A phosphatidylinositol 3-kinase of Candida albicans influences adhesion, filamentous growth and virulence

Astrid Bruckmann1, Waldemar Künkel1, Albert Härtl2, Reinhard Wetzker3 and Raimund Eck1

Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology1 and Department of Drug Testing2, Beutenbergstrasse 11, D-07745 Jena, Germany
Friedrich Schiller University, Medical Faculty, Department of Molecular Cell Biology, Drackendorfer Strasse 1, D-07747 Jena, Germany3

Author for correspondence: Raimund Eck. Tel: +49 3641 656852. Fax: +49 3641 656652. e-mail: reck{at}pmail.hki-jena.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To determine if cellular functions of the phosphatidylinositol 3-kinase CaVps34p are related to processes governing Candida albicans pathogenicity, both copies of the gene were sequentially disrupted. Homozygous deletion of C. albicans VPS34 resulted in a mutant strain which exhibited defects not only in intracellular vesicle transport processes but also in morphogenesis. The CaVPS34 null mutant was unable to form hyphae on different solid media whilst showing a significantly delayed yeast-to-hyphae transition in liquid media. In addition, the mutant was rendered hypersensitive to temperature and osmotic stresses and had a strongly decreased ability to adhere to mouse fibroblast cells compared to the wild-type strain SC5314. Finally, evidence was obtained that CaVPS34 is essential for pathogenicity of C. albicans as the CaVPS34 null mutant was shown to be avirulent in a mouse model of systemic infection. C. albicans pathogenicity was restored to a near wild-type degree upon reintroduction of CaVPS34 into the chromosome of the null mutant, demonstrating that the observed avirulence corresponded to the loss of CaVPS34. Thus, the results suggest that CaVPS34 may serve as a potential target for antifungal drugs.

Keywords: phosphatidylinositol 3-kinase, Candida albicans, gene disruption, virulence factors, pathogenicity

Abbreviations: FCS, foetal calf serum; PI, phosphatidylinositol


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans is the major fungal pathogen in humans (Odds, 1988 ). This dimorphic yeast is capable of causing life-threatening infections in immunocompromised patients, and also a variety of mucosal infections in healthy individuals. Present evidence suggests that virulence of C. albicans is dependent on several properties, including the ability to switch between different morphogenetic forms, host epithelial and endothelial cell recognition and adhesion, as well as secretion of proteinases and phospholipases (Cutler, 1991 ; Köhler & Fink, 1996 ; Lo et al., 1997 ; Odds, 1994 ). Whilst a number of virulence factors of C. albicans have been characterized, the mechanism which enables the opportunistic fungus to become pathogenic has not yet been unravelled.

Recent data suggest that signal transduction via MAP kinases and cAMP-regulated events in pathogenic fungi are of importance for virulence (Lo et al., 1997 ; Madhani & Fink, 1998 ). In higher eukaryotic organisms, phosphoinositide-based signal transduction mechanisms play an important role in mediation of cellular response to extracellular signals. Phosphatidylinositol (PI) 3-kinases phosphorylate the 3' OH position of the inositol ring of phosphoinositides, generating the second messengers PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. PI 3-kinases have been shown to be involved in a wide variety of cellular processes, including mitogenesis, protection from apoptosis, growth factor receptor downregulation, stimulation of glucose uptake, endocytosis, actin cytoskeleton rearrangement and intracellular protein/membrane trafficking (DeCamilli et al., 1996 ; Toker & Cantley, 1997 ). Other phospholipids, such as PI(4,5)P2 and PI(3,5)P2, have been implicated in exocytosis, membrane trafficking and osmotic stress responses (DeCamilli et al., 1996 ; Eberhard et al., 1990 ).

