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1 University of Applied Sciences, Department of Medical Engineering, Carl-Zeiss-Promenade 2, D-07745 Jena, Germany
2 Leibniz-Institute for Natural Products Research and Infection Biology/Hans-Knöll-Institute, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany
3 Department of Hygiene, Microbiology and Social Medicine, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
Correspondence
Raimund Eck
raimund.eck{at}hki-jena.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Present evidence suggests that several factors control virulence of C. albicans, including the ability to switch between different morphogenetic forms, adherence to host epithelial and endothelial cells, and secretion of proteinases and phospholipases (Cutler, 1991; Köhler & Fink, 1996; Lo et al., 1997; Odds, 1994). So far, a number of virulence factors of C. albicans has been characterized. However, the mechanism that enables this opportunistic fungus to become pathogenic has not been unravelled yet. Several classical virulence factors have been identified so far; however, it seems relevant to analyse virulence-determining signalling pathways as well. These define the molecular steps during switching from the non-pathogenic commensal form to the life-threatening pathogenic form. Several signalling pathways have been linked to virulence, including hyphal induction pathways (Ernst, 2000a, b), and the vacuolar transport pathways (Bruckmann et al., 2000, 2001; Eck et al., 2000; Palmer et al., 2003).
The phosphatidylinositol (PI) 3-kinase Vps34p of C. albicans is a key enzyme of the vacuolar protein transport, and is required for virulence. A vps34 null mutant strain shows several defects related to virulence, including avirulence in a mouse model of systemic candidiasis, inability to form hyphae on different solid media, delayed yeast-to-hyphae transition in liquid media, hyperfilamentation under microaerophilic/embedded conditions, hypersensitivity to high temperature and hyperosmotic stress, and reduced adherence to human cells (Bruckmann et al., 2000; Eck et al., 2000). In addition, the vps34 null mutant displays enlarged and electron-transparent vacuoles (Bruckmann et al., 2001). The Vps34p protein of C. albicans interacts with Vma7p, a component of the Candida V-ATPase complex (Eck et al., 2005). vma7 null mutants are also avirulent (Poltermann et al., 2005). Moreover, the secretion of aspartyl proteinases (Saps) and the resistance to toxic metal ions and fungistatic compounds are decreased in the vps34 null mutant strain (Kitanovic et al., 2005). The Vps34p protein exhibits lipid kinase and autophosphorylation activity. The characterization of a specific Vps34p lipid kinase defective mutant strain showed that the lipid kinase activity is essential for the role of Vps34p in pathogenicity (Günther et al., 2005). Thus, lipid substrates of Vps34p, such as phosphatidylinositol 3-phosphate [PtdIns(3)P], are candidates to connect Vps34p signalling and virulence. Therefore, we speculated that the PtdIns(3)P-binding proteins Fab1p and Vac1p play a role in virulence.
PtdIns(3)P-binding proteins were identified in the yeast Saccharomyces cerevisiae and in mammalian cells. They contain a conserved FYFE finger domain named after the first four identified FYFE finger proteins Fab1p, YOTB, Vps27p and EEA1 (human homologous protein of Vac1p) (Burd & Emr, 1998; Stenmark & Aasland, 1999). In S. cerevisiae the binding protein Fab1p functions downstream of Vps34p as a PI(3) 5-kinase, which regulates vacuolar membrane turnover through the production of PdtIns(3,5)P2 (Gary et al., 1998). The PtdIns(3)P-binding protein Vac1p functions in Golgi-to-endosome vesicular protein transport (Weisman & Wicker, 1992). Because the lipid kinase function of Vps34p is essential for virulence of C. albicans, and the PdtIns(3)P-binding protein Fab1p is not involved in virulence, we focused on the functions of another PdtIns(3)P-binding protein, namely Vac1p (Augsten et al., 2002; Günther et al., 2005).
