.

Construction of C. albicans GPR1 deletion strains. The plasmid as well as the procedure to generate the disruption of the GPR1 gene was described previously (21). Both the heterozygous and homozygous TPS2 deletion strains were used to delete both copies of GPR1. The method to obtain the reintegrated GPR1 allele was also described previously (21). All the strains were checked by diagnostic PCR as well as by Southern blotting as previously described. Briefly, genomic DNA (10 µg) from the different strains was digested with EcoRI, and the fragments were separated on a 0.8% agarose gel. After transfer to a nylon membrane the DNA was hybridized using the XbaI-PstI CaGPR1 promoter fragment as a probe. With this probe, a 7,868-bp fragment is detected in the wild-type strain. In an allele where the GPR1 gene has been replaced by the HisG-URA3-HisG cassette, a fragment of 5,335 bp is detected. After selection on 5-fluoroorotic acid medium, an allele with only one HisG sequence is left, which results in a fragment of 4,269 bp. In the reintegrated strains, the CaGPR1 promoter probe will hybridize to a 6,194-bp EcoRI fragment. In a gpr1{Delta} strain this probe will hybridize to the 5,335-bp or 4,269-bp fragment, depending on the presence of a HisG-URA-HisG cassette or a HisG fragment, respectively.

Trehalose and T6P levels and heat shock response. Trehalose and T6P levels as well as the heat shock response on plates were determined as described previously (32).

Determination of T6P synthase activity. T6P synthase activity was measured by a coupled-enzyme assay as described previously (34). Specific activity was expressed as µkat/g protein. Protein concentration was determined as described by Bradford (7).

Peritoneal macrophages and cell line cultures. (i) Culture medium and reagents. RPMI 1640 medium, fetal calf serum, L-glutamine, and antibiotics (penicillin-streptomycin) were obtained from Gibco BRL. Lipopolysaccharide (LPS) from Escherichia coli serotype 055:B55 was purchased from Sigma Chemical. Murine recombinant gamma interferon (IFN-{gamma}) was purchased from Prepro Tech. Both peritoneal and cell line macrophages were resuspended in RPMI medium supplemented with glutamine (2 mM), antibiotics (penicillin [100 U/ml]-streptomycin [100 µg/ml]), and 10% heat-inactivated fetal bovine serum (complete medium).

(ii) Peritoneal macrophages. Thioglycolate (endotoxin free; Difco)-elicited murine (BALB/c) peritoneal macrophages were harvested by washing the peritoneal cavity 4 days after injection. Peritoneal cells were plated at a concentration of 2 x 105 cells per well. After 4 h of incubation, adhering macrophages were selected by washing with prewarmed complete medium and aspirating nonadhering peritoneal cells. Only wells that were totally covered with macrophages were used in the studies.

(iii) Macrophage cell line. The RAW 264.7 cell line was obtained from the American Type Culture Collection. Cells were grown in complete medium in a 5% carbon dioxide incubator at 37°C and maintained at low densities (75% confluence). They were passaged until the confluent state was reached, usually every 3 to 4 days, on sterile culture plates. RAW 264.7 cells were plated 18 to 24 h prior to initiation of the experiments.

(iv) Determination of the candidacidal activity of macrophages. C. albicans strains were harvested from the YPD plates, washed twice with phosphate-buffered saline (PBS), and diluted to the desired density in RPMI medium supplemented with glutamine (2 mM), antibiotics, and 10% heat-inactivated fetal bovine serum (complete medium). For the determination of anti-Candida activity, macrophages were infected with 0.1 ml of C. albicans strains (1 x 106 cells/ml; multiplicity of infection, 1:1). Parallel control wells (without macrophages) were used. After 3 h of incubation at 37°C in 5% CO2, Triton X-100 (0.1% final concentration) was added to the wells and the plates were shaken vigorously. The contents of the wells were removed by pipetting and washed with sterile distilled water. Serial dilutions from each well were made up in distilled water and were plated (triplicate samples) on YPD plates. Results were expressed as percentages of CFU inhibition activity according to the formula [1 – (CFU of experimental group/CFU of control cultures)] x 100.

Anti-Candida activity of RAW 264.7 macrophages was evaluated under basal conditions (previously described) and in the presence of IFN-{gamma} (200 U/ml) plus LPS (100 ng/ml) for 18 h prior to assessment.

In vivo pathogenicity assay. To determine the virulence of C. albicans, 70 female BALB/c mice (20 g) were injected in the caudal vein with 5 x 105 cells (10 mice/strain). Survival was determined every day.