In the yeast Saccharomyces cerevisiae, the gene product of VPS34 (vacuolar protein sorting) represents the only detectable PI 3-kinase activity (Vanhaesebroeck et al., 1997 ). By interaction with the Vps15p serine/threonine protein kinase, Vps34p is recruited to the membrane and activated (Stack et al., 1995 ). Together, Vps15p and Vps34p play a crucial role in vesicle-mediated sorting of vacuolar hydrolases in both late-Golgi-to-prevacuolar-endosome and endosome-to-vacuole transport pathways (Gammie et al., 1995 ; Herman et al., 1991 ; Munn & Riezman, 1994 ; Odorizzi et al., 1998 ; Piper et al., 1995 ; Schu et al., 1993 ; Stack et al., 1995 ). Vps34p was also shown to be involved in the delivery of cargo along the endocytic pathway (Gammie et al., 1995 ; Munn & Riezman, 1994 ; Wurmser & Emr, 1998 ). Recently, target proteins of PI(3)P were identified in the yeast Sacch. cerevisiae and mammalian cells as proteins that contain a PI(3)P-binding RING-FYVE finger domain (Burd & Emr, 1998 ). Two of these proteins, Vac1p and Vps27p, are known to function in Golgi-to-endosome and endosome-to-vacuole transport processes (Piper et al., 1995 ; Weisman & Wicker, 1992 ). Another effector protein, Fab1p, functions downstream of ScVps34p as a PI(3)P 5-kinase which regulates vacuolar membrane turnover via the production of PI(3,5)P2 (Gary et al., 1998 ).

We have previously cloned a PI 3-kinase gene (CaVPS34) which encodes a 1020 amino acid protein with 47% sequence identity with Sacch. cerevisiae Vps34p. The gene product of CaVPS34 exhibits in vitro PI 3-kinase activity. Complementation experiments with Sacch. cerevisiae vps34 suggested a functional conservation in intracellular trafficking in C. albicans (Eck et al., 2000 ).

In this study, we describe the construction of a CaVPS34 null mutant and subsequent analysis of the cellular functions of C. albicans Vps34p, particularly regarding its influence on virulence determinants.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
The C. albicans strains used during this study are listed in Table 1. Strains were grown either in YPD medium [2% (w/v) glucose, 2% (w/v) peptone, 1% (w/v) yeast extract], SD [0·7% (w/v) yeast nitrogen base without amino acids (Difco), 2% glucose, 1 M sorbitol], YNB (SD without 1 M sorbitol) or Sabouraud dextrose broth (Difco) at 28 °C. SD, YNB and Sabouraud dextrose media were supplemented with 20 µg uridine ml-1 for Ura- strains. Selection of uridine auxotrophs was carried out on YNB plates containing uridine and 1 mg 5-fluoroorotic acid ml-1 (Sigma). Growth was monitored by counting cell numbers using a haemocytometer. Hyphal growth was induced by diluting late-exponential-phase cultures grown at 28 °C tenfold, either into fresh YPD or Sabouraud dextrose medium supplemented with 10–15% (w/v) foetal calf serum (FCS), or into Spider medium [1% (w/v) nutrient broth, 0·2% (w/v) K2HPO4, 1% (w/v) mannitol] at 37 °C. To induce hyphal growth on solid medium, cells were grown overnight in YPD at 28 °C, washed, diluted and spread either on Spider plates [1% nutrient broth, 0·2% K2HPO4, 1·35% (w/v) agar, 1% mannitol] or on YPD plates supplemented with 10 % FCS. Between 20 and 100 cells per plate were incubated at 37 °C for at least 7 d. Sensitivities of the mutants to ions were tested on YPD plates. 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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Table 1. Plasmids and strains used

 
Construction of plasmids pVA0, pVA1 and pKUE1.
We amplified the 5' region of CaVPS34 by PCR using chromosomal DNA of C. albicans SC5314 as a template and primers 1 and 2 (primer 1, 5'-GCGAGCTCCAACTGATATTATGCACTGTTAC-3', position -108 to -85; primer 2, 5'-GCGGTACCGTTGATAATGGGACTAAAGAGAC-3', position +152 to +130; underlined sequences are complementary to the genomic sequence of the CaVPS34 gene). The resulting 274 bp PCR product was SacI/KpnI-digested and cloned into the disruption vector pMB7, yielding pVA0 (Fonzi & Irwin, 1993 ). Primer 3 (5'-GCGTCGACAGTTTAGCACAGTATTGGAGAGCT-3', position +3037 to +3060) and primer 4 (5'-GCCTGCAGATGTGCTCAAAAGGGAAA CAATGT-3', position +3261 to +3238) were used to amplify the 3' region. This yielded a 241 bp product, which was digested with SalI and PstI and cloned into plasmid pVA0, resulting in pVA1 (Fig. 1a). For gene disruption, plasmid pVA1 was cut with SacI and PstI (Fig. 1a). To construct the template for homologous reintegration of the CaVPS34 gene, the 1·3 kb RsaI/XbaI fragment of plasmid pMB-7 containing the URA3 gene was blunt-ended by treatment with the Klenow fragment and cloned into the blunt-ended BspMI site 1148 bp downstream from the stop codon of CaVPS34 in pKE2, yielding pKUE1 (Fig. 1a). Plasmid pKE2 contains the 4·9 kb EcoRI/HindIII fragment from plasmid pKE1. After digestion with BbvI (319 bp upstream from the start codon of CaVPS34) and HindIII, a 6·0 kb insert was isolated harbouring the CaVPS34 gene with upstream and downstream sequences for homologous recombination, as well as the URA3 gene as a selectable marker.