In S. cerevisiae Vac1p is a peripheral membrane protein, which belongs to the class D vps group, and plays a role in vesicle docking at the late endosome (multivesicular body). Mutations in the FYFE domain of the Vac1p protein result in mis-sorting of vacuolar proteins and temperature-sensitive growth (Burd et al., 1997). Vac1p is an essential component of the endosomal SNARE (SNAP receptor) complex, which is responsible for the interaction and fusion of Golgi-derived vesicles with the late endosome (Burd et al., 1997; Webb et al., 1997). Furthermore, Vac1p links the Vps34p-signalling pathway to the fusion of transport vesicles with the endosome (Burd & Emr, 1998). This step is regulated by direct interaction with Rab GTPase signalling protein Vps21p; the fusion is mediated by Pep12p and Vps45p (Horazdovsky et al., 1994; Peterson et al., 1999; Peterson & Emr, 2001; Tall et al., 1999). In addition, the class C vps proteins Vps18p and Vps11p interact with Vac1p (Peterson & Emr, 2001; Srivastava et al., 2000).
In this paper we show that Vac1p of C. albicans contributes to virulence, and that a vac1 null mutant is avirulent. In addition, Vac1p affects several important virulence parameters, thus demonstrating that in C. albicans this PdtIns(3)P-binding protein acts as a central component of the virulence-determining Vps34p pathway.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Strains were grown in YPD medium [2 % (w/v) glucose, 2 % (w/v) peptone, 1 % (w/v) yeast extract], YP-sucrose medium [2 % (w/v) sucrose, 2 % (w/v) peptone, 1 % (w/v) yeast extract], SD medium [0.7 % (w/v) yeast nitrogen base without amino acids (Difco), 2 % (w/v) glucose, 1 M sorbitol], YNB medium (SD without 1 M sorbitol) or Sabouraud medium [2 % (w/v) glucose, 1 % (w/v) peptone (casein)] at 28 °C. SD and YNB were supplemented with 20 µg uridine ml–1 for the growth of Ura– strains. Uridine auxotrophs were selected on YNB plates containing uridine and 1 mg 5-fluoroorotic acid (5-FOA) ml–1 (Sigma). Growth was monitored by measuring optical density at 600 nm in a SpectraMax 190 spectrophotometer. To induce hyphal growth on solid medium, cells were grown overnight in YPD at 30 °C, then washed with 0.9 M NaCl, diluted and spread either on Spider plates [1 % (w/v) nutrient broth, 0.2 % (w/v) K2HPO4, 1.35 % (w/v) agar, 1 % (w/v) mannitol] (Lee et al., 1975), or on YPD plates supplemented with 15 % (w/v) fetal calf serum (FCS) at a density of 20 to 100 cells per plate. Plates were incubated at 37 °C for at least 7 days. Hyphal growth in liquid media was induced by diluting late exponential-phase cultures grown at 30 °C tenfold either into fresh YPD supplemented with 10 % (w/v) FCS, or into Spider medium at 37 °C. Sensitivities of the mutants to various metal ions were assayed on YPD plates supplemented with the appropriate salts. Escherichia coli XL-1 Blue {supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac F'[proAB+ lacIq 
M15 Tn10(tetr)]} (Stratagene) was used for cloning.
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). Primers 3 and 4 were used to amplify the 3' region. Primer 3: 5'-ACGCGTCGACCAAAGCGAGACGGTTTGATG-3' (positions +1232 to +1252); primer 4: 5'-GCCTGCAGCAAGCAGAACAAGTTATGACAGC-3' (positions –178 to –156; downstream of VAC1) yielded a 311 bp product, which was digested with SalI and PstI and cloned into plasmid pV0, resulting in pV1 (Fig. 1a). For gene disruption, plasmid pV1 was cut with SacI and PstI. The digested plasmid pV1 was transformed to C. albicans. For homologous reintegration of the VAC1 gene, VAC1 was amplified by PCR with primer 5 [5'-GCGAGCTCTGAGAGTAATCCAAAGCAGTTAC-3' (–695 to –672, upstream of VAC1)] and primer 6 [5'-GCGGTACCACCCATGCGTGTCCAACAACT-3' (–905 to –884, downstream of VAC1)]. The 2961 bp PCR product was restricted with SacI/KpnI and cloned into pUC19 (Yanisch-Perron et al., 1985), resulting in pUCV. The URA3 gene was amplified with primer 7 [5'-ACGCCCAAGGAATTGATTTGGATGGTATA-3' (–411 to –393, upstream of URA3)] and primer 8 [5'-ACGCCCTTGGACCACCTTTGATTGTAAATA-3' (–120 to –101, downstream of URA3)]. The 1364 bp PCR product was restricted with StyI and cloned into the StyI site (positions –244 to –249, upstream of VAC1) of pUCV, resulting in pUCVU. After digestion of pUCVU with SacI and KpnI a 4.3 kb insert was isolated that harbours the VAC1 gene, upstream as well as downstream regions for homologous recombination, and the URA3 gene as selectable marker.
