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Infection and Immunity, December 2005, p. 7977-7987, Vol. 73, No. 12
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.12.7977-7987.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Deletions of Endocytic Components VPS28 and VPS32 Affect Growth at Alkaline pH and Virulence through both RIM101-Dependent and RIM101-Independent Pathways in Candida albicans

Muriel Cornet,1,2,{dagger} Frédérique Bidard,1,{dagger} Patrick Schwarz,3 Grégory Da Costa,1 Sylvie Blanchin-Roland,1 Françoise Dromer,3 and Claude Gaillardin1*

Microbiologie et Génétique Moléculaire, Institut National Agronomique Paris-Grignon and Institut National de la Recherche Agronomique UMR1238, Centre National de la Recherche Scientifique UMR2585, 78850 Thiverval-Grignon, France,1 Microbiologie, Hôtel-Dieu, 1 place du parvis Notre-Dame, 75181 Paris Cedex 04, France,2 Unité de Mycologie Moléculaire, CNRS FRE2849, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France3

Received 26 April 2005/ Returned for modification 31 May 2005/ Accepted 7 September 2005


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ABSTRACT
 
Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi Aspergillus nidulans, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida albicans suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with C. albicans and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the PHR1 and PHR2 genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two VPS genes, particularly VPS32, has a more drastic effect than a RIM101 deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both vps null mutants were significantly less virulent than a rim101 mutant, suggesting that VPS28 and VPS32 gene products affect virulence both through Rim-dependent and Rim-independent pathways.


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INTRODUCTION
 
Candida albicans is an opportunistic fungal pathogen that causes infections ranging from mucocutaneous illnesses to invasive processes that may involve any organ. The development of new therapies such as intensive chemotherapy for cancer or immunosuppressive regimens for transplantations, the increasing use of indwelling catheters and prosthetic devices, and progress in the treatment of bacterial infections have led to an increase in susceptible patients and cases of invasive Candida infections (12). The ability of C. albicans to colonize a wide range of tissues requires its adaptation to various environmental conditions, involving detection by the fungus of a variety of ambient signals. Extracellular pH is one of the environmental factors that modifies the physiology and morphology of the cell, and mutations in genes responding to external pH have been shown to result in less-virulent strains. For example, the cell wall protein Phr1p is expressed at a pH of ≥5.5 and is required for systemic candidiasis (blood pH is near neutrality), whereas its paralogue Phr2p is expressed only at acidic pH (pH≤ 5) and is required for vaginal candidiasis (vaginal pH is around 4.5) (11, 19, 34).

A conserved fungal ambient pH signal transduction pathway was initially described for Aspergillus and was later extended to several ascomycetes, including C. albicans (9, 10, 13, 16, 30, 31). At alkaline pH, a cascade of six Pal genes in Aspergillus nidulans or five RIM genes in Saccharomyces cerevisiae activates the zinc finger transcriptional factor PacC/Rim101p through a C-terminal proteolytic processing event. This PacC/Rim101p short active form is able to activate alkaline pH-responsive genes and to repress acidic genes (15, 29, 35). Defects in this pathway lead to reduced virulence in C. albicans, A.nidulans, and other pathogenic fungi (5, 7-9, 17).

Recent observations made by several groups pointed out a possible contribution of vacuolar protein sorting (VPS) genes, encoding class E factors of the endocytic pathway, to the Rim-dependent pathway of pH signaling. In S. cerevisiae, genome-wide two-hybrid screens revealed that Rim13p and Rim20p interacted with Snf7p/Vps32p (named Vps32p in this paper) and that Rim20p interacted with Vps4p (6, 21). Vps32p forms part of the endosomal sorting complex required for transport (ESCRT) III (2), which acts downstream of ESCRT-I and ESCRT-II complexes in the multivesicular body (MVB) pathway, whereas the Vps4p AAA ATPase acts at the end of the endocytic cycle to dissociate ESCRT complexes from the endosomal membrane (3). In addition, both Vps32p and Vps4p were shown to interact with Bro1p/Vps31p (18), another soluble class E factor associated with endosomes and required for carboxypeptidase S sorting by the MVB pathway (28). The MVB pathway is conserved from yeast to higher eukaryotes and is required for a growing list of cellular functions that includes cell surface receptors and transporters degradation, regulation of the immune response, and even budding of certain viruses like human immunodeficiency virus (for reviews, see references 1 and 23).

Direct evidence for MVB class E factor involvement in ambient pH signaling was recently obtained for Yarrowia lipolytica (20; S. Blanchin-Roland et al., unpublished data), S. cerevisae, and C. albicans (24, 39). Current models suggest that ESCRT complexes, mainly through their Vps32p moiety, function at neutral and/or alkaline pH as an assembly platform for the recruitment of Rim components leading to Rim101p activation (24, 29, 39). In this paper, we confirm the above conclusions and show that VPS genes may play additional roles in the control of the alkaline response and that vps deletions result in more pronounced defects in pathogenesis than would be expected with a simple interruption of the Rim pathway.


