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
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ABSTRACT |
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Keywords: phosphatidylinositol 3-kinase, Candida albicans, gene disruption, virulence factors, pathogenicity
Abbreviations: FCS, foetal calf serum; PI, phosphatidylinositol
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INTRODUCTION |
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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.
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METHODS |
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Disruption and reintegration of CaVPS34.
To disrupt CaVPS34, the previously described hisGURA3hisG 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 hisGURA3hisG 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 Students t-test. A P value
0·025 was considered as significant.
Virulence studies.
Male outbred NMRI mice (HarlanWinkelmann; 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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 6 April 2000;
revised 3 July 2000;
accepted 31 July 2000.