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; Eck et al., 1997).
Disruption and reintegration of VAC1.
To disrupt the VAC1 gene, the previously described hisG-URA3-hisG cassette was used in a multistep procedure (Fonzi & Irwin, 1993). A 4.6 kb SacI–PstI fragment of pV1 containing the URA-blaster flanked by short sequences from the upstream and downstream sequences of VAC1 and portions of the promoter and terminator, respectively, was used to transform C. albicans Ura– strain CAI-4. To confirm proper deletion of the VAC1 ORF (1338 bp, 445 aa, Stanford's Candida albicans sequencing project Assembly 19, ORF19.5662 on Contig231; http://sequence-www.stanford.edu/group/candida/index.html), the resulting Ura+ transformants were examined by Southern analysis using HindIII/PstI-digested chromosomal DNA and a labelled 300 bp SacI–KpnI fragment of pV1 as probe. Southern hybridization was also applied to evaluate segregants and transformants of the later disruption steps, according to standard protocols. In the first step one VAC1 allele was replaced by the hisG-URA3-hisG cassette (CV1). Strain CV1 was plated on 5-FOA-containing medium for isolation of Ura– segregants (CV2). A second transformation with the same disruption construct led to the isolation of a vac1 null mutant strain (CV3). Again, Ura– segregants were selected (CV4).
VAC1 was reintroduced into the null mutant strain CV4 by transformation with the 4.3 kb SacI/KpnI insert of pUCVU. Chromosomal DNA from selected clones was digested with HindIII/PstI and analysed by Southern blotting and hybridization (CV5).
Fluorescent labelling with FM4-64 and microscopy.
Yeast cells were stained with the dye FM4-64 as described previously (Günther et al., 2005; Vida & Emr, 1995). For microscopy, cells were placed on slides that were covered with agarose. Transport of FM4-64 was visualized microscopically using a BP510-550 excitation filter (U-MWG2), BA590 beam splitter and DM570 emission filter (Olympus BX51TF).
Hyphal induction within agar matrix.
For filamentous growth within an agar matrix the various C. albicans strains were grown overnight in YPD, diluted to a concentration of 5x106 cells ml–1, and cultivated for 4 h at 30 °C. Then 100 µl of the diluted cells (103 cells ml–1) was mixed in YP-sucrose agar (YP-sucrose medium supplemented with 2 % agar), plated, and incubated for 120 h at 22 °C. Colonies were examined microscopically, and the percentage of filamentous colonies was determined.
Hyphal induction by growth at neutral pH.
C. albicans cells (100 µl of an overnight culture) were added to 20 ml acidic Soll's medium (Swoboda et al., 1994), pH 4.5, and incubated for 12 h at 30 °C. Then 20 ml neutral Soll's medium, pH 6.5, was supplemented with 500 µl from the acidic culture. The hyphal growing cells were counted using a microscope.
Chlamydospore formation.
Rice agar (2 %, w/v, agar; 40 g rice per litre H2O) was melted and after a short centrifugation the agar was plated onto a slide. C. albicans cells from an overnight culture were streaked onto this agar and covered with a coverslip. The slides were incubated in the dark at 25 °C in a damp environment. Chlamydospore formation was evaluated microscopically after 96 h. For growth in liquid medium the rice agar was diluted 1 : 1 (v/v) with water.
