Molecular and Cellular Biology Program1, Division of Mycology2, Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, PO Box 250504, Charleston, SC 29403, USA
Author for correspondence: Joseph W. Dolan. Tel: +1 843 792 3104. Fax: +1 843 792 2464. e-mail: dolanjw{at}musc.edu
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ABSTRACT |
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Keywords: dimorphic transition, phospholipase D, PLD1, MSS4
Abbreviations: DAG, 1,2-diacylglycerol; LPP, lipid phosphate phosphohydrolases; PA, phosphatidic acid; PI4P5K, phosphatidylinositol-4-phosphate 5-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PLD1, phospholipase D1
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INTRODUCTION |
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PIP2 is synthesized by the sequential phosphorylation of phosphatidylinositol by phosphatidylinositol-4-kinase and phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) at the D-4 and D-5 position of the inositol ring, respectively. PIP2 can also be synthesized by phosphatidylinositol-5-phosphate 4-kinase (from phosphatidylinositol 5-phosphate) (Rameh et al., 1997 ). Originally, type I and type II PI4P5Ks were described in mammals (Loijens & Anderson, 1996
); however, the substrate specificity of these enzymes has revealed that the type II enzymes are phosphatidylinositol-5-phosphate 4-kinases (Rameh et al., 1997
). In mammals, different isoforms of PI4P5K have been identified and designated
, ß and
(Ishihara et al., 1996
, 1998
; Loijens & Anderson, 1996
; Loijens et al., 1996
). Acidic phospholipids such as phosphatidic acid (PA) (Ishihara et al., 1996
, 1998
; Jenkins et al., 1994
; Moritz et al., 1992
) and phosphatidylserine (Cochet & Chambaz, 1986
) have been shown to stimulate PI4P5K in mammalian cells. Additional regulators of PIP2 synthesis include the small GTP-binding proteins Rho and Rac (Chong et al., 1994
; Tolias et al., 2000
). Honda et al. (1999)
demonstrated recently that PI4P5K
is a downstream effector of ADP-ribosylation factor ARF6 and is involved in membrane ruffling.
The S. cerevisiae PI4P5K is encoded by the gene MSS4 and is a 779 aa protein (Desrivieres et al., 1998 ; Homma et al., 1998
). The yeast PI4P5K is required for actin cytoskeleton assembly as mutants fail to form normal actin filaments and fail to localize the cytoskeleton properly during polarized cell growth (Desrivieres et al., 1998
; Dove et al., 1997
; Homma et al., 1998
). Loss of PI4P5K is suppressed by overexpression of RHO2, a Rho-type GTPase involved in the regulation of the actin cytoskeleton, implicating Rho2p as a downstream effector of PI4P5K (Desrivieres et al., 1998
). As in mammalian systems, PI4P5K in S. cerevisiae has been reported to be stimulated by PA (Homma et al., 1998
). PA is a putative second messenger that has been implicated in the regulation of lipid metabolism (Liscovitch et al., 1999
), cell proliferation (Kaszkin et al., 1996
), differentiation (Amsterdam et al., 1994
), secretion (Siddhanta & Shields, 1998
) and actin polymerization (Ha & Exton, 1993
). PA can be produced by the hydrolysis of the major membrane phospholipid, phosphatidylcholine, by signal-activated PLD1. Since PI4P5K catalyses the production of PIP2, which is required for the maximal activity of PLD1, and PLD1 catalyses the production of PA, which is required for the maximal activity of PI4P5K, the sequential action of PLD1 and PI4P5K could result in a feedback loop that would stimulate the production of both PA and PIP2 (Liscovitch et al., 1994
).
C. albicans is the most frequently isolated fungal pathogen in humans. Infections caused by this organism are a major cause of morbidity and mortality among immunocompromised patients, women of childbearing age, infants and the elderly. Antifungal antibiotics are available; however, many have the potential to cause liver and/or kidney toxicity in humans. Strains of C. albicans that are resistant to the most commonly used antifungals are appearing with increasing frequency (Rex et al., 1995 ). It is therefore essential that the mechanisms involved in C. albicans virulence be elucidated. This organism can grow as budding yeast-form cells or as elongated hyphae, depending on growth conditions. Virulence has been associated with the ability to undergo conversion from the round yeast-form to the elongated hyphal form in a morphogenetic process termed dimorphic transition (Cutler, 1991
; Navarro-Garcia et al., 2001
). High temperature, neutral pH and serum are potent inducers of dimorphic transition (Ernst, 2000
; Odds, 1988
). The initial stage of dimorphic transition is the formation of a germ tube, which is an elongated daughter cell formed by an existing yeast-form cell. It has been proposed that hyphae have a mechanical advantage in the penetration of host-cell membranes and the destruction of phagocytes (Odds, 1988
). Consequently, molecules found to regulate the process of dimorphic transition may be effective targets for improved antifungal drugs (Leberer et al., 1997
). In this report, PI4P5K activity in C. albicans is described. This enzyme is stimulated by PA and during temperature-induced morphogenesis. Finally, we present evidence that stimulation during morphogenesis requires PLD1 activity.