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MATERIALS AND METHODS
 
Strains and sequence data. The bacterial strain used for transformation and amplification of recombinant DNA was Escherichia coli DH5{alpha}. C. albicans strains are described in Table 1. All C. albicans sequence data were obtained from http://genolist.pasteur.fr/CandidaDB/.


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  		TABLE 1. Strains used in this study

Culture media and phenotypic tests. For gene expression experiments, synthetic complete medium (SC) was used, consisting of a 0.67% yeast nitrogen base, 2% glucose, and 0.77-g/liter complete amino acid supplement mixture without arginine and histidine, supplemented when required with uridine (Uri; 80 µg/ml), arginine (20 µg/ml), and histidine (20 µg/ml). Growth experiments at various pHs were carried out in SC (pH 5.3) and in SC buffered with 50 mM glycine-NaOH at either pH 9.0 or pH 10.0 and with 0.2 M citrate phosphate at pH 3.0. Droplets of serial dilutions of an exponential-phase culture in yeast extract-peptone-glucose (YPD) medium were spotted on SC, SC (pH 3.0), SC (pH 9.0), or SC (pH 10.0) and incubated at 30°C. For the filamentation assays, we used M199 medium (Invitrogen, Paisley, United Kingdom) buffered at pH 4.0 or pH 7.5 with 150 mM HEPES as previously described (10). Data were averaged from three independent experiments. As the reference strain cells produce hyphae extensively at 37°C and pH 7.5, growth rates under these conditions in both SC and M199 media were determined by dry weight measurements as follows. Cells were grown overnight in SC medium, diluted in 100 ml SC or M199 (pH 7.5) to an optical density at 600 nm (OD600) of about 0.1, and grown at 37°C. Every 2 h, 5 ml of the culture was filtered through glass microfiber filters (Whatman, Maidstone, United Kingdom) that were predried at 80°C overnight and preweighed. Then the filters were dried at 80°C overnight, and dry weights were calculated.

DNA and RNA techniques. Standard recombinant DNA techniques were performed as previously described (32). All transformation events were checked by colony PCR using puRE Taq Ready-To-Go PCR Beads (Amersham, Piscataway, NJ) and confirmed by Southern blot analysis. Sequences were obtained from the DNA sequencing department of Eurogentec (Ivoz-Ramet, Belgium). They were assembled and annotated with the Genetics Computer Group package (Madison, WI).

Gene expression was determined by quantitative real-time PCR using a light cycler (Roche Molecular Biochemicals, Meylan, France). Cells were grown in SC buffered at pH 4.0 or pH 7.5 with 150 mM HEPES at 30°C and harvested in the exponential phase (OD600, ~0.6). Cells were pelleted, frozen in liquid nitrogen, and kept at –80°C until RNA isolation. Total RNA was extracted with the RNeasy Mini-kit (QIAGEN, Courtaboeuf, France) and then treated with DNase I(QIAGEN). The Superscript II RNase H-Reverse Transcriptase kit (Invitrogen) was used for a reverse transcriptase assay from 1 µg of total RNA. The real-time PCR assay was performed using the following primer pairs: OFB16-OFB17 for RIM101, OFB22-OFB23 for PHR1, OFB32-OFB33 for ACT1, and OFB40-OFB41 for PHR2 (primer sequences are shown in Table 2). PCR parameters were 95°C for 8 min, followed by 45 cycles, each consisting of 95°C for 10 s, 55°C for 7 s, and 72°C for 10 s. A negative control with sterile water was performed for each primer set. The threshold cycle was determined as the cycle above which the fluorescence signal, produced by the SYBRgreen I dye, reached a baseline level. The expression levels of the genes were determined relative to the expression of the ACT1 gene. For each gene, experiments were carried out four times, using two cDNA samples from two independent cultures.


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  		TABLE 2. List of primers used in this study

Construction of strains. Complete deletions of VPS28 (vps28–/–; strain MC2) and VPS32 (vps32–/–; strain MC4) were constructed by the UAU1 method (14) with the BWP17 strain (38). The UAU1 cassette was amplified from plasmid pBME101 (14) with primers OFB3-OFB4 for the VPS28 gene and OFB5-OFB6 for the VPS32 gene, using the Expand High Fidelity PCR System kit (Roche Molecular Biochemicals). These primers allow amplification of the UAU1 cassette with 60- and 61-nucleotide (nt)-long flanking sequence homologues to VPS28 and VPS32, respectively. Transformants of the BWP17 strain obtained with the UAU1 cassette were screened by colony PCR: wild-type VPS28 and VPS32 alleles were detected using primers OFB8-OFB9 and OFB10-OFB11, respectively; mutant vps28::UAU1 and vps32::UAU1 alleles were detected using primer OFB8 or OFB10, respectively, with Arg4det (14). Among the nearly 100 transformants obtained for each gene, 5 of 16 (31%) and 3 of 74 (4%) tested by PCR were heterozygous clones VPS28+/– (MC1) and VPS32+/– (MC3), respectively. Five vps28–/– (MC2) and two vps32–/– (MC4) homozygotes were obtained from independent heterozygotes by mitotic recombination after growth on YPD medium at 30°C for 48 h and selected by replication onto SC medium without arginine and without Uri. Arg+ Uri+ colonies were screened by colony PCR, and integration was confirmed by Southern blot analysis.