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Fig. 1. Disruption and reintegration of the VPS34 gene in C. albicans. (a) Restriction maps of the plasmids pKE1, pKUE1 and pVA1 illustrating the strategy for the disruption and reintegration of CaVPS34. To generate pKE1, the CaVPS34 gene (wide black arrow, coding region; narrow open boxes, non-coding regions) was cloned into pUC18. To generate pKUE1, a 1·3 kb RsaI/XbaI fragment (wide open box) containing the URA3 gene was cloned into the BspMI site in pKE2. To generate pVA1, the 5' and 3' regions of CaVPS34 obtained by PCR were cloned in front of or behind the hisG–URA3–hisG cassette (4·1 kb; wide open boxes) in the disruption vector pMB-7. Vertical black arrows show the location of restriction sites as follows: Bb, BbvI; Bs, BspMI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; Sc, SacI; Sl, SalI. (b) Southern analysis of EcoRI-digested chromosomal DNA from the following C. albicans strains: parental strain CAI-4 (lane 1), CAV1 (lane 2), CAV2 (lane 3), CAV3 (lane 4), CAV4 (lane 5), CAV5 (lane 6) and CAV6 (lane 7). The blot was hybridized with the [{alpha}-32P]dCTP-labelled EcoRI/HindIII 4·9 kb insert of plasmid pKE1.

 
Transformation of C. albicans and selection of Ura- auxotrophs.
These were done according to the procedures described previously (Boeke et al., 1984 ; Eck et al., 1997 ; Swoboda et al., 1995 ).

Disruption and reintegration of CaVPS34.
To disrupt CaVPS34, the previously described hisG–URA3–hisG cassette was used in a multistep procedure (Fonzi & Irwin, 1993 ). A 4·6 kb SacI/PstI fragment of pVA1 containing the URA-blaster flanked by short sequences from the 5' and 3' ends of CaVPS34 and portions of the promoter and terminator, respectively, was used to transform C. albicans Ura- strain CAI-4 (Fig. 1a). After selection on SD medium containing 1 M sorbitol, the resulting Ura+ transformants were examined for gene replacement by Southern analysis of EcoRI-digested chromosomal DNA with the 4·9 kb HindIII/EcoRI fragment as a probe. Southern hybridization was also applied to evaluate segregants and transformants of the later disruption steps. In the first step, one allele of CaVPS34 had been replaced by the hisG–URA3–hisG cassette (CAV1). Strain CAV1 was plated on 5-fluoroorotic acid-containing medium for isolation of Ura- segregants (CAV2). A second transformation with the same disruption construct led to the isolation of a CaVPS34 null mutant (CAV3). Again Ura- segregants were selected (CAV4).

CaVPS34 was reintroduced into the null mutant using the 6·0 kb BbvI/HindIII insert of pKUE1 to transform strain CAV4 (Fig. 1a). Chromosomal DNA from selected clones was digested with EcoRI and analysed by Southern hybridization (CAV5). One of the revertants tested (CAV6) exhibited an additional fragment, suggesting that the mutant contained two CaVPS34 genes. This was confirmed by Southern analysis with HindIII-digested DNA.

