1 Leibniz Institute for Natural Products Research and Infection Biology/Hans Knöll Institute, Department of Infection Biology, Beutenbergstrasse 11, D-07745 Jena, Germany
2 University of Applied Sciences, Tatzendpromenade 1b, D-07745 Jena, Germany
3 Friedrich Schiller University, Department of Microbiology, D-07745 Jena, Germany
Correspondence
Raimund Eck
raimund.eck{at}hki-jena.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fungal vacuole is an acidic compartment which is involved in hydrolysis, storage, osmoregulation, homeostasis and detoxification (Teter & Klionsky, 2000). In C. albicans, transport pathways into the vacuole are linked with hyphal growth and virulence (Bruckmann et al., 2000
; Palmer et al., 2003
; Theiss et al., 2002
). The vacuolar H+-ATPase (V-ATPase) is required for vacuole proton transport and is necessary for vacuole acidification.
The V-ATPase is located in endomembranes of eukaryotic cells and the amino acid sequences are highly conserved between yeasts, mammals and plants. Saccharomyces cerevisiae serves as a model organism for vacuolar transport. In S. cerevisiae, the V-ATPase regulates transport of small molecules and ions across the vacuolar membrane and provides an acidic environment that is required for protein degradation in the vacuole and intracellular targeting of vacuolar proteins (Forgac, 2000). The V-ATPase of S. cerevisiae comprises a multiprotein complex which is formed by two subunits with 13 proteins altogether; the soluble, cytoplasmic V1 subunit is composed of eight proteins and the membrane-bound V0 subunit contains five proteins (Graham et al., 2000
). The V-ATPase component Vma7p controls the assembly of the V0 and V1 subunits and the targeting of the V-ATPase to the vacuolar membrane (Graham et al., 1994
, 2000
; Nelson et al., 1994
; Tomashek et al., 1996
).
The yeast vacuole acts as a storage and sequestration site of metal ions and regulates ion homeostasis. The balance between storage and sequestration is controlled by a H+ gradient that is generated by the V-ATPase (MacDiarmid et al., 2002). Toxic metal ions are inactivated directly in the vacuole (e.g. Mn2+, Ni2+, Zn2+, Co2+) or in the cytosol (e.g. Cu2+, Cd2+). In addition, the V-ATPase controls the distribution of plasma membrane ATPase, as demonstrated for S. cerevisiae (Perzov et al., 2000
). In C. albicans, a plasma membrane ATPase mediates high copper tolerance (Weissman et al., 2000
). Therefore, it is possible that the V-ATPase influences indirectly the tolerance to copper ions through regulation of the plasma membrane ATPase assembly.
We have recently shown that the phosphatidylinositol (PI) 3-kinase Vps34p, a key enzyme of vacuolar protein transport, interacts physically with Vma7p, a component of the Candida V-ATPase complex (Eck et al., 2005). The PI 3-kinase Vps34p controls virulence of C. albicans. The vps34 null mutant strain is avirulent in a mouse model of systemic candidiasis, unable to form hyphae on different solid media, shows delayed yeast-to-hyphae transition in liquid media, is hypersensitive to high temperature and hyperosmotic stress and exhibits reduced adherence to human cells (Eck et al., 2000
; Bruckmann et al., 2000
). In addition, the vps34 mutant displays enlarged and electron-transparent vacuoles (Bruckmann et al., 2001
).
Given the formation of a complex between the V-ATPase component Vma7p and the virulence-regulating Vps34p, we were interested to characterize the function of Vma7p in C. albicans. C. albicans vma7 null mutants were generated and these mutant strains were assayed for vacuole acidification, detoxification, endosomal transport, pH-dependent growth, hyphal development and virulence.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Fluorescent labelling and microscopy.
Quinacrine staining was performed as described by Augsten et al. (2002). Images were acquired using a fluorescence microscope (Olympus BX51TF) equipped with a BP450490 excitation filter (U-MSWB2), BA520 beam splitter and DM500 emission filter. FM4-64 staining was carried out essentially as described previously (Vida & Emr, 1995
). For microscopy, cells were placed on slides covered with a thin 1 % agarose film. Transport of FM4-64 was viewed using a BP510-550 excitation filter (U-MWG2), BA590 beam splitter and DM570 emission filter (Olympus BX51TF).