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RESULTS
 
Construction of heterozygous and homozygous gpr1 tps2 mutants. To investigate the effect of additional deletion of GPR1 in a tps2 background, we first generated a set of mutant strains. We have used the KAR5 (TPS2/tps2{Delta}) and EL17 (tps2{Delta}/tps2{Delta}) strains (32) as backgrounds to delete one or both copies of GPR1, using the Ura blaster cassette as previously described (21). In this way we have generated strains which are either heterozygous or homozygous for the deletion of one or both genes. A Southern blot for the different deletion strains used in this study is shown in Fig. 1. The lengths of the hybridizing fragments are 7,868 bp, 5,335 bp, and 4,269 bp for the wild type, URA+, and ura– strains, respectively, as previously described (21). We also used the CaGPR1 reintegration construct (21) to generate reconstituted strains, and these were also checked by Southern blotting (data not shown). The different deletion and reintegration strains were checked by Northern blotting using a CaGPR1 probe. The results show complete absence of expression in the deletion strains. There is no detectable difference in expression between the heterozygous strains and the reconstituted strains (data not shown).


Figure 1
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  		FIG. 1. Southern blot of genomic DNA from different Candida albicans strains. The DNA was digested with EcoRI, and the PstI-XbaI fragment of pUC19/GPR1 was used as the probe. This fragment contains part of the CaGPR1 promoter. Strains: 1, CAI4; 2, EL17; 3, E2; 4, E2-5; 5, EN2; 6, EN2-5; 7, KAR5; 8, K1; 9, K1-11; 10, KM1; 11, KM1-1. The molecular weight marker is MW VII from Roche. WT, wild type.

Phenotypic characterization of hetero- and homozygous TPS2 and GPR1 deletion strains. (i) Morphogenesis on solid media is strongly affected in the double mutant. A major virulence factor of C. albicans is its ability to undergo a dimorphic switch from budding yeast to hyphal or pseudohyphal filamentation (3). Deletion of TPS2 has only a marginal effect on morphology when cells are incubated on various hypha-inducing media (32) (Fig. 2A). Deletion of GPR1, however, has a strong effect on solid media, causing in most cases smooth colonies and a wrinkled phenotype only on serum-containing media (21, 22) (Fig. 2A). Combined deletion of TPS2 and GPR1 shows an enhanced effect, with no hypha formation on Lee's, serum-containing, or SLAD medium (Fig. 2A). On serum-containing YPD plates, the gpr1 mutant never forms radial hyphae but on top of the colony a wrinkled phenotype can be observed. We have obtained similar results when cells are incubated under embedded conditions. The tps2{Delta} strain is only marginally affected, whereas the gpr1{Delta} mutant shows a strong defect in hypha formation, especially at 25°C (Fig. 2B). The double-deletion strain again has a stronger defect for morphogenesis under these conditions. In reintegrant strains, such as LR2 or KM1, the heterozygosity phenotype is partially restored. Under liquid hypha-inducing conditions (serum or Spider medium) we did not observe any defect in morphogenesis in the double-deletion strain (data not shown). This confirms our previous results on the single gpr1 or tps2 mutants (21, 32).


Figure 2
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  		FIG. 2. Colony morphology. (A) Colony morphology on different hypha-inducing media. Candida cells were grown overnight in YPD medium. The cells were diluted, and approximately 20 cells were plated on plates containing SLAD medium, Lee's medium, YPD medium, or YPD medium containing 10% serum. The plates were incubated at 30°C for 5 days, and one representative colony on each plate was photographed. (B) Colony morphology under embedded conditions. Candida cells were mixed with YP sucrose agar medium, and colonies were photographed after 1 and 3 days after incubation at 25°C (top two rows) or 30°C (bottom two rows). WT, wild type.