Adherence assays.
C. albicans cells obtained from an overnight YPD culture at 30 °C were diluted into 20 ml fresh YPD medium (pH 6.0) to a concentration of 5x105 cells ml–1. Following incubation for 16 h at 30 °C, the cells were washed and diluted in YPD medium, pH 4.5, to a concentration of 2x107 cells ml–1. Human vaginal SK-LMS-1 cells (leiomyosarcosoma) were cultivated in microtest plates in RPMI medium (Serva) to a concentration of 104 cells per well. The tissue culture cells were washed with PBS (140 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2), and then 4x106 C. albicans cells were added to each well. After cultivation at 37 °C for 2 h, C. albicans cells were stained with Calcofluor white (12.5 µg ml–1; Sigma) for 30 min, and non-adherent C. albicans cells were removed by washing with PBS. Finally, the fraction of adherent fluorescent yeast cells was determined in a fluorescence reader, using 360 nm for excitation and 460 nm for emission (FluoroScan, Labsystems). The wild-type C. albicans strain SC5314 was set to 100 %. For statistical analysis Student's t-test was used. A P value 
0.025 was considered significant.
Sap ELISA assay.
This assay was carried out essentially as described previously (Kitanovic et al., 2005). The primary mouse antibody (FX 7-10, which preferentially detects Sap2p but also other Saps) was kindly provided by Dr Borg-von Zeppelin, Göttingen, Germany.
Virulence studies.
Male outbred NMRI mice (Harlan-Winkelmann), 6 weeks old, were housed at five per cage and checked daily. The various strains of C. albicans were grown in Sabouraud dextrose [glucose] broth at 28 °C until late exponential phase. Cells were washed three times and resuspended in 0.9 % NaCl. Samples (200 µl) of suspension containing 5x106 cells were used to infect immunocompetent mice by intravenous injection into the lateral tail vein. Survival of the animals was monitored for 20 days. For comparison of survival curves the log-rank test was used (Peto et al., 1977). A P value 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used to transform the C. albicans Ura– strain CAI-4. Southern blot analysis of C. albicans DNA from a heterozygous vac1 mutant (CV1) identified two bands (Fig. 1c). The 2.7 kb fragment represents the VAC1 wild-type allele (Fig. 1c, lane 1), while the 5.7 kb fragment can be identified as the hisG-URA3-hisG cassette that replaced the VAC1 gene (lane 2). The excision of one copy of hisG and the URA3 gene in the Ura– derivatives results in the 2.9 kb band (CV2, lane 3). A second transformation with the same disruption construct generated vac1 null mutant strain CV3. The loss of the 2.7 kb wild-type fragment and the presence of 5.7 kb and 2.9 kb bands are consistent with the replacement of the second VAC1 allele (lane 4). Southern blot analysis of a representative Ura– segregant identified a 2.9 kb band that represents two hisG disrupted alleles (CV4, lane 5). For reintegration of VAC1, the 4.3 kb SacI–KpnI fragment of plasmid pUCVU (VAC1 and URA3) was used to transform strain CV4. Ura+ clones were selected, and proper introduction of VAC1 and URA3 was recognized by the appearance of a 3.6 kb PstI–HindIII fragment, which represents the integrated VAC1 and URA3 genes (CV5, lane 6).