Virulence studies.
Six-week-old male outbred NMRI mice (Harlan-Winkelmann) were housed five per cage and checked daily. The various strains of C. albicans were grown in Sabouraud dextrose broth at 30 °C until late exponential phase. Cells were washed three times and resuspended in 0·9 % NaCl. Aliquots (200 µl) of suspensions containing 5x106 or 5x105 cells were used to infect immunocompetent mice by intravenous injection into the lateral tail vein. Survival 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 as significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the heterozygous strain VU, the second VMA7 allele was replaced by the HIS1 gene resulting in VUH and in the VH strain by the URA3 gene resulting in VHU (Fig. 1a). Proper deletion of the VMA7 genes in all strains was confirmed by Southern analysis and by PCR (Fig. 1b
, data not shown). Chromosomal DNA of the C. albicans strains SC5314 (wild-type), VU, VH, VUH and VHU was isolated and restricted with the restriction enzyme AflII. Following separation of the restricted DNA by agarose gel electrophoresis and Southern hybridization, the correct 8·45 kb wild-type fragment was identified. Replacement of one VMA7 allele by the URA3 gene (VU) or the HIS1 gene (VH) resulted in an additional fragment of 9·45 kb. The replacement of the second VMA7 allele by the HIS1 or URA3 gene led to two vma7 null mutants (VUH, VHU). The loss of the 8·45 kb fragment is consistent with the replacement of the second allele (Fig. 1a, b
).
|
vma7 null mutant strains show defective vacuolar acidification and pH-dependent growth
The acidic environment in the vacuoles of the vma7 mutants was tested by staining with the fluorescent dye quinacrine, which is a marker for acidic pH (Roberts et al., 1991). Both vma7 null mutants (VUH and VHU) lacked vacuolar staining, thus indicating an alkaline vacuolar milieu. The wild-type strain SC5314, the Arg strain CNC44 and the heterozygous mutant strain VU showed stained vacuoles, indicative of an acidic vacuolar pH (Fig. 2
). C. albicans CNC44 was used as an additional control as this strain, similar to the vma7 null mutants, contains a reintegrated URA3 and HIS1 gene and both alleles of the ARG genes are deleted. In addition, the vps34 null mutant strain CAV3 also showed alkaline vacuolar pH, thus indicating similar roles for Vma7p and Vps34p in vacuolar acidification.
|
|
After an initial pulse and a chase of 60 min, FM4-64 was present predominantly in the vacuole in C. albicans wild-type strains and the heterozygous vma7 mutant strain VU and stained the vacuole membrane exclusively (ring staining pattern). The vma7 null mutants, however, showed accumulation of FM4-64 in the membrane of putative endosomal structures of approximately 0·51·0 µm in diameter in the lumen of the vacuole (Fig. 4, left panels). The same patterns were observed by differential interference contrast (DIC) microscopy (Fig. 4
, right panels). Thus, the defect in the vma7 null mutants indicates a block in the degradation of putative endosomal intravacuolar structures. In contrast, the vps34 null mutant showed accumulation of prevacuolar endosomal vesicles in the cytoplasm (Fig. 4
).
|
|
Vma7p is involved in serum- and mannitol (Spider medium)-regulated induction of filamentous growth
Vacuolar transport is involved in germ tube formation (Palmer et al., 2003). To this end, we tested the vma7 null mutants for hyphal development using different conditions for induction (Fig. 6
). Hyphal development was induced with serum and in Spider medium with mannitol as sole carbon source. Both vma7 mutant strains VHU and VUH did not form hyphae in either liquid medium supplemented with serum or in liquid Spider medium (Fig. 6a
). However, both mutants showed filament development when grown on solid Spider medium, but not on solid medium supplemented with serum (Fig. 6b
). These results indicate that the V-ATPase component Vma7p regulates hyphal growth upon induction by either serum or liquid medium with mannitol (Spider medium), but not by solid Spider medium.