Adherence assays.
The adherence fluorescence assay was carried out essentially as described by Borg-von Zepelin & Wagner (1995 ). Briefly, C. albicans cells from an overnight culture in Sabouraud dextrose medium at 28 °C were washed and diluted into fresh medium containing 10% FCS. A suspension containing 2x106 cells ml-1 was then preincubated for 1 or 3 h at 37 °C. Microtest plates containing 1x104 mouse L929 fibroblast cells per well were washed once with 1xPBS buffer (140 mM NaCl, 2·7 mM KCl, 10 mM Na2HPO4, 1·8 mM KH2PO4, pH 7·4) and then filled with 200 µl culture suspension. After incubation at 37 °C for 2 or 4 h, C. albicans cells were stained by adding 25 µg Calcofluor white (Sigma) ml-1 and further incubated for 30 min. Non-adherent cells were then removed by washing twice with 1xPBS. Finally, the number of adherent fluorescent cells was determined using an automatic fluorescence reader (FluoroScan; Labsystems) with a set of 360 nm excitation filters and 460 nm emission filters. For comparison, adherence of the wild-type strain SC5314 was set to 100%. Significance of the observed differences between the strains tested was determined by the Student’s t-test. A P value<=0·025 was considered as significant.

Virulence studies.
Male outbred NMRI mice (Harlan–Winkelmann; Borchen), 6 weeks old, were housed five per cage and checked daily. Strains of C. albicans were grown in Sabouraud dextrose broth at 28 °C until late-exponential phase. Cells were washed three times and resuspended in 0·9% NaCl. Two hundred microlitres of suspensions containing 5x106, 5x105 and 5x104 cells were used to infect immunocompetent mice by intravenous injection into the lateral tail vein. Survival was monitored for 20 d. For comparison of survival curves, the log-rank test was used (Peto et al., 1977 ). A P value<=0·05 was considered as significant. To quantify kidney colonization of C. albicans, mice were sacrificed either 72 h or 20 d after injection and kidneys were homogenized in 3 ml physiological NaCl buffer. Serially diluted suspensions were then plated on YPD agar. After 3 d growth at 28 °C, numbers of C. albicans colonies were counted. Homogenized kidney material was also fixed with 10% formaldehyde and stained with 25 mg Calcofluor white ml-1 to detect C. albicans cells.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Disruption and reintroduction of CaVPS34
As an approach to characterization of the function of CaVPS34, null mutants were constructed and their phenotypes analysed (see also Methods). The 4·6 kb SacI/PstI fragment of pVA1 (Fig. 1a) was used to transform C. albicans Ura- strain CAI-4. Southern analysis of EcoRI-digested DNA from a representative Ura+ transformant (Fig. 1b) resulted in three bands in the case of heterozygous CaVPS34 mutants (CAV1). The 7·0 kb fragment represented the CaVPS34 wild-type allele (as seen in Fig. 1b, lane 1), whereas the 5·8 kb and 2·4 kb fragments confirmed that a 2·9 kb fragment of one CaVPS34 allele had been replaced by the 4·1 kb hisG–URA3–hisG cassette (lane 2). The excision of one copy of hisG and the URA3 gene in the Ura- derivatives resulted in a 5·3 kb band (lane 3). A second transformation with the same disruption construct led to the isolation of a CaVPS34 null mutant (CAV3). The loss of the 7·0 kb fragment and the sizes of the two remaining fragments are consistent with the replacement of the second CaVPS34 allele (lane 4). Southern analysis of a representative Ura- segregant showed only a 5·3 kb hybridizing DNA fragment, indicating that two hisG disrupted alleles were present (CAV4, lane 5). For reintegration of CaVPS34, the 6·0 kb BbvI/HindIII fragment of plasmid pKEU1 was used to transform strain CAV4 (Fig. 1a). Ura+ clones were selected and correct reintroduction of CaVPS34 and the URA3 gene was recognized by the appearance of an additional 3·2 kb fragment (CAV5, lane 6). This fragment consists of 0·8 kb of URA3 and a 2·4 kb fragment of chromosomal DNA including 0·3 kb of the 4·9 kb EcoRI/HindIII probe. The second band of 5·2 kb is not distinguishable from the disrupted allele (5·3 kb). However, analysis of HindIII-digested DNA proved the desired recovery of CaVPS34. In this case, an 8·4 kb fragment results from the integration of the CaVPS34–URA3 cassette and the 5·3 kb band represents the disrupted allele (data not shown). In lane 7, strain CAV6 exhibits a 7·0 kb wild-type allele band created by recombination without URA3, as well as the integration pattern of the CaVPS34–URA3 cassette.