To differentiate haplotypes in the VPS28 and VPS32 regions, we used the SC5314 genome assembly (http://www.candidagenome.org/). We aligned contig 9-10045 and contig 19-20045 on chromosome 2 for CaVPS28, and contigs 19-10237 and 19-20237 on chromosome 1 for CaVPS32. Both alleles of CaVPS28 and CaVPS32 lie in 100% identical regions, flanked by regions where numerous insertions and deletions differentiate both haplotypes. Two oligonucleotides (Table 2) hybridizing in conserved regions were designed to amplify the right border of the CaVPS28 haplotypes: OMC15 (nt 26453-26475 in contigs 19-10045 and 19-20045) and OMC16 (nt 29069 to 29047 in contig 19-10045 and nt 28363 to 28385 in contig 19-20045). Amplicons of 2,616 and 1,932 bp were predicted for each haplotype. Similarly, two primers, OMC7 (nt 53278 to 53299 in contig 19-10045 and nt 52589 to 52610 in contig 19-20045) and OMC8 (nt 53678 to 53658 in contig 19-10045 and nt 53106 to 53086 in contig 19-20045), were used to amplify the CaVPS28 left border. Amplicon sizes of 400 and 520 bp were expected. In the case of CaVPS32, primers OMC9 (nt 57459 to 57480 in contig 19-10237 and nt 57512 to 57533 in contig 19-20237) and OMC10 (nt 58440 to 58419 in contig 19-10237 and nt 58435 to 58414 in contig 19-20237) were used for the left border; amplicons of 980 and 922 bp were expected. Primers OMC11 (nt 153337 to 153356 in contig 19-10237 and nt 153348 to 153367 in contig 19-20237) and OMC12 (nt 157452 to 157431 in contig 19-10237 and nt 153477 to 153456 in contig 19-20237) were used for the right border. Amplicons of 4,116 and 130 bp were expected.

Finally, pINA1337 (F. Bidard, unpublished data), a derivative of pFLAG-MET3 (36) and carrying the HIS1 cassette and the MET3 promoter, was linearized by SwaI and targeted into MC2 and MC4 at the HIS1 locus to yield prototrophic strains MC2H and MC4H, respectively.

For complementation, the wild-type VPS28 and VPS32 genes were amplified from BWP17 genomic DNA with primers OFB57-OFB58 and OFB59-OFB60, respectively. For the VPS28 gene, the resulting PCR product was inserted into pDDB78 carrying the HIS1 cassette and digested with SmaI to construct pDO14 (26). The VPS32 gene was first inserted into pGEM-T-Easy (Promega, Charbonnières, France) and then into pDDB78 digested with NotI to give pDO15. The absence of mutation in each open reading frame was checked by DNA sequencing. NruI-digested pDO14 and pDO15 were targeted to the HIS1 locus of MC2 and MC4, respectively, to construct the vps28–/–+VPS28 (MC5) and vps32–/–+VPS32 (MC6) strains. Correct integration was confirmed by Southern blot analysis for both complemented strains.

A C-terminal truncated form of RIM101, encoding a 415-residue protein, constitutively active, called Rim101SLp, was created by a G-to-T substitution at position 1246, thus creating an in frame amber codon (D. Onesime, unpublished data); all coordinates start at the ATG of the full-length open reading frame as defined previously (13). The truncation site was determined by hydrophobic cluster analysis as for the YlRIM101-1119 allele (25). Plasmid pINA1353 was generated from plasmid pINA1341 (Bidard, unpublished), carrying RIM101SL under the control of the MET3 promoter, by digestion with StuI and HpaI to remove the MET3 promoter. PpuMI-digested pINA1353 was targeted to the RIM101 locus of strains DAY5, MC2, and MC4, generating a tandem structure of RIM101 genes which restored the wild-type 5' upstream sequence in front of RIM101SL and deleted the promoter of the resident RIM101 copy. This resulted in strains MC13, MC14, and MC15, respectively. Integration was confirmed by colony PCR using primers OFB48-OFB50 and Southern blot analysis.

Staining with FM4-64. To analyze yeast vacuolar morphology and dynamics, FM4-64 localization experiments were performed with a protocol derived from Vida and Emr (37). Yeast cells were grown in YPD to an OD600 of 0.5 to 0.7. Three OD units of cells were harvested, incubated in 150 µl of YPD containing 40 µM FM4-64 (Molecular Probes, Eugene, OR) for 30 min at 0°C, washed three times in phosphate-buffered saline at 0°C, and further incubated in 200 µl of YPD for 20 min at 18°C. Cells were centrifuged, resuspended in water, and visualized by differential interference contrast (DIC) optics and by fluorescence microscopy using an Olympus U-RFL-T microscope equipped with a CoolSNAP camera.