Vac1p is required for the transport and fusion of prevacuolar endocytic compartments
Vps34p is required for the transport of prevacuolar vesicles to the vacuole in both C. albicans and S. cerevisiae. This function plays a role in endocytosis and in the CPY (carboxypeptidase Y) protein transport pathway from the late Golgi to the vacuole (Bruckmann et al., 2001; Wurmser & Emr, 1998). This action can be analysed by following the distribution of the fluorescent lipophilic dye FM4-64, as it is transported in yeast cells via the endocytotic uptake and vesicle-mediated transport to the vacuole (Vida & Emr, 1995). The endocytic transport of the lipophilic dye was analysed in the vac1 null mutant strain CV3 by fluorescence microscopy (Fig. 2). The null mutant strain showed weak staining of the vacuolar membrane, whereas prevacuolar endocytic compartments in the cytoplasm were stained. The heterozygous mutant strain CV1 and the revertant strain CV5 showed similar staining to the wild-type strain SC5314, where the dye is contained in the vacuolar membrane and shows a typical ring staining pattern. Therefore we conclude that deletion of VAC1 in the C. albicans vac1 null mutant strain results in defective transport/fusion functionality of the prevacuolar endocytic vesicle.
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).
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). Filamentous growth of C. albicans is induced by several signalling pathways that are triggered by several environmental signals, and which are activated experimentally by Spider medium supplemented with mannitol as a carbon source, serum, microaerophilic conditions, growth in a solid agar matrix, and pH-shift. These various methods were used to induce hyphal growth of the vac1 null mutant strain CV3. When solid Spider medium was used for hyphal induction, the colonies of the vac1 null mutant strain did not form mycelial structures. However, the colonies of the heterozygous mutant strain CV1 and the revertant strain CV5 were similar to the wild-type strain SC5314, which showed mycelial structures indicating hyphal growth (Fig. 4a). The hyphal cells in the colonies were counted microscopically. The colonies of the null mutant strain contained nearly 100 % yeast cells. Those of SC5314, CV1 and CV5 contained about 95 % true hyphae.
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). The different cell types that form these colonies were counted. About 20 % of the null mutant cells formed true hyphae, 60 % pseudohyphae, and 20 % yeast cells (data not shown). Again, colonies of the wild-type strain and strains CV1 and CV5 showed about 95 % true hyphae. In liquid Spider medium as well as liquid medium supplemented with serum, hyphal growth of the C. albicans vac1 null mutant strain was induced and the time-course of hyphal development did not show significant differences from the wild-type strain (data not shown).
Hyphal development is also induced by microaerophilic conditions in an agar matrix. Following growth in such a matrix for 120 h, the vac1 null mutant strain CV3 showed hyperfilamentation (Fig. 4c): 100 % mycelium-forming colonies were observed. The wild-type strain SC5314 showed 12 %, the heterozygous mutant strain CV1 8 %, and the revertant strain CV5 6 % mycelium-forming colonies (Fig. 4c).
In addition, a shift in pH was used for hyphal induction. Under these conditions the vac1 null mutant strain CV3 showed delayed hyphal induction. After 2 h the null mutant strain showed 25 % true hyphae, as compared to 85 % for the wild-type strain. After 3 h the percentages of hyphae for the vac1 null mutant strain (81 %) and wild-type strain (95 %) were similar (Fig. 4d).
The dimorphic growth was restored in all tested conditions by reintroducing one VAC1 allele into the vac1 null mutant strain (CV5, Fig. 4a–d). This reconstitution clearly confirms that the defective phenotypes of the vac1 null mutant strain CV3 are associated with deletion of VAC1. The nearly identical phenotype of the wild-type strain, the heterozygous mutant strain and the revertant strain indicates that the different position of the URA3 gene and a possible reduction of transcription of URA3 gene does not affect the phenotypes of the null mutant strain, because the position of the URA3 gene in the null mutant strain is identical to the position in the heterozygous strain.
Vac1p contributes to chlamydospore formation in C. albicans
The affected hyphal growth of the vac1 null mutant strain inspired us to investigate the potential involvement of Vac1p in another morphogenetic switch in C. albicans. Nutrient-limited conditions induce the formation of thick-walled cells, called chlamydospores (Odds, 1988); these form at the end of elongated suspensor cells that are attached to hyphae. Growth of chlamydospores is induced in the dark under nutrient-deprived oxygen-limited conditions at low temperature on rice agar. In addition, chlamydospore induction occurs upon blockage of hyphae formation (Torosantucci & Cassone, 1983; Nobile et al., 2003). The vac1 null mutant CV3 was completely unable to form either chlamydospores or hyphae on rice agar (Fig. 5). These morphogenetic defects were also observed in liquid rice agar. The heterozygous mutant strain CV1 was similar to the wild-type strain SC5314 in chlamydospore formation, and the ability to form chlamydospores was restored by introduction of the VAC1 gene into the mutant strain (CV5).