The CaVPS34 null mutant exhibits an abnormal vacuole morphology
Morphological examination of blastospores of the CaVPS34 null mutant revealed considerably enlarged vacuoles. In a population of cells, giant vacuoles occupied approximately 80% of the total cell volume (Fig. 2a). No morphological differences were observed in the heterozygous CaVPS34 mutant CAV1 or the revertant strains CAV5 and CAV6. Yeast-phase growth of strains SC5314, CAV1 and CAV3 was compared in YPD medium at 28 °C. There was no difference in growth rates of the heterozygous CaVPS34 mutant and the wild-type strain SC5314. The CaVPS34 null mutant, however, grew at a 1·8-fold-reduced growth rate.



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Fig. 2. Morphology of C. albicans VPS34 null mutant strain CAV3. (a) The use of Nomarski optics clearly revealed enlarged vacuoles and cells in strain CAV3 during yeast-phase growth compared to wild-type strain SC5314. (b) Yeast-to-hyphae transition of SC5314 and CAV3 after 1·5 h induction in Sabouraud dextrose medium plus 10% FCS. (c) Calcoflour white staining of SC5314 and CAV3 after 3 h (SC5314) and 5 h (CAV3) growth in YPD plus 10% FCS. Hyphal growth of the CaVPS34 null mutant consists only in part of true hyphae and shows a high number of pseudohyphae-like structures. Bar, 13 µm (applies to all photographs).

 
Disruption of CaVPS34 results in increased sensitivity to high temperature and hyperosmotic stresses
To analyse the role of CaVPS34 in high temperature stress sensitivity, we tested the growth of the C. albicans strains SC5314, CAV1, CAV3, CAV5 and CAV6 on YPD plates incubated at 30, 37, 40 and 42 °C. The parental strain SC5314, the heterozygous mutant strain CAV1, the heterozygous revertant CAV5 and the homozygous revertant CAV6 grew well at all temperatures tested. Only the homozygous mutant strain CAV3 showed attenuated growth at 40 °C and was unable to grow at 42 °C, whilst growing normally at 30 and 37 °C (Fig. 3a). Although the C. albicans vps34 null mutant was shown to be stress-sensitive to elevated temperatures, this effect was less dramatic than in Sacch. cerevisiae vps34, which is unable to grow even at 37 °C.



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Fig. 3. Stress sensitivity of C. albicans VPS34 null mutant. Growth of C. albicans strains CAV1, CAV3, CAV5 and CAV6 in comparison to wild-type strain SC5314 (a) at 30, 37, 40 and 42 °C; and (b) on plates containing 1·0 M KCl, 1·5 M KCl, 1·0 M NaCl and 1·5 M NaCl. WT, C. albicans SC5314; CAV1, CaVPS34 heterozygous mutant; CAV3, CaVPS34 null mutant; CAV5, heterozygous revertant; CAV6, homozygous revertant.

 
VPS34 mutants of Sacch. cerevisiae and Schizosaccharomyces pombe are sensitive to hyperosmotic pressure (Herman & Emr, 1990 ; Kimura et al., 1995 ). To see whether this was also true for C. albicans vps34, we tested the effects of osmotic pressure on the mutants. Growth of the CaVPS34 null mutant after 3 d at 28 °C was delayed in the presence of 1·0 M NaCl and almost inhibited in the presence of 1·5 M NaCl. The same effect was caused by 1·0 and 1·5 M KCl (Fig. 3b). At 37 °C the growth inhibition effect was even more pronounced (data not shown).

CaVPS34 influences dimorphic growth of C. albicans under different conditions
Dimorphism is considered to be an important virulence factor of C. albicans (Lo et al., 1997 ). Consequently, signal transduction cascades leading to morphogenetic changes are the subject of intense study (Brown & Gow, 1999 ). Concerning the possibility that PI 3-phosphate may act as a second messenger on an as yet undefined signalling component or may otherwise influence dimorphism, we investigated the yeast-to-hyphae transition of the CaVPS34 null mutant. Hyphal growth of C. albicans vps34 (CAV3) was induced in YPD medium with 15% FCS at 37 °C. We observed that the induction of hyphae was significantly delayed compared to the wild-type strain SC5314. Whereas 55% of wild-type yeast cells had formed germ tubes after 30 min, it took 120 min for 60% of mutant cells to form germ tubes (Fig. 4). A similar effect was found upon induction with liquid Spider medium at 37 °C (data not shown). Fig. 2(b) shows that after 1·5 h growth in serum containing Sabouraud dextrose medium, most cells of the wild-type strain SC5314 had formed true elongated hyphae whereas cells of the CaVPS34 null mutant had only formed short pseudohyphal/hyphal cells or remained in the yeast form.