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METHODS |
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Morphogenesis assay.
To measure the effect of the yeast-to-hyphae transition on enzyme activity, PI4P5K and PLD1 in vitro assays were performed with extracts from wild-type cells induced to form hyphae by temperature shift from 30 to 37 °C. Overnight cultures of CAF2-1 and JDC12 in YPD at 30 °C were diluted 1:100 in fresh YPD medium and incubated at 37 °C for 90 min. Samples were taken at increasing times post-induction and whole-cell extracts were prepared and assayed for both PI4P5K and PLD1 activities. Where indicated, morphogenesis was induced by temperature shift and the addition of fetal bovine serum to a final concentration of 5% (v/v).
PI4P5K assay.
PI4P5K activity was assayed according to published methods used for the S. cerevisiae enzyme (Homma et al., 1998 ). Briefly, C. albicans whole-cell extracts containing 55 µg protein, as determined by the Bradford assay, were incubated with 80 µM PI4P (Sigma) and 5 µCi [
-32P]ATP (NEN) for 10 min at 30 °C. Reactions were incubated with or without 50 µM PA where explicitly stated. The reactions were terminated and the lipids extracted by the addition of chloroform/methanol according to the method of Bligh & Dyer (1959)
. The lipid products were separated on a silica gel thin layer chromatography plate impregnated with 1·2% potassium oxalate (v/v) and developed with chloroform/methanol/sodium hydroxide/water (40:42:5:10, by vol.) (Homma et al., 1998
). Quantification of radioactivity incorporated into products was performed with a Molecular Dynamics PhosphorImager. A second assay for PI4P5K used the substrate NBD-PI4P (Echelon Laboratories), which is a fluorescent analogue of the in vivo substrate for PI4P5K. Micelles containing the substrate were prepared as described for the fluorescent PLD1 substrate by McLain & Dolan (1997)
and the reactions were performed as with the radioactive assay. Control samples were always run on the same TLC plate as the experimental samples and both control and experimental samples were scanned at the same time. Values were normalized to controls on the same TLC plate and these normalized values were used to compare results between plates. Statistical analysis was performed using the Students t-test for three independent experiments with duplicate samples in each.
Fluorescent PLD1 assay.
PLD1 activity was assayed using whole-cell extracts and the fluorescent substrate BODIPY-phosphatidylcholine as described previously (McLain & Dolan, 1997 ).
DNA sequence analysis.
Homology searches were conducted with the BLAST algorithm. Sequence alignments were performed with DNASTAR MEGALIGN software version 1.05 (DNASTAR) using the CLUSTAL W 1.6 method. A rooted phylogenetic tree was generated using the CLUSTAL W method with the PAM250 residue weight table.
Construction of plasmids.
The DNA encoding the putative C. albicans Mss4p was amplified from C. albicans SC5314 genomic DNA by PCR. The primers amplifying the upstream untranslated and 5' coding region of MSS4 included restriction sites EcoRI and XbaI at the 5' ends for cloning. Primer sequences were 5'-GCATATGAATTCCATCGCTCTCCATCAACACC-3' and 5'-GAACATCTAGAAACTGCCACTC-3' which contained the restriction sites EcoRI and XbaI (bold type), respectively. The digested PCR product was cloned into the EcoRIXbaI sites of the S. cerevisiae/E. coli shuttle vector YEp352, generating pCW10. The 3' coding region of MSS4 and its terminator were amplified using primers 5'-GCAGTTTCTAGATGTTCTGGAG-3' and 5'-CATGGCAAGCTTTCGGTTGGTCTGAATCAC-3' which contained the restriction sites XbaI and HindIII (bold type), respectively. The digested PCR product was inserted into the XbaI/HindIII sites of pCW10 to generate plasmid pCW20.