Virulence assay. All strains were grown on Sabouraud agar and subcultured on YPD medium at 30°C for 24 h. Cells were harvested, washed twice in sterile physiological saline, counted with a hemocytometer, and adjusted to 2 x 106 or 2 x 107 CFU/ml in sterile physiological saline. BALB/c male mice, 7 weeks old (Charles River, Les Oncins, France), were housed in groups of seven mice per cage and were inoculated by injection of 100 µl with one of the above yeast suspensions into the lateral tail vein (final amount, 2 x 105 or 2 x 106 CFU per mouse). Dilutions of the suspensions were plated on Sabouraud agar to confirm the inoculum size. Survival was monitored twice daily until day 30 postinfection. The log rank test was used to determine significant differences in survival time between groups using GraphPad Prism 3.0 software (GraphPad, San Diego, CA). A P value of <0.05 was considered significant.

Fungal burden and morphological analysis. To assess fungal burden and morphology in infected tissues, a less acute infection was required. We therefore used an inoculum of 5 x 105 CFU per mouse. Fungal cells were prepared and injected as described above. Five mice were infected with each strain and euthanized at day 4 postinfection. The kidneys being the target organs in this model (27), the left kidney was aseptically removed, weighted, and homogenized in 2 ml of sterile physiological saline; serial dilutions were plated on Sabouraud chloramphenicol agar for determination of the CFU per g of kidney. Statistical analysis was performed using the nonparametric Dunn's test for multiple comparisons. A P value of <0.05 was considered significant. Morphology of C.albicans cells recovered from infected kidneys was assessed using calcofluor white (Sigma, St. Quentin Fallavier, France) that stains chitin within the cell wall. Thus, 50 µl of each kidney homogenate was stained with 10 µl of a solution of 0.1% calcofluor white and 1% KOH. Slides were observed by fluorescence microscopy with a 340- to 380-nm filter and the same microscope as for FM4-64 staining. To quantitate the morphogenesis defects, we used the morphological index (MI) described previously by Odds et al. (27): cells were categorized as yeasts showing a spherical shape (MI score = 1), elongated yeasts showing ovoid shape and a length up to twice the diameter of the cell (MI score = 2), pseudohyphae (MI score = 3), or true hyphae (MI score = 4). For each stained homogenate, 50 cells were scored; data are the average of five mice infected with each strain. The nonparametric Kruskal-Wallis test was used for statistical analysis.


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RESULTS AND DISCUSSION
 
Isolation of vps mutants. Recently and while this work was in progress, studies with S. cerevisiae established that Rim101p processing was dependent on class E VPS factors belonging to the three ESCRT complexes required for sorting membrane proteins into the MVB pathway (39). Conservation of this process in C. albicans was assumed following the observations that class E vps mutants inhibit alkaline-induced filamentation and that this inhibition is relieved by expression of a constitutively active form of Rim101p (39). A direct requirement of Vps32p for Rim101p processing in C. albicans was shown independently (24). To confirm this link in C. albicans, we checked the effects of the deletion of two VPS genes on the expression of alkaline and acidic genes and on the growth and hyphal development at various pHs.

The VPS28 and VPS32 genes were chosen as components of the ESCRT-I and ESCRT-III complexes, respectively (2, 22) and strains carrying heterozygous and homozygous null mutations in the BWP17 background were generated by the UAU1 method (14) (see Materials and Methods and Table 1). Confirmation of the vps28 and vps32 deletions by Southern analysis is shown in Fig. 1. The vps32 homozygous mutant that exhibited an unexpected hybridization with the VPS32 probe was not considered in this study (Fig. 1D, lane 3). This method involves spontaneous transfer of the disruption cassette from one chromosome to its homologue. We thus checked whether homogenotization of a large chromosomal segment occurred simultaneously. According to sequence data available for SC5314, the ancestor of the BWP17 strain used here (38), the CaVPS28 and CaVPS32 alleles lie in 24-kb and 96-kb homozygous regions of chromosome 2 and 1, respectively, flanked by heterozygous regions. The status of the flanking regions in the parental strain was checked by PCR (see Materials and Methods), as well as in the single and double disruptants. As shown on Fig. 2, lanes 2 to 4, and as expected, heterozygocity of the VPS28 and VPS32 flanking regions was conserved in BWP17 and in all single disruptants checked. Expected sizes of amplicons were observed, except in the case of the VPS28 right border; this may reflect genomic variations between BWP17 and SC5314 generated during the successive transformations that lead to this strain (38). All double CaVPS32 disruptants retained the chromosomal structure of the parental strain, whereas one of the CaVPS28 double disruptants lost heterozygocity at the left border (Fig. 2, panel PCR2, lane 6). This strain was discarded. Haplotypes initially present on chromosomes 1 and 2 were thus conserved, except for the 24- and 96-kb regions separating the heterozygous markers where extensive homogenotization may have occurred. We therefore cannot rule out that some of the phenotypes displayed by our mutants may result from unmasking of preexisting or transformation-induced mutations in these regions. The fact that most phenotypes could be completely complemented by insertion of an ectopic copy of the wild type argues against such a hypothesis (see below).