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Fig. 4. Hyphal growth of C. albicans SC5314 and CAV3 in liquid media. Disruption of CaVPS34 caused a significant delay in hyphal formation after induction in YPD medium with 15% FCS. Data are taken from one representative experiment. A value of 100% hyphae means that 100% of yeast cells had formed true hyphae or pseudohyphae. Black bars, C. albicans SC5314 (wild-type); open bars, CAV3 (CaVPS34 null mutant).

 
In addition, after 5 h induction, the mutant showed a considerable degree of pseudohyphae-like structures and only approximately 30% true hyphae (Fig. 2c). The filaments considered as pseudohyphae exhibit constrictions between cells and start growing with an apical bud. In contrast, true hyphae are formed by continuous apical extension (Odds, 1988 ). The revertants CAV5 and CAV6 showed the same phenotype of filamentous growth after 1·5 and 5 h as the wild-type strain or the heterozygous mutant strain.

It is known that filamentous growth of C. albicans is induced by a variety of environmental signals, which are mediated by at least two parallel signal transduction pathways. Therefore, we additionally tested the mutant strains for hyphal induction on solid Spider medium containing mannitol as carbon source at 37 °C. As shown in Fig. 5, nutritional starvation was sufficient to induce hyphal differentiation in the wild-type but not in the mutant strain. Moreover, CAV3 had also completely lost its ability to form hyphae on YPD-agar plates containing 10% FCS (Fig. 5). We were able to restore dimorphic growth under these conditions by reintroducing one allele of CaVPS34 into its native locus (CAV5, Fig. 5).



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Fig. 5. Phenotypes of C. albicans VPS34 mutant strains on solid hyphae-inducing media. (a) Spider medium containing mannitol as carbon source. (b) YPD medium containing 10% FCS. WT, C. albicans SC5314; CAV1, CaVPS34 heterozygous mutant; CAV3, CaVPS34 null mutant; CAV5, CaVPS34 revertant. Strains were grown for 7 d at 37 °C. Bar, 2·7 mm (applies to all photographs).

 
Adhesion of the CaVPS34 null mutant to fibroblast cells is strongly decreased
The adhesion of C. albicans to host epithelial and endothelial cells is thought to be a prerequisite for virulence. To investigate a possible involvement of CaVps34p functions in adhesion processes, we tested the adhesion of CaVPS34 mutant and revertant strains on mouse L929 fibroblasts using an adherence fluorescence assay (Borg-von Zepelin & Wagner, 1995 ). The mutant strains CAV1 and CAV3 showed 80±10% and 20±7% adhesion compared to the wild-type strain SC5314 (set at 100%); these values were significantly different according to the Student’s t-test (P<=0·025). In particular, the strong attenuation of adhesion of the CaVPS34 null mutant CAV3 to fibroblast cells indicates a role of CaVps34p in cell-to-cell interactions. Revertants containing either one or both CaVPS34 alleles regained the ability to adhere to a near heterozygous mutant (CAV5, 64±15%) and wild-type degree (CAV6, 83±20%), respectively.

The delay in hyphal/pseudohyphal formation of the CaVPS34 null mutant did not influence adhesion as was shown by repeating the assay with prolonged times of preincubation. After a 3 h preincubation, strain CAV3 had reached approximately 80% hyphae/pseudohyphae; however we did not find a higher degree of adhesion compared to 1 h preincubation (approx. 20% hyphae/pseudohyphae). In addition, we extended the co-incubation time of fibroblasts and C. albicans cells from 2 to 4 h. Again, there was no difference in adhesion.

CaVps34p activity is required for virulence of C. albicans in a mouse model of systemic candidosis
Since at least two factors which play an important role in the pathogenesis of a C. albicans infection are affected in the CaVPS34 null mutant, we tested the virulence of CAV1, CAV3, CAV5 and CAV6 compared with the wild-type strain SC5314 in a mouse model of systemic candidosis (Fig. 6). We observed no significantly reduced virulence of the heterozygous CaVPS34 mutant (P<0·05), whereas the mutant defective in both alleles of CaVPS34 was avirulent (P<0·05) (Peto et al., 1977 ). In the revertants CAV5 and CAV6, virulence was completely restored.