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RESULTS |
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PI4P5K activity is stimulated during morphogenesis
Earlier experiments performed in this laboratory have shown that PLD1 plays a significant role in the morphogenetic process (Hube et al., 2001 ; McLain & Dolan, 1997
). Given that the product of PLD1 catalysis is PA and that PA stimulates PI4P5K, we aimed to assess a possible correlation between PI4P5K and PLD1 during the morphogenetic switch. C. albicans whole-cell extracts were assayed during morphogenesis to determine the level of PI4P5K activity at various time points following induction. These experiments were performed with a fluorescent assay for PI4P5K activity using NBD-PI4P as the substrate for the reaction. The results indicated a maximum level of PI4P5K activity at the 20 min time point, yielding a 2·5-fold increase in activity (Fig. 2a
); this increase over the zero time point was statistically significant (P<0·01; n=6). Maximum PI4P5K activity was coincident with the initiation of germ tube emergence. At 90 min post-induction there was another increase in PI4P5K activity when
90% of cells exhibited germ tubes. In a parallel experiment, cells were inoculated into fresh medium and incubated at 30 °C to detect changes in PI4P5K due to a return to growth. No significant changes were detected in PI4P5K activity under these conditions (data not shown). The small magnitude of stimulation raises the question of physiological impact. By way of comparison, PLD1 activity is stimulated only 60% during morphogenesis, but the null mutant is unable to form hyphae under specific conditions and is avirulent in a mouse model system (Hube et al., 2001
; McLain & Dolan, 1997
). Thus, there is a solid precedent, supported by both biochemical and genetic analysis, for small changes in activity having relevant and significant physiological impact in the process of morphogenesis in C. albicans.
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Propranolol inhibits PI4P5K induction during morphogenesis
To determine whether PA was activating PI4P5K in vivo, propranolol was added to cultures coincident with the induction of morphogenesis. Propranolol has been used at low concentrations (0·10·2 mM) to inhibit mammalian lipid phosphate phosphohydrolases (LPPs) that convert PA into DAG. The LPPs in C. albicans are not affected by low concentrations of propranolol (C. A. Baker, K. Desrosiers & J. W. Dolan, unpublished); consequently, higher concentrations (15 mM) were used. At the higher concentrations, propranolol can act by binding to the PA and rendering it inaccessible to downstream targets of PA (C. A. Baker, K. Desrosiers & J. W. Dolan, unpublished; Surewicz & Leyko, 1981 ). As expected, propranolol attenuated PI4P5K stimulation throughout the dimorphic transition (Fig. 3a
) relative to untreated controls (Fig. 2a
). The addition of 2 mM propranolol also eliminated germ-tube formation in these cultures (Fig. 3a
). To rule out the possibility that the inhibition of germ-tube formation was the result of reduced proliferation, growth rates were determined at increasing propranolol concentrations. The addition of propranolol in concentrations up to 1 mM did not affect growth rates. However, 2 mM caused a slight increase in generation time (Fig. 3b
). Thus, propranolol reduced PI4P5K activation and germ-tube formation without substantially affecting the growth rate.
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To determine whether PLD1 was playing a role in the stimulation of PI4P5K, a homozygous pld1 knockout strain, JDC12 (Hube et al., 2001
), was assayed for PI4P5K activity during morphogenesis. PI4P5K activity was observed to decline steadily during morphogenesis in the pld1
cells (Fig. 4
). Although PA can be generated by the phosphorylation of DAG by diglyceride kinase (Morlock et al., 1991
; Wu et al., 1993
) and by acylation of monoacyl-glycerol 3-phosphate (Athenstaedt et al., 1999
), this complete lack of induction of PI4P5K activity in pld1
cells indicates that PLD1 supplies PA necessary for PI4P5K stimulation during dimorphic transition to elongated hyphae.
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C. albicans MSS4 can complement a S. cerevisiae mss4ts
To determine whether the cloned ORF encodes a functional homologue of the S. cerevisiae PI4P5K Mss4p, we constructed a plasmid (pCW20) carrying this gene under the transcriptional control of its own putative promoter. The amino acid sequence has a serine residue in the C terminus at position 463 that is encoded by non-standard CUG codon usage in C. albicans (Ohama et al., 1993 ; Santos et al., 1993
). pCW20 was transformed into S. cerevisiae strains SEY6210.1 (MSS4) and AAY202.1 (mss4
::HIS3 carrying YEplac-mss4-102ts) and the transformants were tested for the ability to suppress the temperature sensitivity of the mss4-102 allele (Fig. 6
and Table 2
). The temperature-sensitive transformants carrying pCW20, which contains the URA3 gene as a selectable marker, were unable to grow on plates containing 5-fluoroorotic acid, indicating that the Candida MSS4 gene was required for viability at the non-permissive temperature (38 °C). Since these strains carried the mss4-102 temperature-sensitive allele, the cells should be able to lose pCW20 and grow on 5-fluoroorotic acid at the permissive temperature of 26 °C. The pCW20 transformants were able to grow on 5-fluoroorotic acid at 26 °C, forming colonies after extended incubation (data not shown). The slow growth suggests that even at the permissive temperature, the mss4-102 cells benefit from the added activity provided by the C. albicans MSS4 gene on pCW20. Plasmid pCW20 was also used to transform S. cerevisiae strain 23567, a diploid that is heterozygous for MSS4 (MSS4/mss4::KanR). These transformants showed an approximately 40% increase in PI4P5K activity that returned to untransformed levels following loss of pCW20. Taken together, these results confirm that the C. albicans gene designated MSS4 encodes a functional PI4P5K. The presence of additional C. albicans genes encoding PI4P5K activity cannot be excluded, but no other regions with significant homology to PI4P5Ks have been identified in the genome sequence.