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  		FIG. 1. Confirmation of the vps deletions by Southern analysis. (A) Schematic restriction map of the VPS28 and VPS32 wild-type and deleted alleles. Vertical bars denote EcoRV restriction sites, and numbers on their right indicate predicted sizes obtained with the VPS28 or the VPS32 probe (probe1) for the wild-type alleles and the URA3 probe (probe 2 is a 0.8-kb URA3 fragment obtained by XhoI and PvuII digestion of pBME101 (14) for the deleted alleles. (B and C) Southern blot analysis of EcoRV-digested genomic DNA from the five vps28–/–-deleted strains (lanes 2 to 6) and the BWP17 parent strain (lane 7), using the VPS28 probe (B) or the URA3 probe (C). (D and E) Southern blot analysis of EcoRV-digested genomic DNA from six vps32–/–-deleted strains (lanes 2 to 7), two heterozygote-deleted strains (lanes 8 and 9), and the BWP17 parent strain (lane 10), using the VPS32 probe (D) or the URA3 probe (E) Lanes 2 to 5 are double deletants obtained from the lane 8 heterozygote, whereas lanes 6 and 7 show double deletants obtained from the other heterozygote (lane 9). Lane 1 is a lambda DNA-BstEII digest marker for all panels.



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  		FIG. 2. PCR amplifications of the two haplotypes in heterozygous regions flanking VPS28 and VPS32. (Top) Right and left borders of VPS28 (PCR1 and PCR2, respectively). Lane 1, 1-kb Plus DNA ladder; lane 2, BWP17; lanes 3 and 4, two VPS28+/– single disruptants; lanes 5 to 7, three independent vps28–/– double disruptants. (Bottom) Left and right borders of VPS32 (PCR3 and PCR4, respectively). Lane 1, 1-kb Plus DNA ladder; lane 2, BWP17; lanes 3 and 4, two VPS32+/– single disruptants; lanes 5 to 8, vps32–/– double disruptants (strains lanes 5 and 6 were obtained from the lane 3 single disruptant, and strains 7 and 8 were obtained from the other single disruptant).

FM4-64 localization experiments indicated that double disruptants for CaVPS28 or CaVPS32 displayed defects in endocytosis (24). Twenty minutes after being stained, FM4-64 dye could clearly be seen on the vacuole membrane in the reference strain (DAY185) cells, while it remained in small compartments adjacent to the vacuole in the two vps mutant cells (Fig. 3). These structures were highly reminiscent of class E compartments, which accumulate proteins destined for the vacuole in class E vps mutants in S. cerevisiae (33, 37). This phenotype was lost upon introduction of a wild-type copy of the disrupted alleles (Fig. 3).



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  		FIG. 3. FM4-64 staining in the C. albicans mutants. The strains were DAY185 (reference strain), MC2H (vps28–/–), MC5 (vps28–/–+VPS28), MC14 (vps28–/–+RIM101SL), MC4H-(vps32–/–), MC6 (vps32–/–+VPS32), and MC15 (vps32–/–+RIM101SL). They were incubated with 40 µM FM4-64 as described in Materials and Methods and visualized by fluorescence microscopy. The left side of each panel is FM4-64 fluorescence, while the right side of each panel shows differential interference contrast optics.

Role of the VPS28 and VPS32 genes in alkaline and acidic genes regulation. The effects of vps28 and vps32 deletions on the transcription of the alkaline-induced genes (RIM101 and PHR1) and of the acid-induced and alkaline-repressed gene PHR2 were estimated by quantitative real-time PCR of transcripts extracted from cells grown at pH 4.0 and pH 7.5, using actin transcripts as a reference (Fig. 4).



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  		FIG. 4. Expression patterns of alkaline-induced genes (RIM101 and PHR1) and acid-induced gene (PHR2) in C. albicans strains at pH 4.0 and pH 7.5. The strains were (from left to right) DAY185 (reference strain), DAY25 (rim101–/–), MC2H (vps28–/–), MC4H (vps32–/–), MC5 (vps28–/–+VPS28), MC6 (vps32–/–+VPS32), MC13 (rim101–/–+RIM101SL), MC14 (vps28–/–+RIM101SL), and MC15 (vps32–/–+RIM101SL). Expression levels were calculated relative to the expression of the ACT1 reference gene. Data are the average of four experiments.