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Fig. 6. Pathogenicity of C. albicansVPS34 mutant strains. C. albicans SC5314 (wild-type), CAV1 (CaVPS34 heterozygous mutant), CAV3 (CaVPS34 null mutant), CAV5 (CaVPS34 heterozygous revertant) and CAV6 (CaVPS34 homozygous revertant) were tested in a mouse model of systemic candidosis. Survival of mice infected with 5x104 (circles), 5x105 (triangles) and 5x106 (squares) cells was monitored for 20 d. SC5314, n=15, except 5x106 where n=10; CAV1, n=10; CAV3, n=15; CAV5, n=15; CAV6, n=10.

 
A systemic candidosis is often associated with colonization of internal organs such as kidneys, lung or liver. It is known that in animal models of disseminated candidosis C. albicans exhibits a high predilection for the kidneys, which leads to late fatalities in the course of the infection (Odds, 1988 ). We examined kidney colonization of mice infected with 5x105 cells of C. albicans strains SC5314, CAV1 or CAV3 3 d post infection. Three independent experiments were performed with three mice each for strains SC5314 and CAV3. Strain CAV1 was examined in two experiments with three mice each. Of nine mice infected with 5x105 cells of strain SC5314, five had died by day three, whereas, as expected, all of the CAV3-infected mice could be analysed. Four of six mice infected with the same dose of strain CAV1 survived until examination. Kidneys of mice bearing either strain SC5314 or CAV1 exhibited a fungal burden of 2·0–6·2x104 or 0·4–3·1x104 c.f.u. (g kidney tissue)-1, respectively, whereas C. albicans was not found in the kidneys of mice infected with the CaVPS34 null mutant strain CAV3. After 20 d, survivors of the experiment were checked for kidney colonization. Again, kidneys of CAV3-infected mice were C. albicans-free. In one experiment, in addition to kidneys, liver and spleen were also examined. In the CAV3-infected mice, these organs also showed no colonization by C. albicans at either time point.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To date, very little is known about the impact of components of the secretory as well as other protein trafficking pathways on the pathogenicity of C. albicans. Here, we present evidence that a protein involved in the protein/lipid transport from the Golgi/endosome to the vacuole also plays a crucial role for virulence of C. albicans. Intriguingly, in our mouse model of systemic infection C. albicans vps34 null mutants completely lost virulence. In the course of our work, we have ascertained CaVPS34 functions with regard to possible effects on virulence determinants, such as dimorphic growth and adhesion to host cells. Both processes were shown to be strongly affected by the deletion of both alleles of CaVPS34, indicating a role of PI 3-kinase in morphogenesis and host–pathogen interactions.

Upon deletion of both copies of VPS34, C. albicans vps34 entirely lost its ability to form true hyphae on both solid Spider medium and solid serum-containing medium. In liquid Spider or serum-containing media, a significant delay in hyphal formation was seen. In addition, a high number of the hyphae formed in liquid media were pseudohyphae. Thus, CaVPS34 seems to be necessary for hyphal formation under certain growth conditions. It is known that induction of filamentous growth of C. albicans in response to multiple environmental signals is mediated by at least two parallel signal transduction pathways, the mitogen-activated protein kinase (MAPK) signalling cascade and a cAMP-dependent mechanism (Köhler & Fink, 1996 ; Leberer et al., 1996 ; Liu et al., 1994 ; Lo et al., 1997 ; Navarro-Garcia et al., 1998 ; Stoldt et al., 1997 ). Components of the MAPK pathway function to signal filamentation on solid surfaces in response to certain carbon sources as well as nitrogen limitation. The Ras-cAMP-dependent mechanism responds to serum in liquid and solid media, resulting in reduced hyphal growth. Inactivation of the two corresponding transcription factors Cph1p (MAPK pathway) and Efg1p (Ras-cAMP pathway) results in elimination of hyphal formation (Lo et al., 1997 ), suggesting a convergence of multiple signals on these two control elements. Our results suggest that both known signal transduction pathways are possibly affected in the CaVPS34 null mutant. With our current knowledge, it is difficult to speculate about the mechanism underlying this observation. However, one interpretation could be that morphogenetic signal transduction via as yet undefined cell-surface receptors may be affected. It is known that plasma membrane receptors need to be delivered to the vacuole for recycling and turnover. In Sacch. cerevisiae, recycling and turnover of the a-factor receptor Ste3p is blocked due to the loss of Vps34p activity (Munn & Riezman, 1994 ). We recently obtained experimental support for a function of CaVps34p in the endocytic pathway to the vacuole (unpublished). Thus, an impaired receptor downregulation may contribute to the observed defects in dimorphic transition. However, we cannot exclude the possibility that PI 3-phosphate may act as a second messenger on an as yet undefined signalling component.