|
Vma7p is required for virulence of C. albicans
The vma7 null mutants show defective hyphal development. Therefore, the virulence of the vma7 heterozygous and homozygous mutants (VU, VUH and VHU) and the VMA7 revertant strain (VUR) was tested in a mouse model of systemic candidiasis. Both vma7 null mutants were avirulent in this animal model. All mice infected with 5x106 or 5x105 mutated Candida cells survived during the complete course of the experiment. In contrast, the heterozygous vma7 mutant strain VU and the VMA7 revertant strain VUR showed nearly the same, high virulence as the two control strains SC5314 and CNC44 (Fig. 7).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C. albicans vma7 null mutants were generated and showed defective proton transport into the vacuole, as indicated by the alkaline vacuolar pH. This demonstrates a decisive role of Vma7p in the functioning of C. albicans V-ATPase. This effect is in agreement with data obtained from the apathogenic yeast S. cerevisiae, where Vma7p affects both V0 and V1 subunits, reflecting a role in bridging the subunits to form a functional V-ATPase complex (Graham et al., 2000).
The C. albicans vma7 null mutants also showed defective endosomal protein sorting. In the first step of the endocytotic pathway, transmembrane proteins and lipids are sorted into vesicles. These vesicles are invaginated into the lumen of the early endosomes. The endosomal vesicles are transported into multivesicular bodies. Upon fusion of multivesicular bodies with the vacuole, the vesicles and their contents are degraded by hydrolases and lipases (Katzmann et al., 2002). In the C. albicans vma7 null mutant cells, the vacuoles accumulated putative endosomal compartments. This accumulation indicates defective degradation of intravacuolar structures. The alkaline vacuolar lumen may prevent the degradation of intravacuolar structures by hydrolases and lipases, which are only active in an acidic vacuolar milieu.
Copper resistance is most likely a prerequisite for the survival of C. albicans in the digestive tract of its host, as Cu2+ concentrations, at approximately 10 µM, are relatively high in the stomach and duodenum (Underwood, 1977). Copper tolerance in C. albicans is mediated by a plasma membrane ATPase (Weissman et al., 2000
). The influence on the targeting of plasma membrane ATPase of the V-ATPase has been shown in S. cerevisiae (Perzov et al., 2000
). We demonstrated that the wild-type C. albicans strain SC5314 tolerates Cu2+ concentrations of 8 mM. The same concentration was toxic for the vma7 null mutants, which tolerated lower concentrations, of 4 mM. The increased Cu2+ sensitivity of the vma7 null mutants may cause the avirulent phenotype, as the toxicity of copper is increased under the acid, anaerobic conditions which exist in the digestive tract. We hypothesize that a defective V-ATPase affects the distribution of plasma membrane ATPase in the vma7 null mutants and thus leads to decreased copper resistance.
The vma7 null mutants showed increased sensitivities to other metal ions (Zn2+, Mn2+, Co2+, Ni2+), linking Vma7p activity to the detoxification of metal ions. Detoxification depends on the proton gradient of the vacuole membrane, which is defective in the vma7 null mutant strains. Thus, defective detoxification is likely caused by the low proton concentration in the vacuole.
In addition, the vma7 null mutants showed no hyphal growth in liquid medium. Defective hyphal formation of the vma7 null mutant strains may cause the avirulent phenotype, as the transition from the ellipsoidal yeast form to the filamentous form is important for virulence of C. albicans.
Both the mitogen-activated protein (MAP) kinase pathway and the cAMP-regulated cascade are major signalling pathways of hyphal induction (Köhler & Fink, 1996; Sonneborn et al., 2000
). The defective hyphal development of the vma7 null mutants in both serum and Spider medium is explained by a defective cAMP signalling pathway, because nutrient starvation in Spider medium induces hyphal development via the MAP kinase and cAMP pathways whereas serum-induced hyphal growth is controlled exclusively by the cAMP pathway (Ernst, 2000
). The vma7 null mutants show defective hyphal growth in liquid Spider medium, but not on solid Spider medium. This effect is explained by derepression of hyphal development in the vma7 null mutants by specific growth conditions on solid Spider medium which activate a so-far uncharacterized pathway of hyphal induction.