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DISCUSSION |
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Our results suggest that PI4P5K might play a role in C. albicans transition to polarized growth in response to some, but not all, inducing stimuli. The exact role PI4P5K would play in this process is unknown; however, the change of shape from an ellipsoid to an elongated germ tube involves actin cytoskeleton reorganization. The product of PI4P5K activity, PIP2, could affect cell polarization in a number of ways. For example, PIP2 has been identified as a lipid that dissociates capping proteins from the barbed ends of actin filaments, thus promoting actin assembly (Heiss & Cooper, 1991 ; Janmey et al., 1987
; Janmey & Stossel, 1987
). Profilin is an actin-binding protein that binds PIP2. It is required for the maintenance of cell polarity and is essential for the proper formation of actin cables that occur at areas of active cellular growth (DiNubile & Huang, 1997a
, b
). Thus, PI4P5Ks in yeast and murine models have been shown to be critical for actin assembly (Audhya et al., 2000
; Desrivieres et al., 1998
; Homma et al., 1998
; Tolias et al., 2000
). It is therefore plausible that C. albicans PI4P5K is stimulated to provide increased levels of PIP2 for the actin remodelling process.
PI4P5K stimulation during morphogenesis is attenuated by the addition of propranolol (Fig. 3). Propranolol is typically used in mammalian cells at low concentrations (100200 µM) to inhibit LPP, the enzyme that hydrolyses PA to DAG (Meier et al., 1998
; Morlock et al., 1991
; Wu et al., 1993
). Frequently, inhibition by propranolol suggests that DAG, rather than PA, is the essential molecule in a cellular process. In contrast to mammalian LPP, yeast LPP is relatively insensitive to propranolol (C. A. Baker, K. Desrosiers & J. W. Dolan, unpublished; Ella et al., 1995
). An earlier study using fluorescent spectroscopy and millimolar concentrations of propranolol revealed that the drug bound directly to several phospholipids, particularly PA and phosphatidylserine (Surewicz & Leyko, 1981
). In addition, fluorimetry studies have shown that the intrinsic fluorescence of propranolol is quenched by PA in a concentration-dependent mode (C. A. Baker, K. Desrosiers & J. W. Dolan, unpublished). The change in fluorescence at high PA concentrations suggests that propranolol is acting by binding to and sequestering PA, making the PA unavailable for subsequent activity by LPP, PI4P5K or any other downstream target. Furthermore, when DAG levels were measured in wild-type C. albicans cells in response to millimolar concentrations of the drug, total cellular DAG levels remained unchanged compared to untreated cells (C. A. Baker, K. Desrosiers & J. W. Dolan, unpublished). These results, taken together, indicate that PA, rather than DAG, is the important molecule derived from PLD1 activity during morphogenesis.
In this study, the data suggest PLD1 is the source of PA required to stimulate PI4P5K during morphogenesis. Our results provide further evidence for a role of PLD1 in polarized growth (Hube et al., 2001 ; McLain & Dolan, 1997
) and a possible role for PI4P5K in this process. As shown in Fig. 2(a)
, PI4P5K is highly activated when germ tubes begin to emerge from pre-existing cells, whereas PLD1 activity does not peak until later. This timing would suggest that PIP2 formed by the action of PI4P5K stimulates PLD1. There is, however, a highly reproducible 10% increase in PLD1 activity 10 min after induction of morphogenesis before any germ tubes have emerged (Fig. 2a
; McLain & Dolan, 1997
). It is possible that PA is generated by stimulation of a highly localized pool of PLD1 to initially activate PI4P5K. Supporting this idea, PI4P5K activity was attenuated in extracts prepared from pld1
null mutant cells and by the addition of propranolol. Interestingly, germ-tube formation in pld1
cells was unaffected by the lack of PLD1 signalling and PI4P5K stimulation. Similarly, germ tubes were formed normally in response to induction by both serum and temperature, even though PI4P5K activity was relatively unaffected. These results were not unexpected, due to the multiple pathways that can induce hyphal development (Ernst, 2000
). Thus, PIP2 may be important to morphogenesis in response to some stimuli and irrelevant in response to other stimuli. Further studies are needed to determine whether the increase in PI4P5K activity is a cause or consequence of temperature-induced morphogenesis.
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ACKNOWLEDGEMENTS |
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Received 7 August 2001;
revised 11 February 2002;
accepted 25 February 2002.
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