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a). This attenuation indicates a role of Vac1p in yeast adhesion to host cells. The adhesion of the heterozygous mutant strain CV1 (90 %±25 %) and the revertant strain CV5 (80 %±16 %) did not show significant differences from the wild-type strain (Fig. 6a).
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; Schaller et al., 2003). Therefore, secretion of Saps, mainly Sap2p, was analysed in the vac1 null mutant strain CV3. Sap secretion was reduced in this strain, whereas the heterozygous mutant strain CV1 and the revertant strain CV5 showed similar secretion to the wild-type strain SC5314 (Fig. 6b). These results suggest that Vac1p affects the secretion of Sap proteinases, probably by regulating their vesicular transport and/or exocytosis.
Vac1p is required for virulence of C. albicans in a mouse model of systemic candidiasis
Three factors that play an important role in pathogenesis of C. albicans are affected in the vac1 null mutant strain. The virulence of the vac1 mutant strains CV1, CV3 and CV5 was therefore assayed in a mouse model of systemic candidiasis. The vac1 null mutant strain CV3 was avirulent: mice infected with 5x106 CV3 cells survived during the complete course of the experiment (Fig. 7a). In contrast, all mice infected with the wild-type strain SC5315 died after 8 days. The heterozygous mutant strain CV1 and the revertant strain CV5 showed virulence comparable to that of the wild-type strain (Fig. 7a).
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). Similarly, identical growth was observed in YPD medium (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The lipid kinase, but not the protein kinase activity of the multifunctional vacuolar transport protein Vps34p affects virulence of C. albicans (Günther et al., 2005). In order to elucidate the signalling pathway that mediates virulence, we analysed whether proteins with high homology to PtdIns(3)P-binding proteins of S. cerevisiae like Fab1p and Vac1p are associated with virulence. A C. albicans fab1 null mutant strain has the same virulence as the wild-type strain (Augsten et al., 2002). Our work shows that another potential lipid-binding protein, Vac1p, is involved in virulence. The vac1 null mutant strain is avirulent and shows defective yeast-to-hyphae transition.
Vac1p has been shown to mediate endosomal docking of vesicles in S. cerevisiae (Peterson & Emr, 2001). We show here that Vac1p also affects the docking of prevacuolar vesicles to the vacuole in C. albicans. This is concluded from the accumulation of the endocytotic fluorescent dye marker in prevacuolar vesicles in the vac1 null mutant strain. At the stage of prevacuolar vesicles, endocytosis is linked to the CPY pathway from the Golgi to the vacuole, transiting through the prevacuolar vesicles. Thus in C. albicans Vac1p may influence both the CPY pathway and the endocytosis.
The role of Vac1p in endocytosis as shown here for C. albicans is in agreement with results reported for the human Vac1p homologue, which regulates fusion of endosomal vesicles with the early endosome (Stenmark et al., 1996). Further evidence for a role of Vac1p in endocytosis comes from the interaction of S. cerevisiae Vac1p and Vps33p, a protein involved in endocytosis (Subramanian et al., 2004).
A vps34 null mutant strain of C. albicans exhibits an increased sensitivity to metal ions (Kitanovic et al., 2005). The vac1 null mutant strain also shows an increased metal ion sensitivity. Thus, Vac1p and Vps34p, and consequently the vacuolar protein transport, evidently play a role in resistance to metal ions.