In heterozygous vps-deleted strains vps28+/– and vps32+/– at pH 4.0 and pH 7.5, RIM101, PHR1, and PHR2 expression levels were comparable to those observed for the reference strain (data not shown). In the vps28–/– and vps32–/– homozygous backgrounds and at pH 4.0, the RIM101, PHR1, and PHR2 mRNA levels were not significantly affected (Fig. 4). At pH 7.5, both homozygous deletions of VPS28 and VPS32 significantly decreased RIM101 expression and abolished PHR1 expression, whereas PHR2 expression derepressed up to twice its level at acidic pH in the reference strain. These effects are completely similar to those observed with the rim101 homozygous deletion (Fig. 4). Complementation of the vps28–/– and vps32–/– homozygous deleted strains with one copy of their corresponding wild-type allele completely restored a wild-type phenotype.

These results confirm that Vps28p and Vps32p are required at alkaline pH for PHR1 and RIM101 induction and for PHR2 repression, two processes known to require Rim101p activation. To further confirm this hypothesis, we checked whether expression of a constitutively active form of Rim101p was able to bypass the vps defects. To this end, we integrated a plasmid carrying RIM101SL, encoding a 415-residue C-terminally truncated form of Rim101p, in the various mutants at the RIM101 locus (see Materials and Methods). Transcript levels of RIM101SL made under these conditions were similar to those driven by the native RIM101 gene (compare RIM101 expression in the reference strain and in the rim101–/–+RIM101SL backgrounds at ph 7.5) (Fig. 4). On the contrary, and as shown in Fig. 4, expression of RIM101SL in vps null mutants led to partial derepression of PHR1 and partial repression of PHR2 at both pHs. These effects are qualitatively similar to those observed after the suppression of a rim101 homozygous deletion (rim101–/–+RIM101SL) (Fig. 4); they suggest that our truncated allele does not perfectly mimic the physiologically active form of Rim101p and show that regulation of PHR1 and PHR2 is restored at alkaline pH in vps mutants to the same extent as it would be in a rim mutant. However, the suppression induced by the RIM101SL truncated form in the vps mutants, even partial, suggests that Vps28p and Vps32p act in the Rim pathway upstream from the processing of Rim101p. As the expression of this RIM101SL truncated allele has no effect on the endocytic phenotype (Fig. 3), the role of the two Vps factors in the Rim pathway appears separate from their role in the MVB transport machinery.

Role of VPS28 and VPS32 on growth and morphology at acidic and alkaline pH. On solid SC medium, two independent clones of each vps28–/– and vps32–/– homozygous deletion grew as well as the reference strain at pHs 3.0 and 5.3 (Fig. 5). At pH 9.0, the vps32–/– strain was affected more drastically than the vps28–/– and the rim101–/– null homozygotes that still showed a growth pattern near the reference strain. At pH 10.0, the vps32–/– strain was unable to form colonies, whereas both vps28–/– and rim101–/– were severely affected, with vps28–/– being slightly more sensitive than rim101–/–. Growth inhibition at alkaline pH was totally repaired after integration of one copy of the corresponding wild-type allele at the HIS1 locus (vps28–/–+VPS28 and vps32–/–+VPS32) (Fig. 5). The suppression by the constitutively active RIM101SL gene was nearly complete in the case of the rim101–/–+RIM101SL strain but only partial in strains vps28–/–+RIM101SL and vps32–/–+RIM101SL. This suppression by the RIM101SL gene was confirmed by growth experiments in liquid SC medium buffered at pH 9.0 (data not shown).



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  		FIG. 5. Sensitivity of C. albicans strains to ambient pH. The strains were (from left to right) DAY185 (reference strain), DAY25 (rim101–/–),MC2H (vps28–/–), MC4H (vps32–/–), MC5 (vps28–/–+VPS28), MC6 (vps32–/–+VPS32), MC13 (rim101–/–+RIM101SL), MC14 (vps28–/–+RIM101SL), and MC15 (vps32–/–+RIM101SL). Droplets of two dilutions (105 and 104 cells/ml) were spotted on SC medium (pH 5.3) and SC medium buffered at pHs 3.0, 9.0, and 10.0. Plates were incubated at 30°C for 48 h except for the plate at pH 10.0, which was incubated for 5 days.

These results show that the VPS factors have a significant function in the alkaline response and suggest that this function is not only Rim dependent.

The Rim pathway is required in C. albicans on several media for hypha formation, which normally occurs only at neutral and/or alkaline pH (9). Recently, several vps mutants were shown to affect hypha formation in C. albicans at alkaline pH, and these effects were partially relieved by expressing a Rim101p constitutively active form (39). To confirm the role of the ESCRT complexes in the Rim-dependent induced filamentation, we compared the vps mutants to the rim101 mutant for filamentation at pH 4.0 and pH 7.5.

After 4 h at 37°C in M199 at pH 4.0, cells of all strains were in the yeast form, as were those of the reference strain (Fig. 6A), except for the RIM101SL suppressed strains (see below). At pH 7.5, the reference strain showed an average of 92% ± 2% of the cells producing hyphae (Fig. 6E). All deleted rim101–/–, vps28–/–, and vps32–/– strains were defective in filamentation, showing <5% hyphae (Fig. 6F to H) even after 36 h of incubation (data not shown). This filamentation defect was totally suppressed in the vps28–/–+VPS28 and vps32–/– +VPS32 complemented strains, which exhibited 92% ± 2% and 89% ± 3% of hyphal cells, respectively (Fig. 6I and J).