(ii) Temperature-sensitive growth and survival. We grew the wild type and heterozygous and homozygous tps2, gpr1, and tps2 gpr1 deletion mutants in glucose-containing medium at 30°C and 43°C in a Bioscreen apparatus (Life Sciences). At 30°C, the growth rates of all strains, except the tps2{Delta} gpr1{Delta} strain, are very similar (Fig. 3A). The double-deletion strain has a 20% longer doubling time at this temperature than the wild type. On incubation at 43°C, however, there is a strong difference in growth between the different strains (Fig. 3B). As we showed before, a tps2{Delta} strain is strongly affected for growth at this temperature. This is probably due to accumulation of high levels of T6P under this stressful condition (32). A gpr1{Delta} strain grows like a wild-type strain at 30°C but has a slight growth defect at 43°C. Deletion of one allele of GPR1 in the tps2{Delta} background does not cause an additional growth effect. However, the homozygous gpr1{Delta} tps2{Delta} strain has a very strong growth defect at this temperature (Fig. 3B). To further explore the cause of this growth phenotype, we have incubated exponentially growing cultures of the different strains for different time periods at 44°C, after which serial dilutions were plated on YPD plates and incubated further at 30°C. Figure 3C shows that the tps2{Delta} mutant is temperature sensitive, as was described previously (32). Similar to what we have observed in S. cerevisiae (16), the C. albicans gpr1{Delta} strain is more thermotolerant than the wild type. Deletion of one or two copies of TPS2 in a gpr1{Delta} strain results in a strain that is still more resistant than the wild-type control. A strain that lacks both Gpr1 and Tps2 is more resistant than a strain that only lacks Tps2, and it is slightly less resistant than a gpr1{Delta} mutant. Hence, the results obtained with the Bioscreen seem to be different from those obtained with the spot assay. In the Bioscreen, the strongest effect is seen with the tps2{Delta} gpr1{Delta} strain, whereas in the spot assay, the tps2{Delta} strain gives the strongest phenotype (cells are dying after 2 h at 44°C). We have measured T6P and trehalose levels in the different strains to determine whether there is a correlation between the levels of these metabolites and heat stress tolerance.


Figure 3
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  		FIG. 3. Growth curves of Candida albicans strains at 30°C (A) and 43°C (B). Overnight cultures of YPD-grown Candida albicans cells were diluted to an OD600 of 0.2 in YPD. The growth of 300-µl cultures was monitored using a Bioscreen. Shown are wild-type (•), CC17 (tps2{Delta}/tps2{Delta}; {circ}), LDR8 (gpr1{Delta}/gpr1{Delta}; {blacktriangleup}), K1 (GPR1/gpr1{Delta} TPS2/tps2{Delta}p; {triangleup}), E2 (GPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacksquare}), KM1 (gpr1{Delta}/gpr1{Delta} TPS2/tps2{Delta}; {square}), and EN2 (gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacklozenge}) cultures. (C) Growth on plates after heat stress treatment. Candida albicans cells were grown overnight in YPD medium and diluted to an OD600 of 0.1, and 300-µl aliquots were then incubated at 44°C in a water bath for the indicated times. Serial dilutions were plated on YPD plates and further incubated for 2 days at 30°C. WT, wild type.

Additional deletion of GPR1 in a TPS2 deletion background enhances the T6P level. Trehalose accumulation is under negative control of the cAMP-PKA pathway in S. cerevisiae. In an overactive pathway (e.g., in a strain containing a dominant mutation in GPA2), no trehalose can be detected, whereas inactivation of the glucose-sensing complex results in higher levels of trehalose (16).

Deletion of TPS2 results in accumulation of T6P when cells are under stress (32). Based on these data, additional deletion of GPR1 in a tps2{Delta} mutant should result in higher levels of T6P. This is indeed the case, as shown in Fig. 4A. When cells are grown at 30°C and then shifted to 43°C, we see a rapid accumulation of T6P in a tps2{Delta} mutant. Deletion of one allele of GPR1 in this background further increases the T6P level. A homozygous gpr1{Delta} tps2{Delta} mutant accumulates even higher levels of T6P. This seems to indicate that in C. albicans the trehalose metabolism pathway is also downstream of the Gpr1-Gpa2 pathway. One copy of TPS2 is apparently sufficient to sustain dephosphorylation of T6P. These data would predict that the tps2{Delta} gpr1{Delta} strain is more thermosensitive than a tps2{Delta} strain. This is not what we have seen in the spot assay (Fig. 3C), where it is clear that a gpr1{Delta} tps2{Delta} strain is more resistant than a tps2{Delta} strain containing one or two alleles of GPR1. The reason for this discrepancy may be that, although cells accumulate large amounts of T6P, they undergo only growth arrest and do not die, possibly because they also accumulate more trehalose. We have previously shown that, when cells accumulate high levels of T6P (as in a tps2{Delta} mutant), they can still produce relatively high levels of trehalose, apparently because of the activity of nonspecific phosphatases. These phosphatases could have a higher Km for T6P than Tps2 or could be activated only under these conditions (32). The inset in Fig. 4B shows that a tps2{Delta} strain indeed produces trehalose at 43°C, but less than a strain with an additional deletion of one or two alleles of GPR1. A similar observation is seen for a heterozygous tps2 mutant. This strain accumulates about 60 nmol/mg cells, but additional deletion of one or two alleles of GPR1 doubles the trehalose level when cells are incubated at 43°C. Hence, the higher stress tolerance of the tps2{Delta} gpr1{Delta} strain is probably caused by higher accumulation of trehalose.