Vac1p influences virulence by affecting the virulence factors hyphal growth, chlamydospore formation, adhesion to host cells, and secretion of Saps. In this work we have shown that Vac1p is involved in control of hyphal growth under microaerophilic/embedded conditions. In addition, Vac1p has a mild effect on hyphal growth induced by serum or mannitol (Spider medium), or by shift to neutral pH. However, when vac1 null mutant cells were cultivated in liquid media no significant reduction in hyphal growth was observed, indicating that Vac1p-dependent hyphal induction pathways are induced by environmental stress, such as starvation, limited supply of oxygen or shift in pH. The response of yeast cells to such stress conditions requires the transport and accumulation of compounds in the vacuole. The defective last step in vacuolar transport, the fusion of prevacuolar vesicles to the vacuole, in vac1 null mutant cells may prevent induction of hyphal development and of several other stress-induced signalling pathways. The in vitro stress conditions that induce Vac1p-dependent pathways of hyphal growth are also found during pathogenesis of candidiasis. Therefore, the affected adaptation of the vac1 null mutant to these stress conditions, and the defect in hyphal growth, results in the avirulence of the null mutant in the mouse model of systemic candidiasis.
In addition, Vac1p is required for the morphogenetic pathway of chlamydospore formation. The vac1 null mutant strain is completely unable to form chlamydospores either on a slide with rice agar covered with a coverslip or in liquid rice agar. This result, together with the hyperfilamentation of embedded cells of the vac1 null mutant, may indicate that the morphogenetic regulator Efg1p is affected in the vac1 null mutant, because Efg1p is required for chlamydospore development and repressed the filamentation of embedded cells (Sonneborn et al., 1999; Giusani et al., 2002). In addition, hyphal growth in response to alkaline pH requires Efg1p (El Barkani et al., 2000). Therefore, the affected hyphal growth under alkaline conditions seems to further indicate that Vac1p influences pathways that are regulated by Efg1p.
Vac1p plays a decisive role in the secretion of Saps. The defective Saps activity in the vac1 null mutant strain is probably caused by defects in the secretory pathway from the Golgi apparatus, or the pathway from the vacuole to the cell surface, because in S. cerevisiae the secretory pathway leads to both the Golgi and the vacuole (Gurunathan et al., 2002). Such a link between the vacuolar transport and exocytosis was also shown in the C. albicans vps34 null mutant strain, where exocytotic vesicles accumulated in the periplasm (Bruckmann et al., 2001). In addition, the Vps34p and Vps11p vacuolar transport proteins are also involved in Sap secretion in C. albicans (Kitanovic et al., 2005; Palmer et al., 2003). In S. cerevisiae Vps11p, as well as Vac1p, mediates the docking of transport vesicles to the vacuolar and endosomal membrane. The synthetic interaction between a vps11ts allele and VAC1 in S. cerevisiae (Srivastava et al., 2000) indicates a cooperation of Vac1p and Vps11p in Sap secretion of C. albicans. Sap2p contributes to both tissue damage and invasion during systemic candidiasis (Schaller et al., 2003). Therefore, the defective Sap2p secretion very probably contributes to the avirulence of the vac1 null mutant strain.
The vac1 null mutant strain is avirulent in the mouse model of systemic candidiasis. This avirulence phenotype is independent of defective cell growth, since the null mutant shows almost identical growth to the wild-type strain SC5314.
In summary, the potential PtdIns(3)P-binding protein Vac1p is involved in the vacuolar vesicle transport and probably acts downstream of the PI 3-kinase Vps34p. Both the vps34 and vac1 null mutants are avirulent, and they show some similar phenotypes. Together with knowledge from S. cerevisiae these results indicate that Vac1p and Vps34p are involved in the same virulence-determining pathway in C. albicans.
Environmental stress affects protein transport into the vacuole of C. albicans. This process may induce signals for the activation of virulence signalling pathways and the start of the pathogenic life cycle. Therefore we conclude that as key regulators of vacuolar transport, Vac1p, Vps34p and Vma7p could influence virulence of C. albicans. Such proteins can be termed virulence-determining signal proteins.
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ACKNOWLEDGEMENTS |
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Received 4 May 2006;
revised 14 July 2006;
accepted 17 July 2006.
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