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  		FIG. 6. Morphology of C. albicans cells in M199 medium adjusted to pH 4.0 or pH 7.5. The strains were DAY185 (reference strain) at pH 4.0 (A) and pH 7.5 (E); deleted strains DAY25 (rim101–/–), MC2H (vps28–/–), and MC4H (vps32–/–) at pH 7.5 (F to H); complemented strains MC5 (vps28–/–+VPS28) and MC6 (vps32–/–+VPS32) at pH 7.5 (I and J); and suppressed MC13 (rim101–/–+RIM101SL), MC14 (vps28–/–+RIM101SL), and MC15 (vps32–/–+RIM101SL) strains at pH 4.0 (B to D) and at pH 7.5 (K to M).

The introduction of the RIM101SL allele into the rim101–/–, vps28–/–, and vps32–/– mutants restored complete hypha production at pH 7.5 in 91% ± 4%, 92% ± 2%, and 92% ± 5% of the cells, respectively (Fig. 6K to M). Filamentation was also induced in these three strains at pH 4.0 with averages of 62%± 3%, 66% ± 4%, and 76% ± 4% of the cells with hyphae, respectively (Fig. 6B to D).

These results indicate that in C. albicans, both VPS28 and VPS32 are required for alkaline-induced Rim101p-dependent hypha formation and that the 415-residue truncated form of RIM101 induces filamentation under both acidic and alkaline conditions. Since the VPS deleted strains suppressed by the RIM101SL carry both the wild-type and the truncated alleles at the RIM101 locus, the RIM101SL truncated allele appears at least partially dominant.

Deletions in VPS28 and VPS32 affect virulence, kidney fungal burden, and morphogenesis in vivo. To determine whether the MVB pathway plays a role in C. albicans virulence, we analyzed the vps28–/– and vps32–/– mutants in the hematogenously disseminated candidiasis model (32). Reference and isogenic strains rim101–/–, vps28–/– and vps32–/–, as well as complemented strains (vps28–/–+VPS28 and vps32–/–+VPS32), were injected into the tail veins of mice at a dose of 2 x 106 CFU/mouse. All groups were compared by the log-rank test with highly significant differences in survival time (P < 0.0001) (Fig. 7). As previously shown (9), the virulence of the rim101–/– strain was reduced compared to the reference strain (median survival time of 8 days compared to 2 days; P = 0.0003). The deleted strains vps28–/– and vps32–/– appeared even more attenuated than the rim101–/– strain, with >50% of the mice surviving 29 days after inoculation (Fig. 7) (P = 0.006 for vps28–/– and P = 0.0008 for vps32–/–, each compared to rim101–/–). The dramatic virulence defect of the two vps mutants might be related to a growth defect in vivo. We noticed, however, that the growth rate of the vps28–/– mutant was comparable to that of the reference strain and that the vps32–/– mutant showed only a slight growth defect when strains were tested on M199 or SC at 37°C and pH 7.5, a pH comparable to the physiological pH of mammalian blood (data not shown). Complementation of the mutants with the wild-type allele repaired virulence, as all mice of the complemented groups died by 6 days for vps32–/–+VPS32 and 8 days for vps28–/–+VPS28, a significant difference from their respective null mutants (P < 0.001 for both complemented strains). The restored virulence showed by the complemented strains, particularly vps28–/–+VPS28,the was not complete, suggesting a gene dosage effect on virulence, due to the restitution of a single copy of the gene (4). However, we cannot exclude the formal possibility that part of the phenotype of the vps28–/– mutant resulted from additional mutations in the 28-kb region surrounding VPS28 that may have undergone homogenotization during mutant construction (see above). Similar trends in virulence were observed in mice infected with 2 x 105 CFU/mouse with each of the above strains (P < 0.0001; data not shown).



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  		FIG. 7. Survival curves of groups of seven BALB/c male mice infected intravenously with 2 x 106 cells of DAY185 reference strain (+); MC2H (vps28–/–) ({square}); MC4H (vps32–/–) ({diamond}); MC5 (vps28–/– +VPS28) ({blacktriangleup}); MC6 (vps32–/–+VPS32) (•); and DAY25 (rim101–/–) ({circ}).

To assess fungal burden in kidneys, mice infected with 5 x 105 CFU/mouse were sacrificed 4 days postinfection. Figure 8 shows that mice challenged by the C. albicans reference strain were heavily infected with a fungal burden of 5.6 log10 CFU/g of kidney, whereas mice infected with the two deleted strains vps28–/– and vps32–/– had a reduced fungal burden of 4.4 and 4.1 log10 CFU/g of organ, respectively (P < 0.05 for both strains, each compared to the reference strain). The complemented strains vps28–/–+VPS28 and vps32–/–+VPS32 colonized kidneys as efficiently as the reference strain (P > 0.05 for both strains compared to the reference strain).