Figure 4
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  		FIG. 4. (A) Tre6P levels in different C. albicans strains after transfer from 30°C to 43°C. Wild-type (•), CC17 (tps2{Delta}/tps2{Delta}; {circ}), LDR8 (gpr1{Delta}/gpr1{Delta}; {blacktriangleup}), K1 (GPR1/gpr1{Delta} TPS2/tps2{Delta}; {triangleup}), E2 (GPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacksquare}); KM1 (gpr1{Delta}/gpr1{Delta} TPS2/tps2{Delta}; {square}), EN2 (gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacklozenge}); CC5 (TPS2/tps2{Delta}; +), and LR2 (GPR1/gpr1{Delta}, x) strains are shown. ww, wt/wt. (B) Trehalose levels in different C. albicans strains after transfer from 30°C to 43°C. Wild-type (•), CC17 (tps2{Delta}/tps2{Delta}; {circ}), LDR8 (gpr1{Delta}/gpr1{Delta}; {blacktriangleup}), K1 (GPR1/gpr1{Delta} TPS2/tps2{Delta}; {triangleup}), E2 (GPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacksquare}), KM1 (gpr1{Delta}/gpr1{Delta} TPS2/tps2{Delta}; {square}), EN2 (gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacklozenge}); CC5 (TPS2/tps2{Delta}; +), LR2 (RiGPR1/gpr1{Delta}; x), and E2R3 (RiGPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}; {diamond}) strains are shown. The inset shows in more detail the trehalose levels of the strains with a complete deletion of TPS2.

To investigate whether there is a direct effect on the trehalose biosynthesis activity, we have determined the Tps1 enzyme activities at 37°C and 43°C in the different strain backgrounds (Table 2). The main conclusions from these measurements are that (i) independent of the background, deletion of both copies of TPS2 strongly reduces the Tps1 enzyme activity (about 50%), (ii) additional deletion of one or two alleles of GPR1 in a heterozygous TPS2 mutant significantly increases the Tps1 enzyme activity (about 20%), and (iii) Tps1 activity is strongly induced in a GPR1 deletion strain at 43°C compared to 37°C (about 30%).


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  		TABLE 2. T6P synthase activity measurementsa

Additional deletion of GPR1 in the tps2 mutant does not increase killing by macrophages. The initial handling of the pathogen by macrophages plays a central role in the resolution of systemic candidiasis (30). To determine the resistance of the tps2, gpr1, and tps2 gpr1 C. albicans mutants to macrophage clearance, we tested the anti-Candida activity of peritoneal macrophages (Fig. 5). Peritoneal macrophages were directly isolated from mice and were coincubated at a 1:1 ratio with the different C. albicans strains for 3 h of interaction. Killing by peritoneal macrophages of wild-type cells was about 10%; higher killing efficiency was detected using the gpr1 mutant (30% killing). The tps2 mutant, however, is more susceptible to macrophage activity (70% killing). However, a similar behavior was detected by employing the gpr1 tps2 double-deletion strain (Fig. 5). Recently it has been shown that mutants with a deletion of TPS1, encoding the first enzyme of the trehalose biosynthesis pathway, are more easily killed by macrophages than wild-type control cells (23).


Figure 5
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  		FIG. 5. Killing of Candida cells by peritoneal macrophages. Cells of the wild-type, gpr1{Delta}/gpr1{Delta}, tps2{Delta}/tps2{Delta}, and gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta} strains were coincubated with macrophages for 3 hours. The number of CFU after this 3-hour incubation is compared to the number of CFU obtained with the same dilution of C. albicans cells in the absence of macrophages. Data shown are the means and standard deviations of three independent experiments. SC, SC5314 strain.