Deletion of both copies of CaVPS34 resulted in hypersensitivity to osmotic stress. In the yeasts Sacch. cerevisiae and Schiz. pombe, the RING-FYVE protein Fab1p converts PI(3)P to PI(3,5)P2 when the cells are stressed hyperosmotically (Dove et al., 1997 ). This Vps34p-dependent accumulation of PI(3,5)P2 occurs independent of the high osmolarity glycerol response (HOG1) pathway, which is also considered to play a role in adaptation to osmotic stress in C. albicans (Alonso-Monge et al., 1999 ). Vacuoles of the CaVPS34 null mutant were considerably enlarged, similar to those of a Sacch. cerevisiae fab1 mutant (Gary et al., 1998 ; Yamamoto et al., 1995 ). Since the PI(3)P 5-kinase Fab1p acts downstream of Vps34p in controlling vacuolar size and membrane homeostasis especially during an acute osmotic adaptation response, it seems possible that the filamentation defect seen with C. albicans vps34 on solid surfaces may at least partially be an osmotic-like effect (Alex et al., 1998 ). A similar phenotype comprising osmosensitivity and defects in hyphal formation on solid surfaces has been described for CaCOS1, a two-component histidine kinase (Alex et al., 1998 ).

Surface proteins of micro-organisms play a pivotal role as sensors of environmental signals and in interactions with other cells. Adhesion to epithelial and endothelial cells is the first step in the interaction process between the pathogen and host tissues (Odds, 1988 ). When C. albicans vps34 strains were tested in a mouse fibroblast adherence assay, they exhibited markedly lower adherence compared to the wild-type strain. This adhesion deficiency may be caused by a lack of receptor and/or adhesion proteins due to the loss of a functional CaVps34p, a situation similar to that discussed above for plasma membrane receptor recycling. However, we cannot at present exclude the possibility that the defects in morphogenesis were partly responsible for the lower adherence of mutant cells. Poor adhesion of C. albicans to host cells has for example been reported in strains defective in protein O-mannosylation (pmt1) or deleted for an integrin-like protein (int1), both of them linked to the cell wall structure (Gale et al., 1998 ; Timpel et al., 1998 ).

The cell surface protein CaInt1p, which is similar to CaVps34p, has recently been shown to influence not only adhesion but also dimorphic transition under certain conditions. It was suggested that integrin may act as a morphogenetic sensor for a subset of environmental conditions (Gale et al., 1998 ).

Avirulence of C. albicans vps34 in the mouse model of systemic infection was connected with failure of kidney colonization. Rapid clearance of C. albicans cells by immune defence mechanisms may account for this phenomenon, as well as the impaired adherence properties that may prevent dissemination. Another conceivable possibility is that the osmotic instability of the null mutant contributes to its elimination and/or interferes with spreading or adaptation to different microenvironments.

In this study, a protein likely to be involved in regulation of intracellular protein/lipid traffic events was shown to be essential to C. albicans pathogenicity. Further investigation is needed to elucidate the mechanisms by which Vps34p is able to affect a broad spectrum of cellular processes, as seen in C. albicans. The characterization of Vps34p in C. albicans demonstrates that proteins involved in signalling intracellular transport processes may be promising new targets for antifungal therapy.


   ACKNOWLEDGEMENTS
 
We are greatly indebted to A. Hartmann, E. Franzl and M. Nguyen for technical assistance. We would also like to thank B. Frais, U. Stöckel and B. Weber for technical assistance in the virulence test. We thank H.-M. Dahse for providing us with mouse L929 fibroblast cells and W. A. Fonzi for the gift of plasmid pMB-7 and C. albicans CAI-4. We are grateful to C. Bergmann for helpful discussions and critical reading of the manuscript. We thank J. Ernst, J. Hacker and A. Hinnen for comments on the manuscript. This work was supported by Grants EC 182/1-1 and WE 1565/4-1 from the Deutsche Forschungsgemeinschaft.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 6 April 2000; revised 3 July 2000; accepted 31 July 2000.