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  		FIG. 8. Fungal burden in the kidney of BALB/c male mice infected intravenously with 5 x 105 cells of DAY185 reference strain (+), MC2H (vps28–/–) ({square}), MC4H (vps32–/–) ({diamond}), MC5 (vps28–/–+VPS28) ({blacktriangleup}), and MC6 (vps32–/–+VPS32) (•). Each strain was inoculated in five mice.

We have shown above that the vps-deleted strains were defective in vitro for hyphal formation. We analyzed, by calcofluor staining, the morphology of the fungal elements present in the kidneys 4 days after infection (Fig. 9), and we used the MI to quantitate defects (Table 3). In mice infected with the reference strain, 91% of the cells had an MI score of 4, showing numerous clusters of long true hyphae (Table 3 and Fig. 9A). Conversely, only 7% and 4% of the cells had an MI score of 4 in mice infected with the vps28–/– and vps32–/– mutant strains, respectively (Table 3) (P < 0.01 for both strains, each compared to the reference strain). For the mutant strains, cells were predominantly had an MI score of 3 with pseudohyphal forms (Table 3; Fig. 9D, vps28–/– and Fig. 9E and F, vps32–/–), other shapes had an MI score of 1 or 2 with yeast forms and rare germ tubes (Fig. 9B, vps28–/–; Fig. 9C, vps32–/–). Each vps28–/–+VPS28 and vps32–/–+VPS32 complemented strain formed comparable rates of cells with an MI score of 4, compared to the reference stain (Table 3; Fig. 9G, vps28–/–+VPS28; Fig. 9H, vps32–/–+VPS32) (P > 0.05 for both strains compared to the reference strain).



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  		FIG. 9. Morphology of fungal elements present in the kidneys of mice infected intravenously with 5 x 105 yeast cells of DAY185 (reference strain) (A), deleted strains MC2H (vps28–/–) (B and D), and MC4H (vps32–/–) (C, E, and F), and complemented strains MC5 (vps28–/–+VPS28) and MC6 (vps32–/–+VPS32) (G and H). Kidney homogenates obtained at day 4 postinfection were stained with 0.1% calcofluor white and 1% KOH and examined with an epifluorescence microscope.


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  		TABLE 3. Morphology of the cells recovered from the kidneys of the mice infected intravenously with 5 x 105 cells

These results show that both Vps28p and Vps32p factors are required in vivo for pathogenesis. The vps mutants showed inadequate hyphal development and failed to colonize the kidneys as efficiently as the control strain. Such defects have already been reported for mutants of the Rim pathway (7). We demonstrate here that both vps28 and vps32 mutants are significantly less virulent than the rim101 mutant, suggesting that activity of the ESCRT-I and the ESCRT-III complexes is required for virulence not only through their effect on the Rim pathway.

Such an increased effect may be related to the general role of the MWB pathway in cell physiology. The main function of this machinery in S. cerevisiae is to sort endosomal transmembrane proteins directed to the lumen of the vacuole away from proteins delivered to the membrane of the vacuole or to be recycled back to the plasma membrane or Golgi complex (1, 5). This control of membrane protein recycling or degradation is essential for the regulation of the cell surface composition and may thus affect various cell sensing and adaptation processes besides external pH sensing. It is thus not unexpected that defects in the MVB pathway may have a more dramatic impact than crippling the Rim pathway on cell adaptation to the extracellular environment and thus on cell survival in the host. Comparison of global changes elicited by Rim and Vps mutations may shed light on these additional processes.


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ACKNOWLEDGMENTS
 
This work was supported by the Direction de la Recherche Clinique of the Assistance Publique-Hôpitaux de Paris, the Centre National de la Recherche Scientifique, and grants from Merck & Company and Fujisawa, Inc., through funding of M.C.; and by the European Commission (QLK2-2000-00795, Galar Fungail Consortium), through funding of F.B.

Sequence data from C. albicans were obtained from the Stanford DNA Sequencing and Technology Center, with the support of the NIDR and the Burroughs Wellcome Fund.

Plasmids carrying the MET3 promoter (pFLAG-MET3) were kindly donated by Y. Uehara, and strains deleted for RIM101, BWP17, pDDB78, and UAU1-carrying plasmids were a generous gift from A. P. Mitchell. Helpful discussions with members of the lab, particularly Mathias Richard, are greatly acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Microbiologie et Génétique Moléculaire, INRA, CBAI, 78850 Thiverval-Grignon, France. Phone: 33 1 30 81 54 52. Fax: 33 1 30 81 54 57. E-mail: claude{at}grignon.inra.fr. Back

Editor: T. R. Kozel

{dagger} Both authors contributed equally to this work. Back


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Infection and Immunity, December 2005, p. 7977-7987, Vol. 73, No. 12
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.12.7977-7987.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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