To investigate the cause for these differences in killing by the peritoneal macrophages, we performed in the same conditions a phagocytosis assay. The tps2, gpr1, and tps2 gpr1 C. albicans mutants were phagocytosed to the same extent (data not shown). That indicates that differences in killing are not due to differences in phagocytosis. Candidacidal activity of macrophages is due to oxidative/nonoxidative mechanisms. In order to further investigate the possible contribution of the oxidative/nitrosative mechanisms to the detected killing rate of the mutants by macrophages, we used macrophage cell line RAW 264.7. This cell line was chosen because, when activated with IFN-{gamma} and LPS, it produces higher titers of NO than peritoneal macrophages (10, 35). Nonactivated macrophages showed similar percentages of killing between the strains, with minor differences. Upon activation there was an increased killing for all the strains but in particular for the tps2 and gpr1 tps2 mutants (data not shown). These data suggest that the lower virulence of these two mutants may be due to a higher sensitivity to oxidative/nitrosative molecules secreted by macrophages.

The gpr1 tps2 mutant is avirulent in a mouse model for systemic infection. To determine whether deletion of both GPR1 and TPS2 affects the virulence of C. albicans, we injected 70 mice (10 per strain) in the caudal vein with 5 x 105 cells of the wild-type, TPS2/tps2{Delta}, tps2{Delta}/tps2{Delta}, gpr1{Delta}/gpr1{Delta}, GPR1/gpr1{Delta} (reconstituted strain), GPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}, and gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta} strains. As shown in Fig. 6, mice injected with wild-type C. albicans, with the double-deletion strain containing one allele of TPS2, or with the gpr1{Delta} stain containing the reintegrated GPR1 gene died within a period of 10 to 15 days. As previously shown, a gpr1{Delta}/gpr1{Delta} strain is only slightly affected in virulence (10 to 20% of mice survive), whereas a tps2{Delta}/tps2{Delta} strain is strongly affected in virulence (50% of mice survive). Here we show that additional deletion of one allele of GPR1 in a TPS2 deletion background further lowers the virulence. A homozygous double-deletion strain is avirulent in this mouse model system for systemic infection. We have performed this experiment twice and obtained similar results (not shown).


Figure 6
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  		FIG. 6. Virulence assay. Survival curves for mice (female BALB/c, 20 g, 10 mice/group) systemically infected with 5 x 105 cells of the C. albicans wild-type (•), CC17 (tps2{Delta}/tps2{Delta}; {circ}), LDR8 (gpr1{Delta}/gpr1{Delta}; {blacktriangleup}), E2 (GPR1/gpr1{Delta} tps2{Delta}/tps2{Delta}; {square}), KM1 (gpr1{Delta}/gpr1{Delta} TPS2/tps2{Delta}; {blacksquare}), EN2 (gpr1{Delta}/gpr1{Delta} tps2{Delta}/tps2{Delta}; {blacklozenge}), and LR2 (RiGPR1/gpr1{Delta}; {triangleup}) strains are shown.


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DISCUSSION
 
Trehalose biosynthesis genes have been proposed as interesting targets for novel drugs against a number of pathogens (13, 15, 27, 32, 38, 39). Especially the second enzyme in trehalose biosynthesis, the T6P phosphatase, is attractive, as neither the enzyme itself nor the substrate is present in the human host and accumulation of the substrate (i.e., the intermediate in the trehalose biosynthesis pathway, T6P) deregulates central metabolism. In addition, Tps2 is a phosphatase, which allows easy high-throughput screening for novel drugs. Previously, we and others have shown that deletion of the TPS2 gene strongly reduces virulence in a mouse model for systemic infection (32, 39). Apparently, the reason is the rapid accumulation of the intermediate T6P in these cells under "hostile" environmental conditions. However, the strain is not avirulent.

In this work we have tried to further increase the T6P level in the cells, thereby trying to increase the metabolic imbalance for the cells, with the aim of further reducing virulence. Stronger activation or induction of the first enzyme, Tps1, in a tps2{Delta} strain should further increase the T6P level. In S. cerevisiae, trehalose metabolism is under the negative control of the cAMP-PKA pathway. Here we have shown that this is also the case in C. albicans. Deletion of GPR1, the sensor upstream of the cAMP-PKA pathway, results in higher trehalose levels. As a consequence, the gpr1 tps2 mutant accumulates higher levels of T6P than the single tps2 mutant, resulting in a slower-growth phenotype at higher temperatures. However, the gpr1 tps2 mutant also accumulates more trehalose, and this is probably the reason for its better survival under thermostress conditions.

The high accumulation of T6P does not fit with the Tps1 enzyme activities measured in vitro (Table 2) and also does not fit with the stress tolerance assays shown in Fig. 2C. The Tps1 enzyme activity in a gpr1 tps2 double mutant is much lower (43% of wild type) than that in a single tps2 mutant (65% of wild type). The reason for the lower-than-wild-type activity may be that, similar to what is found for S. cerevisiae, Tps1 activity may depend on proper complex formation between the different subunits of the trehalose synthase complex (2). Apart from the Tps1 and Tps2 genes, the C. albicans genome also contains a gene (ORF19.5348) with homology to the yeast TPS3 gene. The three gene products together may be required to form a fully functional complex. A likely reason for the lower Tps1 activity in strains lacking TPS2 is that the trehalose biosynthesis system is partially inactivated because of lower stability in the absence of Tps2. It seems, however, that in vivo a strain lacking Tps2 still has sufficient Tps1 activity to accumulate high T6P levels. Additional deletion of GPR1 in such a strain will then result in higher T6P levels. This is supported by the fact that a single gpr1 mutant has a much higher in vitro Tps1 activity (116% of wild type). The conclusion so far would be that the gpr1 tps2 mutant is less virulent because of the higher T6P accumulation. However, in the heat stress experiment on plates the gpr1 tps2 mutant has a much higher survival rate than the single tps2 mutant. The reason for this is most probably because a gpr1 mutant is much more resistant than the wild type, and this resistance results not only from the higher trehalose level but apparently also from the fact that other genes involved in stress tolerance are also under the negative control of the Gpr1-cAMP-PKA pathway, similar to the situation in S. cerevisiae (16, 33, 36).

Previously, we showed that a tps2 mutant is not affected in morphogenesis on various hypha-inducing media whereas the gpr1 mutant is strongly affected (21, 22, 32). The morphogenesis phenotype of the gpr1 tps2 mutant is not strongly different from that of the single gpr1 mutant, with a stronger effect on Lee's medium and serum-containing medium and a slightly weaker effect on SLAD medium. This again illustrates the role of the Gpr1 receptor in sensing the environment to induce morphogenesis (3). Based on these in vitro morphogenesis experiments, it does not appear that the difference in virulence between the single mutants and the double mutant is dependent on a difference in morphogenesis.

Using differential-display technology, the group of Prigneau et al. showed that GPR1 is induced upon contact with macrophages (29), indicating that the Gpr1 protein may be important for survival within the macrophage. We have now shown that this is indeed the case, as peritoneal macrophages can kill 30% of gpr1 mutant cells whereas only 10% of the wild-type cells are killed. Interestingly 70% of the tps2 mutant cells are killed. As there are only limited supplies of substrate for the Tps1 enzyme, metabolism changes can be related to fungal death. We have to take into account that, after encountering macrophages, C. albicans cells are mainly found in the phagosome. The molecular composition of the phagosome, in terms of usable nutrients, is likely a poor environment; in particular, it is not expected to contain substantial quantities of glucose or other sugars. As has been described previously, the glyoxylate cycle enables microorganisms to grow on acetate or fatty acids as the sole carbon sources, reflecting the important adaptation of pathogenic microorganisms such as Mycobacterium tuberculosis and C. albicans (19, 24). A recent paper described how the C. albicans cells modify their metabolism after sensing this hostile environment (11). Additional deletion of GPR1 in the tps2 mutant background results in the same killing percentage as that for the single tps2 mutant.

We have shown that combined deletion of TPS2 and GPR1 results in a strain that is avirulent in a mouse model for systemic infection. This indicates that a combination of drugs targeting both the Tps2 and the Gpr1 proteins may result in an interesting antifungal combination therapy. Whereas our original idea was that additional deletion of GPR1 would enhance only the T6P levels in a tps2{Delta} strain background, our results showed that the basis for the avirulence is not dependent on the linear relationship between Gpr1 and Tps2. Each of these proteins seems to control C. albicans virulence by different mechanisms: metabolic imbalance caused by deletion of TPS2 and a defect in sensing certain environmental cues to adopt cell morphology caused by disruption of GPR1.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent technical assistance of Cindy Colombo and Suzanne Marcelis. We also thank Nico Van Goethem for preparing all the figures.

This work was supported by grants from the Research Fund of Flanders (G.0242.04) and from the European Commission, Marie-Curie RTN project CanTrain (MRTN-CT-2004-512481), both to P.V.D.


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FOOTNOTES
 
* Corresponding author. Mailing address: VIB Department of Molecular Microbiology, Laboratory of Molecular Cell Biology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium. Phone: 32 16321512. Fax: 32 16321979. E-mail: Patrick.vandijck{at}bio.kuleuven.be Back

{triangledown} Published ahead of print on 11 February 2008. Back

Editor: A. Casadevall


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Infection and Immunity, April 2008, p. 1686-1694, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01497-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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