The altered position of the URA3 gene or deletion of one URA3 allele may complicate the interpretation of mutant phenotypes (Lay et al., 1998; Cheng et al., 2003
). Therefore, the URA3 gene was integrated at the same locus in the heterozygous and null mutant strains. The related phenotypes of the heterozygous mutant strain and of the wild-type strain argue against a URA3-position effect in the vma7 null mutant strain. In addition, after supplementation of medium with uridine, the mutant phenotypes did not revert (data not shown), which confirms that the null mutant phenotypes do not result from a URA3-position effect.
Recently, we have shown that Vma7p interacts physically with the PI 3-kinase Vps34p, a key enzyme of vacuolar protein transport (Eck et al., 2005). In addition, the C. albicans vma7 and vps34 null mutants show the same defects in vacuolar acidification and detoxification of metal ions. The physical and functional interaction between Vma7p and Vps34p links vacuolar proton transport and vacuole acidification with vacuolar protein transport. Vps34p may control the assembly of the V-ATPase subcomplexes by directly interaction with Vma7p. This assumption is supported by results obtained in human cells and in S. cerevisiae. In human cells, the assembly of V-ATPase subunits V0 and V1 depends on PI 3-kinases (Sautin et al., 2005
) and, in S. cerevisiae, the V-ATPase is involved in intracellular protein transport to the vacuole (Munn & Riezman, 1994
; Stevens & Forgac, 1997
; Bonangelino et al., 2002
; Perzov et al., 2002
).
In this study, we show that the putative V-ATPase component Vma7p of C. albicans influences cellular functions which are connected to the acidification of the vacuole and transport processes into the vacuole. These cellular functions influence hyphal growth and virulence of C. albicans.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bonangelino, C. J., Chavez, E. M. & Bonifacino, J. S. (2002). Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13, 24862501.
Bruckmann, A., Künkel, W., Härtl, A., Wetzker, R. & Eck, R. (2000). A phosphatidylinositol 3-kinase of Candida albicans influences adhesion, filamentous growth and virulence. Microbiology 146, 27552764.[Medline]
Bruckmann, A., Künkel, W., Augsten, K., Wetzker, R. & Eck, R. (2001). The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation. Yeast 18, 343353.[CrossRef][Medline]
Cheng, S., Nguyen, M. H., Zhang, Z., Jia, H., Handfield, M. & Clancy, C. J. (2003). Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus. Infect Immun 71, 61016103.
Cutler, J. E. (1991). Putative virulence factors of Candida albicans. Annu Rev Microbiol 45, 187218.[CrossRef][Medline]
Eck, R., Bruckmann, A., Wetzker, R. & Künkel, W. (2000). A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization. Yeast 16, 933944.[CrossRef][Medline]
Eck, R., Nguyen, M., Günther, J., Künkel, W. & Zipfel, P. F. (2005). The phosphatidylinositol 3-kinase Vps34p of the human pathogenic yeast Candida albicans is a multifunctional protein that interacts with the putative vacuolar H+-ATPase subunit Vma7p. Int J Med Microbiol (in press).
Ernst, J. F. (2000). Transcription factors in Candida albicans environmental control of morphogenesis. Microbiology 146, 17631774.[Medline]
Fonzi, W. A. & Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717728.
Forgac, M. (2000). Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H+-ATPases. J Exp Biol 203, 7180.[Abstract]
Gola, S., Martin, R., Walther, A., Dunkler, A. & Wendland, J. (2003). New modules for PCR-based gene targeting in Candida albicans: rapid and efficient gene targeting using 100 bp of flanking homology region. Yeast 20, 13391347.[CrossRef][Medline]
Graham, L. A., Hill, K. J. & Stevens, T. H. (1994). VMA7 encodes a novel 14-kDa subunit of the Saccharomyces cerevisiae vacuolar H+-ATPase complex. J Biol Chem 269, 2597425977.
Graham, L. A., Powell, B. & Stevens, T. H. (2000). Composition and assembly of the yeast vacuolar H+-ATPase complex. J Exp Biol 203, 6170.[Abstract]
Katzmann, D. J., Odorizzi, G. & Emr, S. D. (2002). Receptor downregulation and multivesicular-body sorting. Nat Rev Mol Cell Biol 3, 893905.[CrossRef][Medline]
Köhler, J. R. & Fink, G. R. (1996). Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc Natl Acad Sci U S A 93, 1322313228.
Lay, J., Henry, L. K., Clifford, J., Koltin, Y., Bulawa, C. E. & Becker, J. M. (1998). Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies. Infect Immun 66, 53015306.
Lee, K. L., Buckley, H. R. & Campbell, C. C. (1975). An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148153.[Medline]
MacDiarmid, C. W., Milanick, M. A. & Eide, D. J. (2002). Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J Biol Chem 277, 3918739194.
Munn, A. L. & Riezman, H. (1994). Endocytosis is required for the growth of vacuolar H+-ATPase-defective yeast: identification of six new END genes. J Cell Biol 127, 373386.[Abstract]
Negredo, A., Monteoliva, L., Gil, C., Pla, J. & Nombela, C. (1997). Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans. Microbiology 143, 297302.[Medline]
Nelson, H., Mandiyan, S. & Nelson, N. (1994). The Saccharomyces cerevisiae VMA7 gene encodes a 14-kDa subunit of the vacuolar H+-ATPase catalytic sector. J Biol Chem 269, 2415024155.
Odds, F. C. (1994). Pathogenesis of Candida infections. J Am Acad Dermatol 31, S2S5.[Medline]
Palmer, G. E., Cashmore, A. & Sturtevant, J. (2003). Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation. Eukaryot Cell 2, 411421.
Perzov, N., Nelson, H. & Nelson, N. (2000). Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants. J Biol Chem 275, 4008840095.
Perzov, N., Padler-Karavani, V., Nelson, H. & Nelson, N. (2002). Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis. J Exp Biol 205, 12091219.
Peto, R., Pike, M. C., Armitage, P. & 7 other authors (1977). Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br J Cancer 35, 139.[Medline]
Roberts, C. J., Raymond, C. K., Yamashiro, C. T. & Stevens, T. H. (1991). Methods for studying the yeast vacuole. Methods Enzymol 194, 644661.[Medline]
Sautin, Y. Y., Lu, M., Gaugler, A., Zhang, L. & Gluck, S. L. (2005). Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells. Mol Cell Biol 25, 575589.
Sonneborn, A., Bockmühl, D. P., Gerads, M., Kurpanek, K., Sanglard, D. & Ernst, J. F. (2000). Protein kinase A encoded by TPK2 regulates dimorphism of Candida albicans. Mol Microbiol 35, 386396.[CrossRef][Medline]
Stevens, T. H. & Forgac, M. (1997). Structure, function and regulation of the vacuolar (H+)-ATPase. Annu Rev Cell Dev Biol 13, 779808.[CrossRef][Medline]
Teter, S. A. & Klionsky, D. J. (2000). Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation. Semin Cell Dev Biol 11, 173179.[CrossRef][Medline]
Theiss, S., Kretschmar, M., Nichterlein, T., Hof, H., Agabian, N., Hacker, J. & Kohler, G. A. (2002). Functional analysis of a vacuolar ABC transporter in wild-type Candida albicans reveals its involvement in virulence. Mol Microbiol 43, 571584.[CrossRef][Medline]
Tomashek, J. J., Sonnenburg, J. L., Artimovich, J. M. & Klionsky, D. J. (1996). Resolution of subunit interactions and cytoplasmic subcomplexes of the yeast vacuolar proton-translocating ATPase. J Biol Chem 271, 1039710404.
Underwood, E. J. (1977). Trace Elements in Human and Animal Nutrition. London: Academic Press.
Vida, T. A. & Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128, 779792.[Abstract]
Vida, T. A., Huyer, G. & Emr, S. D. (1993). Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment. J Cell Biol 121, 12451256.[Abstract]
Weissman, Z., Berdicevsky, I., Cavari, B. Z. & Kornitzer, D. (2000). The high copper tolerance of Candida albicans is mediated by a P-type ATPase. Proc Natl Acad Sci U S A 97, 35203525.
Wendland, B., McCaffery, J. M., Xiao, Q. & Emr, S. D. (1996). A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian Eps15. J Cell Biol 135, 14851500.[Abstract]
Wilson, R. B., Davis, D. & Mitchell, A. P. (1999). Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181, 18681874.
Received 21 July 2004;
revised 2 February 2005;
accepted 14 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |