The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans

International Journal of Medical Microbiology 303 (2013) 257–266

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International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm

The P-type ATPase Spf1 is required for endoplasmic reticulum functions and cell wall integrity in Candida albicans Qilin Yu a , Xiaohui Ding a , Bing Zhang a , Ning Xu a , Xinxin Cheng a , Kefan Qian a , Biao Zhang b , Laijun Xing a , Mingchun Li a,∗ a b

Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin, PR China Tianjin Traditional Chinese Medicine University, Tianjin, PR China

a r t i c l e

i n f o

Article history: Received 13 September 2012 Received in revised form 3 May 2013 Accepted 5 May 2013 Keywords: ER P-type ATPase Spf1 Calcium homeostasis CWI Candida albicans

a b s t r a c t Endoplasmic reticulum (ER) is crucial for protein folding, glycosylation and secretion in eukaryotic organisms. These important functions are supported by high levels of Ca2+ in the ER. We have recently identified a putative ER Ca2+ pump in Candida albicans, called Spf1, which plays key roles in maintenance of cellular Ca2+ homeostasis, morphogenesis and virulence. In this study, we purified Spf1 and confirmed that it is a P-type ATPase, suggesting its role in maintaining high levels of ER Ca2+ . Disruption of SPF1 caused severe defects in glycosylation of the ER-localized protein Cdc101 and secretory acid phosphatase, and a decrease in expression of SEC61 which encodes an important ER protein. Moreover, the spf1/ mutant showed increased sensitivity to cell wall stresses, abnormal cell wall composition, delayed cell wall reconstruction and decreased flocculation and adherence, indicating its defect in cell wall integrity (CWI). We also revealed that disruption of SPF1 has an impact on gene expression related to CWI and morphogenesis. This study provides evidence that Spf1, as a P-type ATPase, is essential for ER functions and consequent CWI, implicating a role of ER Ca2+ homeostasis in C. albicans physiology. © 2013 Elsevier GmbH. All rights reserved.

Introduction Candida albicans is a common fungus residing on the skin, mucosa, and intestinal tract of healthy individuals (Kleinegger et al., 1996). However, it may cause serious mucosal and invasive infections in immunocompromised patients, ranking as the fourth most common pathogens of nosocomial bloodstream infections (Klepser, 2003). The properties of adherence and yeast-to-hyphal switch allow this fungus to effectively invade the host tissues (Gow et al., 2012). Moreover, this fungus may also form biofilms on mucosal surfaces and implanted medical devices, which enhance its capacity to tolerate antifungal agents (Finkel and Mitchell, 2011). These properties contribute to its pathogenicity, and have profound consequence in clinical practice. The C. albicans cell wall is mainly composed of a flexible network of highly branched ␤-(1,3)-glucan and ␤-(1,6)-glucan which are covalently attached to chitin, glycophosphatidyl-inositol-anchored cell wall proteins (GPI-CWP), Pir proteins and phospholipomannan. This network is surrounded by non-covalently connected

∗ Corresponding author at: Department of Microbiology, College of Life Science, Nankai University, Tianjin 300071, PR China. Tel.: +86 22 23508506; fax: +86 22 23508800. E-mail address: [email protected] (M. Li). 1438-4221/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2013.05.003

proteins (Ernst and Pla, 2011; Chaffin, 2008). The cell wall is an important virulence factor because of its pathogen-specific protein components. Several cell wall protein families, such as the Als family and the Hwp family, play key roles in adhesion to host surfaces, hyphal development and biofilm formation, and therefore are closely involved in morphogenesis and pathogenesis in C. albicans (Butler et al., 2009; Hoyer, 2001; Nobile et al., 2006). These proteins are enriched in the cell wall of hyphal cells, and show a high degree of genetic and structural diversity, which is likely required for adaptation to the environment prevailing in the host tissues and pathogenesis (Butler et al., 2009). Therefore, maintenance of cell wall integrity (CWI), especially the biosynthesis, delivery and architecture of these pathogen-specific proteins, is very important for virulence of C. albicans. Stresses that threaten CWI may cause the activation of the CWI pathway (Scrimale et al., 2009). In this pathway, the transcription factors Rlm1 and Cas5 are key regulators governing expression of different CWI genes, such as ECM331, PGA13, DFG5 and CRH11 (Bruno et al., 2006). The endoplasmic reticulum (ER) plays pivotal roles in protein folding, glycosylation and secretion in all eukaryotic cells (Görlach et al., 2006), all of which are associated with cell wall biosynthesis. ER stresses, such as treatment of tunicamycin, lead to cell wall defects in Saccharomyces cerevisiae, indicating a link between ER functions and CWI. High levels of Ca2+ in ER lumen are of central importance to ER functions (Scarborough, 1999). The

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ER Ca2+ levels are maintained by a super-family of Ca2+ pumps known as P-type ATPases (Bayle et al., 1995). In mammals, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), a type II P-type ATPase, is responsible for ER Ca2+ maintenance (Brini and Carafoli, 2009; Axelsen and Palmgren, 2001). Absence of SERCA in Saccharomyces cerevisiae leads to the discovery of another Ptype ATPase, ScCod1/ScSpf1 (Cronin et al., 2002). We have recently identified a ER-localized homologue of ScCod1/ScSpf1 in C. albicans and named it Spf1. The spf1/ mutant shows hypersensitivity to the Ca2+ chelator EGTA, high-level Ca2+ and ER-targeted antifungal agents, such as fluconazole and tunicamycin, suggesting a role of Spf1 in cellular calcium homeostasis and ER functions. Moreover, this protein is required for hyphal development, biofilm formation, invasion, and thus highly affects virulence of C. albicans (Yu et al., 2012). Here, Spf1 is demonstrated as a P-type ATPase. Due to the ER localization and its key role in cellular calcium homeostasis (Yu et al., 2012), we propose that this protein function in supplying the ER with calcium. The spf1/ mutant displays decreased expression of the ER-associated gene SEC61, and has defects in N-glycosylation, an important function of the ER. We further demonstrate that the mutant exhibits hypersensitivity to cell wall stresses, abnormal cell wall compositions and consequent defects in flocculation and adhesion. Taken together, these results indicate that ER calcium homeostasis maintained by Spf1 is required for ER functions, and thus plays an important role in CWI in C. albicans. Materials and methods

Table 1 Strains and plasmids used in this study. Strains and plasmids C. albicans DAY1

NKF111

NKF112

NKF120

NKF121

NKF122

NKF123

NKF124

Strains The C. albicans and S. cerevisiae strains used in this study are listed in Table 1. For constructing NKF120 with one copy of SPF1 tagged by V5-6×His epitope, the fragment with V5-6×His epitope and the selection marker URA3 were amplified from the plasmid pV5-URA (Milne et al., 2011) and transformed into DAY1 (BWP17, Davis et al., 2000). For determining the effect of SPF1 disruption on SEC61 expression, the strains DAY1, NKF111 and NKF112 were transformed by NruI-digested D78-PSEC61 -LacZ (detailed as below), obtaining NKF121, NKF122 and NKF123, respectively. To test the glycosylation extent of the ER-localized protein Cdc101, the strains DAY1, NKF111 and NKF112 were transformed by the HA fragment with URA3 amplified from the plasmid pFA-HA-URA (Lavoie et al., 2008), obtaining NKF124, NKF125 and NKF126 expressing C-terminally HA-tagged Cdc101. Plasmid construction The plasmids used in this study are also listed in Table 1. The plasmid D78-PSEC61 -LacZ was constructed as follows. The SEC61 promoter was amplified from DAY1 genomic DNA, obtaining a 1 kb upstream region of SEC61 coding sequence. This fragment was cloned into pGEM-T easy vector (Promega, USA) to create the plasmid T-PSEC61 . The LacZ fragment was obtained from AseI/MluI-digested pDDB211 (Baek et al., 2008), and cloned into NdeI/MluI-digested pGEM-T-PSEC61 to create the plasmid T-PSEC61 LacZ. DrdI-digested pGEM-T-PSEC61 -LacZ and EcoRI/NotIdigested pDDB78 (Baek et al., 2008) were co-transformed into the S. cerevisiae strain DAY414, obtaining the transformant NKS2 which contains the recombination plasmid D78-PSEC61 -LacZ. Protein extracts and Western blotting To test for glycosylation of Cdc101, cells were grown in liquid YPD medium at 30 ◦ C with shaking and harvested after 6–8 h of

NKF125

NKF126

Genotype

Source

ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200, SPF1 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG SPF1-V5-6 × His::SPF1 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG pDDB211-PSEC61 -LacZ ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200 pDDB211-PSEC61 -LacZ ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200, SPF1 pDDB211-PSEC61 -LacZ ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG CDC101-HA/CDC101 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200 CDC101-HA/CDC101 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG spf1::ARG4/spf1::URA3-dpl200, SPF1 CDC101-HA/CDC101

Dana Davis

Yu et al. (2012)

Yu et al. (2012)

This study

This study

This study

This study

This study

This study

This study

S. cerevisiae DAY414 NKS2

trp1 TRP1 HIS1 PSEC61 -LacZ

Dana Davis This study

Plasmids pGEM-T easy pDDB211 pDDB78 pV5-URA pFA-HA-URA T-PSEC61 T-PSEC61 -LacZ D78-PSEC61 -LacZ

ApR ApR ApR ApR ApR ApR ApR ApR

Promega Dana Davis Dana Davis Steven Bates Linghuo Jiang This study This study This study

LacZ TRP1 HIS1 V5-6×His URA3 HA URA3 PSEC61 PSEC61 -LacZ TRP1 HIS1 PSEC61 -LacZ

incubation. Cells were washed with distilled water, resuspended in RAPI lysis buffer, and vortexed with glass beads. Cell lysate was centrifuged at 31,000 × g for 30 min. Protein contents were quantified by Coomassie protein assays. In endoglycosidase H (EndoH) analysis, total protein extracts were suspended in 20 ␮l of EndoH buffer (NEB, USA), boiled for 10 min, chilled and treated with 2 ␮l of EndoH at 37 ◦ C for 2 h. EndoH-treated or untreated samples were separated by SDS-PAGE on 8% polyacrylamide gels, and then electrophoretically transferred from gels onto PVDF filters at 200 mA for 1.5 h. Filters were probed with rabbit antibodies against HA-epitope (Santa Cruz, USA), followed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz, USA). The peroxidase activity was detected by using the Immobilon Western Kit (Millipore, USA),

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and luminescence was recorded by exposing the filter to radioautographic X-ray films. Purification of Spf1 and ATPase activity assay Spf1 was purified following a modified method of Cronin et al. (2002). Briefly, the strain expressing V5-6×His epitope-tagged Spf1 was cultured in liquid YPD medium at 30 ◦ C with shaking for 10–12 h. Cells were then harvested, resuspended in lysis buffer (4 M sorbitol, 0.1 M phosphate potassium, 1 mM EDTA, pH 7.5) plus protease inhibitors and lysed by vortex with glass beads. Lysates were clarified at 5000 × g for 30 min, and centrifuged at 20,000 × g for 30 min to remove mitochondria and heavy membranes. Crude ER membranes were then collected by centrifugation at 120,000 × g for 1 h (Beckman MAX XP, USA), and resuspended in P&R buffer (50 mM surcrose, 10 mM MES/KOH, 150 mM KCl, pH 6.0) plus protease inhibitors. The suspension was mixed with 20 ml detergent buffer (10 mM imidazole, 20 mM Hepes/Tris, 1% l-␣-phosphatidylcholine, 0.1% Triton-X 100, 6 mM ␤-mercaptoethanol) with protease inhibitors and gently shaken at 4 ◦ C for 2 h. The suspension was then clarified at 20,000 × g for 30 min, and detergent-solubilized Spf1-V5-6×His was obtained. This protein was purified by nickel affinity chromatography, and detected by SDS-PAGE and Western blotting using rabbit antibodies against V5-epitope. ATPase activity of Spf1 was assayed using Xu’s method (Xu and Song, 1986). 1 ml purified Spf1-V5-6×His solution suspension was mixed with reaction solution (2.5 mM Tris/maleate, 1 mM ATP, 100 mM NaCl, 2 mM MgCl2 , 36 ␮M CaCl2 ). 1 ml reaction solution was added to 5 ml AMT solution (1.05% ammonium molybdate, 1 M HCl, 0.04% Malachite Green oxalate, 0.06% Tween-20) at different time. 1 ml 24% sodium citrate solution was then added, and A660 (the absorbance at 660 nm) was determined to assess ATPase activity. The ATPase activity of 1 ml heat-treated Spf1-V5-6 × His solution were also determined as the negative control. ˇ-Galactosidase assays ␤-Galactosidase assays were performed as described previously (Yu et al., 2012). Strains were grown overnight at 30 ◦ C in YPD medium, diluted to an A600 (the absorbance at 600 nm) of 0.1, incubated at 30 ◦ C for 5 h, and treated with tunicamycin (2 ␮g/ml), FK506 (1 ␮g/ml) or TN (2 ␮g/ml) plus FK506 (1 ␮g/ml) for further 2 h. Cells were harvested and suspended in Working Z buffer (60 mM Na2 HPO4 , 40 mM NaH2 PO4 , 10 mM KCl, 1 mM MgSO4 , 38 mM ␤-mercaptoethanol). A600 of 50 ␮l cell suspensions was determined. 150 ␮l suspensions were permeabilized with 20 ␮l 0.1% SDS and 50 ␮l chloroform, mixed with 700 ␮l O-nitrophenylˇ-d-galactopyranoside (ONPG, 1 mg/ml), and incubated at 37 ◦ C for certain time (T). The reaction was stopped by addition of 500 ␮l 1 M Na2 CO3 . Suspensions were spun, and A420 of the supernatant was determined. ␤-Galactosidase activity (Miller units) were calculated as (A420 × 1000)/(A600 × T × 3). Acid phosphatase assay Acid phosphatase activity was assayed by a modified method of Bates (Bates et al., 2005). Strains were cultured in Sabouraud glucose medium (peptone 1%, glucose 4%, pH 5.6) at 30 ◦ C for 12 h to induce expression of acid phosphatase. Cells were harvested, washed with distilled water for 3 times, and resuspended in 500 ␮l lysis buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 6.8) plus protease inhibitors. Cells were then broken by grinding with glass beads and liquid nitrogen. The lysate was centrifuged at 31,000 × g for 30 min to pellet insoluble materials. EndoH-treated or untreated samples were run on 6% native PAGE gels at 100 kV

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for 4 h. The gels were rinsed in 100 mM sodium acetate (pH 5.2) with gentle shaking for 20 min, incubated with 0.05% monosodium 1-naphthyl phosphate (Tokyo Chemical Industry Co., Japan) in 100 mM sodium acetate (pH 5.2) at 37 ◦ C for 30 min, and then stained with 0.05% monosodium 1-naphthyl phosphate plus 0.03% fast blue (Sigma, USA) in 100 mM sodium acetate (pH 5.2) at 60 ◦ C for 30–60 min. Assays of sensitivity to cell wall stresses To test the strains for sensitivity to the cell wall stress agent Calcofluor white (CFW), cells were grown in liquid YPD medium overnight, adjusted to an A600 of 0.1 with distilled water, and spotted on YPD plates containing 0, 20 and 100 ␮g/ml CFW. The plates were incubated at 30 ◦ C for 2 d and observed. Sensitivity to vortex was tested by suspending overnight cells in distilled water and vortexed with glass beads for 1 min. Released proteins were analyzed by Coomassie protein assay reagent and SDS-PAGE. To test the sensitivity to lyophilization, cultures were incubated on polystyrene plates at 37 ◦ C for 12 h, allowing the cells to adhere to the plate surface. The plates were then washed with PBS, sectioned, and lyophilized in vacuum desiccators. The samples were coated with gold and observed under a scanning electron microscope (QUANTA, Czech). Percentage of collapsed cells was calculated as the number of collapsed cells × 100%/the total number of observed cells. Cell wall reconstruction Cell wall reconstruction and sensitivity to zymolyase was assayed using a modified method of Gelis (Gelis et al., 2012). Early log phase cells were suspended in pretreatment buffer (0.05% pronase E, 0.5% ␤-mercaptoethanol, 100 mM EDTA, 10 mM sodium phosphate, pH 8.0) and gently shaken at 30 ◦ C for 30 min. The cells were then washed with 0.6 M KCl, resuspended in zymolyase solution (Zymo Research, USA, 5 U/␮l) and incubated with shaking at 30 ◦ C until all cells formed protoplasts. Serial dilutions of protoplasts and untreated cells were plated with 10-fold gradients on YPD agar containing 1.2 M sorbitol, and incubated at 30 ◦ C for 2 d. Colonies were counted to indicate the ability of cell wall reconstruction. Percentage of reconstructed cells was calculated as the number of colonies formed by zymolyase-treated cells × 100%/the number of colonies formed by untreated cells. Five independent experiments were preformed. Cell wall isolation and composition To isolate the cell wall, strains were cultured in liquid YPD medium at 30 ◦ C for 12 h. Harvested cells were then disrupted as described in acid phosphatase assay. The lysate was suspended in distilled water plus 1 mM phenylmethanesulfonyl fluoride (PMSF) and centrifuged at 160–200 × g to remove glass beads and intact cells, and then centrifuged at 32,000 × g to pellet the crude cell wall. The pellet was washed two times with 20% NaCl plus 1 mM PMSF, and then washed five times with distilled water plus 1 mM PMSF, obtaining the pure cell wall. Cell wall composition was analyzed as described (Perez and Ribas, 2004; Dijkgraaf et al., 1996). To isolate soluble cell wall proteins, the pure cell wall was resuspended in 2% SDS plus 5 mM dithiothreitol and 1 mM PMSF, boiled for 1 h, and centrifuged at 32,000 × g. The relative amount of soluble cell wall proteins in the spf1/ mutant (or the reconstituted strain) was calculated as the decreased cell wall weight of the mutant cell wall (or the reconstituted strain cell wall) × 100%/the decreased cell wall weight of the wild-type strain. To isolate alkali-soluble extracts, the SDSinsoluble fraction was resuspended in 2 M NaOH, boiled for 2 h, and centrifuged at 32,000 × g. The alkali-insoluble fraction was then

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resuspended in 1 ml of zymolyase solution and digested at 37 ◦ C for 12 h. The insoluble fraction was removed at 32,000 × g and weighed as the amount of ␤-(1,6)-glucan. The amount of alkali-insoluble ␤-(1,3)-glucan was calculated as the decreased alkali-insoluble fraction after zymolyase digestion. The relative amount of alkalisoluble extracts, alkali-insoluble ␤-(1,3)-glucan and ␤-(1,6)-glucan in the mutant (or the reconstituted strain) was also calculated. Real-time quantitative PCR (RT-PCR) analysis To test expression of the CWI genes, ECM331, PGA13, DFG5 and CRH11 (Bruno et al., 2006), cultures were grown and treated with drugs as described in ␤-galactosidase assays. Cells were then harvested for RNA extraction. To test expression of the genes related to morphogenesis, HWP1, ECE1 and ALS3, strains were incubated with liquid YPD medium plus 10% fetal bovine serum (FCS) to induce hyphal development, or with polystyrene plates to induce biofilm formation (Yu et al., 2012). Cells were harvested and frozen in liquid nitrogen. Total RNA was extracted from the cells as described (Davis et al., 2000), and genomic DNA was digested with DNaseI (Takara, China) at 37 ◦ C for 2 h. Total RNA was then used for reverse transcriptional synthesis of cDNA with oligo(dT)-primed RT reagent Kit (Promega, USA). RT-PCR analysis for expression of the CWI genes or the genes related to morphogenesis was performed using the RealMasterMix (SYBR Green) Kit (TransGen, China). Transcription levels of these genes were normalized against the levels of ACT1 (Frade et al., 2004). Each sample was analyzed in five independent experiments. The results were expressed as fold change compared with the untreated wild-type strain.

et al., 2012). To assess adhesion to buccal epithelial cells (BEC), C. albicans strains were cultured in NGY medium (0.1% peptone, 0.4% glucose, 0.1% yeast extract) at 37 ◦ C to early log phase, washed with sterile 0.09% (w/v) NaCl and resuspended with 5 × 105 cells/ml. BEC isolated from healthy volunteers were washed and suspended at 5 × 105 cells/ml. The C. albicans cells and BEC were mixed with equal volumes and incubated at 37 ◦ C for 4 h. The suspension was then fixed with 7% (v/v) formalin, stained with eosin and photographed. The BEC adhered with C. albicans cells were counted (Bates et al., 2005). This assay was repeated four times. Results C. albicans Spf1 is a P-type ATPase BLASTP assay indicated that C. albicans Spf1 is a P-type ATPase. To test this, we purified C. albicans Spf1 and studied its biochemical characteristics. The V5-6×His epitope-tagged SPF1 allele was generated in wild-type C. albicans, expressing a modified Spf1 protein tagged with a V5 and 6×His tag after the last carboxyl-terminal alanine. The histidine-tagged Spf1 was then purified using nickel affinity chromatography. SDS-PAGE and Western blotting analysis showed that the purification yielded a single band with ∼140 kD (Fig. 1A). The ATPase activity of the purified protein was further tested. While the negative control led to extremely low-level production of inorganic phosphate, the purified protein showed ATPase activity, hydrolyzing ATP to ADP and inorganic phosphate in a time-dependent manner (Fig. 1B). These results, in combination with our previous study (Yu et al., 2012), demonstrated that C. albicans Spf1 is a P-type ATPase.

Flocculation and adhesion assay Disruption of SPF1 results in decreased expression of SEC61 For the analysis of flocculation, strains were cultured at 30 ◦ C for 12 h, washed with PBS and harvested. Collected cells were suspended in RPMI 1640 medium to an A600 of 0.5, added into glass tubes, and incubated with gently shaking at 37 ◦ C for 6–8 h. The cultures were then vortexed for 5–10 s and photographed (Gelis et al., 2012). Adhesion to polystyrene surface was assessed by incubating cells in RPMI 1640 medium on polystyrene plates at 37 ◦ C for 4 h, washing with PBS to remove non-adhered cells, and staining with 1% (w/v) crystal violet for 2 min. The plates were washed with distilled water and photographed. Crystal violet in adhered cells was extracted with 10% (v/v) acetic acid, and the absorbance of the extract at 595 nm (A595 ) was determined (Yu

Our previous study demonstrated that the disruption of SPF1 caused hypersensitivity to ER stress agents, such as tunicamycin and fluconazole, suggesting an important role of Spf1 in ER functions. It was also indicated that Spf1 functions in maintaining ER and cytoplasmic calcium homeostasis, which is required for normal ER functions (Yu et al., 2012). Here, we further examined whether the disruption of SPF1 also affects the expression of SEC61, a gene essential for ER functions and cell viability. SEC61 encodes an ER-localized protein, Sec61, which is a subunit of ER proteintranslocation complex, and plays an essential role in the secretion of cell surface components and enzymes contributing to pathogenic-

Fig. 1. Purification and ATPase assay of V5-6×His-tagged Spf1. (A) Nickel affinity chromatography-purified Spf1-V5-6×His was detected by SDS-PAGE with Coomassie Blue staining (CS) and Western blotting (WB) using rabbit antibodies against V5-epitope. (B) ATPase assay of Spf1. The purified protein Spf1-V5-6×His (black diamonds) and the heat-treated control (white squares) were incubated with ATP, and inorganic phosphate was detected by ATM solution at different time, respectively. Values represent mean ± SD of three independent tests.

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phosphatase than the control strains. Hence, the spf1/ mutant exhibited a glycosylation defect. The spf1/ mutant is hypersensitive to cell wall stresses

Fig. 2. ␤-Galactosidase assays for expression of SEC61 in the wild-type (WT, NKF121), spf1/ (NKF122) and reconstituted (spf1/+SPF1, NKF123) strains all containing the D78-PSEC61 -LacZ fragment. Early log phase cells were treated without drugs (white), with tunicamycin (TN, light gray), FK506 (dark gray) or TN plus FK506 (black) for 2 h, and then used for assaying the activity of ␤-galactosidase (Miller units). Values represent mean + SD of three independent strains. *Statistically significant differences between the strains with or without drug treatment (each of the columns except the first column) and the wild-type strain without drug treatment (the first column) (unpaired student t-test). P < 0.01.

ity. Consistent with results of transcript profiling (Wimalasena et al., 2008), ␤-galactosidase assays showed that the expression of SEC61 increased when the wild-type or reconstituted strain was treated with the ER stress agent tunicamycin (Fig. 2). However, the expression was about threefold lower in the spf1/ mutant than in the wild-type or reconstituted strain in the absence or presence of tunicamycin (Fig. 2), indicating that the expression of SEC61 decreased when SPF1 was disrupted. Our previous study has demonstrated that Spf1 is involved in cellular calcium homeostasis and regulates the calcineurin signaling pathway (Yu et al., 2012). Here we further investigated whether the decreased expression of SEC61 is related to this pathway. FK506, a specific inhibitor of calcineurin, also caused a significant decrease of SEC61 expression in the absence or presence of tunicamycin (Fig. 2). Therefore, the expression of SEC61 is regulated by the calcineurin signaling pathway, which presumably contributes to the decreased expression of this gene in the spf1/ mutant.

The spf1/ mutant shows defects in glycosylation Glycosylation is one of the important functions of ER. We hypothesized that the disruption of SPF1 may result in ER malfunction and consequent defect in glycosylation. Therefore, we tested the extent of glycosylation in the spf1/ mutant with Cdc101 and secreted acid phosphatase assay. Cdc101, encoded by orf19.4014 and recently characterized by our team, is an ER-localized and Nglycosylated protein (our unpublished data). Whereas Cdc101 in the wild-type and reconstituted strains displayed the same mobility, the protein in the spf1/ mutant showed a shift with smaller molecular weight. This is similar to a shift caused by the digestion of wild-type Cdc101 with EndoH, implying an un-glycosylated form of Cdc101 in the mutant (Fig. 3A). It indicated that N-glycosylation of Cdc101 in the mutant was inhibited. Secreted acid phosphatase is another N-glycosylated protein, and can be detected by ␣-naphthyl phosphate and fast blue because of its phosphatase activity (Bates et al., 2005). Whereas the protein extracted from the wild-type and reconstituted strain appeared as a diffuse band, the secreted acid phosphatase extracted from the spf1/ mutant migrated faster through the native PAGE, and had the same mobility as the EndoH-treated wild-type enzyme (Fig. 3B), indicating that the mutant produced less glycosylated acid

As ER is closely associated with modification secretion of cell wall proteins, we further and hypothesized that ER malfunction resulting from the disruption of SPF1 may affect cell wall synthesis, and alter cell resistance to cell wall stresses. Firstly, we tested the sensitivity of the spf1/ mutant to the well-known cell wall stress agent CFW. Expectedly, the mutant showed increased sensitivity to CFW as compared to the wild-type or reconstituted strain. Especially, 100 ␮g/ml CFW completely inhibited the growth of the mutant, but only showed slight inhibition effect to the growth of the control strains (Fig. 4A). Then we tested the resistance of the mutant cell wall to the physical stresses, vortex and lyophilization. After cells were treated with vortex, the extracellular proteins from the supernatant were determined as the extracted cell proteins. Coomassie protein assays revealed that the extracted cell proteins from the mutant were more than 20 times higher than that from the control strains (Fig. 4B). Extracted proteins from the control strains produced distinct and shallow bands in the SDS-PAGE gel. In contrast, extracted proteins from the mutant produced wider and darker bands than those from the control strains, indicating that the cell wall of the mutant was much more sensitive to vortex than the control stains, resulting in cell disruption and consequent release of abundant intracellular proteins in the mutant cells. Furthermore, SEM showed that abundant mutant cells collapsed after lyophilization, whereas most of control cells maintained integrity (Fig. 4C). The percentage of collapsed mutant cells was 3–5 times higher than the percentage of collapsed control cells (Fig. 4D), indicating the increased sensitivity of the cell wall in the mutant to lyophilization. Taken together, the disruption of SPF1 resulted in increased sensitivity to cell wall stresses, including the cell wall agent CFW and physical stresses. The spf1/ mutant shows abnormal cell wall reconstruction and organization We predicted that the increased sensitivity of the spf1/ mutant might be caused by the defects of cell wall organization and consequent alterations of cell wall composition. After removal of cell walls, while approximately 50% wild-type or reconstituted protoplasts survived, only 15% mutant protoplasts reconstructed cell walls (Fig. 5A). Moreover, no significant difference in the sensitivity to zymolyase, an enzyme digesting the fungal cell wall, was observed between the mutant and the wild-type and reconstituted strains (data not shown), indicating that the reduced recovery of the mutant after protoplastation is due to a defective cell wall reconstruction of the mutant rather than a higher damage of the mutant protoplasts. Analysis of the cell wall composition revealed a 3.5-fold increase of soluble proteins and a twofold decrease of both ␤-(1,3)-glucan and ␤-(1,6)-glucan in the mutant as compared to the wild-type and reconstituted strains, but no statistically significant difference in alkali soluble extracts among these strains (Fig. 5B). These results indicated that disruption of SPF1 caused altered cell wall architecture and delayed cell wall reconstruction, which might be associated with the defects in glycosylation and secretory pathway in the mutant. Disruption of SPF1 affects gene expression in the CWI pathway Since the spf1/ mutant displayed hypersensitivity to cell wall stresses and abnormal cell wall constructions, we further

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Fig. 3. Glycosylation defect of Cdc101 (A) and secreted acid phosphatase (B) in the spf1/ mutant. (A) HA-tagged Cdc101 was detected by Western blotting using rabbit antibodies against HA-epitope. (B) Cells were cultured in phosphate-depleted medium to induce acid phosphatase. Total cell proteins were run on non-denaturing PAGE gels, and acid phosphatase was detected using 0.05% 1-naphthyl phosphate plus 0.03% fast blue. The extracts from the wild-type cells in the last line (A) and the first line (B) were treated with EndoH.

hypothesized that the CWI pathway was affected in this mutant. Here, the expression of CWI genes in the mutant was tested. As predicted, expression of ECM331 was up-regulated in the mutant when compared with the wild-type strain with no drug treatment (Fig. 6A). Moreover, PGA13 was up-regulated in the mutant under the treatment of tunicamycin alone or in combination with the calcineurin inhibitor FK506, and DFG5 was up-regulated in the mutant under the treatment of FK506 (Fig. 6A). These indicated that ER

stress caused by disruption of SPF1 or tunicamycin led to cell wall damage and consequent activation of the CWI pathway, and this activation is regulated by calcineurin. Interestingly, in this mutant, CRH11 were down-regulated with no drug treatment or under the treatment of tunicamycin, and ECM331 and DFG5 was downregulated under the treatment of tunicamycin in combination with FK506 (Fig. 6A), suggesting that the expression of CWI genes is controlled by complicated mechanisms in the spf1/ mutant.

Fig. 4. Tests of cell wall stress sensitivity. (A) Sensitivity of the wild-type (WT), spf1/, and reconstituted (spf1/+SPF1) strains to Calcofluor white (CFW). Overnight cultured cells were spotted on YPD plates containing 0, 20 and 100 ␮g/ml CFW, and the plates were incubated at 30 ◦ C for 2 d. (B) Sensitivity of the strains to vortex. Cells were suspended in distilled water and vortexed with glass beads for 1 min. Released proteins were then analyzed by Coomassie protein assay reagent (left) and SDS-PAGE (right 1, WT; 2, spf1/; 3, spf1/+SPF1; M, protein markers, kD). (C and D) Sensitivity of the strains to lyophilization. Cells were incubated on polystyrene plates at 37 ◦ C for 12 h, lyophilized in vacuum desiccators, and observed with a scanning electron microscope. White arrows indicate the collapsed cells. Percentage of collapsed cells was calculated as the number of collapsed cells × 100%/the total number of observed cells and shown in (D). Values represent mean + SD of five independent tests. *Statistically significant differences between spf1/ (or spf1/+SPF1) and WT (unpaired student t-test). P < 0.01.

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Fig. 5. Tests of cell wall reconstruction and cell wall components. (A) Cell wall reconstruction assays of the strains. Cells were treated with zymolyase solution to form protoplasts, plated on YPD agar containing 1.2 M sorbitol with serial dilutions, and incubated for colony counting. Percentage of reconstructed cells was calculated as the number of colonies of zymolyase-treated cells × 100%/the number of colonies of untreated cells. (B) Cell wall composition. The wild-type (WT, white), spf1/ (gray) and reconstituted (spf1/+SPF1, black) strains were cultured and disrupted for cell wall isolation. The relative amounts of soluble cell wall proteins, alkali-soluble extracts, ␤-(1,3)-glucan and ␤-(1,6)-glucan in spf1/ (the gray bars) and spf1/+SPF1 (the black bars) were calculated as compared to the amounts in WT (the white bars). Values represent mean + SD of five independent tests. *Statistically significant differences between spf1/ (or spf1/+SPF1) and WT (unpaired student t-test). P < 0.01.

Disruption of SPF1 affects gene expression related to morphogenesis The abnormal expression of SEC61 and CWI genes implies a pleiotropic effect of SPF1 disruption on gene expression profiling.

Our previous study also showed defects of the spf1/ mutant in morphogenesis, such as hyphal development and biofilm formation (Yu et al., 2012). We wondered whether gene expression related to morphogenesis was also affected by SPF1 disruption. Under hyphaland biofilm-inducing conditions, HWP1, ECE1 and ALS3, all of which

Fig. 6. RT-PCR analysis for expression of the genes related to the CWI pathway (A) and morphogenesis (B, C). (A) Untreated cells (Control) or cells treated with tunicamycin (TN), FK506 or TN plus FK506 were harvested for RNA extraction. RNA was then reverse-transcribed into cDNA. Expression of ECM331, PGA13, DFG5 and CRH11 was analyzed by RT-PCR. (B) Cells were cultured in liquid YPD medium plus 10% FCS to induce hyphal formation, then RNA was extracted and used for analyzing expression of HWP1 (white), ECE1 (gray) and ALS3 (black) by RT-PCR. (C) Cells were incubated in RPMI 1640 medium with polystyrene plates to induce biofilm formation, and expression of the genes were analyzed as described in (B). Values represent mean + SD of five independent assays. *Statistically significant differences between spf1/ (or spf1/+SPF1) and WT (unpaired student t-test). P < 0.01.

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encode components associated with hyphal and biofilm development, were down-regulated in the mutant as compared to the wild-type and reconstituted strains (Fig. 6B and C).

The spf1/ mutant displays decreased flocculation and adhesion Flocculation and adhesion, two important properties of C. albicans relevant to infection and biofilm formation, are conferred by some special cell wall proteins. Since the spf1/ mutant is deficient in cell wall organization and reconstruction, we predicted that this mutant would display irregular flocculation and adhesion properties. As expected, in liquid RPMI 1640 medium, the mutant showed decreased flocculation as compared to the wild-type and reconstituted strains (Fig. 7A). Furthermore, we tested the ability of the mutant to adhere to microplate surface and buccal epithelial cells (BEC). After 4 h of incubation, substantial hyphal and pseudohyphal cells of the control strains were seen attached to the polystyrene surface. In contrast, very little yeast cells were attached to the surface for the mutant (Fig. 7B), with biomass levels 5-fold lower than those for the control strains (Fig. 7D). Similarly, after 1 h of incubation with C. albicans cells, approximately 90% BECs were adhered by hyphal cells of the control strains, whereas only 50% BECs were adhered by yeast cells of the mutant (Fig. 7C and E). Overall, the mutant showed significantly decreased flocculation and adhesion, which might be attributed to the abnormal cell wall architecture and decreased expression of adhesin-encoding genes in this mutant.

Discussion In our previous study, we found that Spf1, a putative ERlocalized Ca2+ ATPase, plays a major role in maintenance of cell calcium homeostasis, tolerance to antifungal agents, morphogenesis and pathogenesis (Yu et al., 2012). Consistent with ER-related phenotypes of the spf1/ mutant and BLASTP analysis of Spf1 with well-known P-type ATPases, Spf1 is demonstrated as a Ptype ATPase, catalyzing the hydrolysis of ATP to ADP and inorganic phosphate. In combination with the ER-related phenotypes of the spf1/ mutant and the fact that Spf1 is an ER-localized protein (Yu et al., 2012), we propose that this protein mediates the active transport of cytoplasmic Ca2+ across ER membrane, serving to maintain high levels of ER Ca2+ . Ca2+ sequestration into the ER is required for regulation of posttranslational modification and protein quality control in the ER (Görlach et al., 2006; Brostrom and Brostrom, 2003). Several key ER chaperones also require the high Ca2+ levels for their normal activity of protein folding (Torres et al., 2011). Here, we further show that the spf1/ mutant has severe defects in glycosylation of both the ER-localized protein Cdc101 and secretory acid phosphatase. A possible explanation is that several ER-localized enzymes essential for glycosylation, similar to ER chaperones, are supported by high levels of Ca2+ . Alternatively, the defect of ER protein folding, caused by chaperone malfunction because of depletion of Ca2+ , may contribute to a delay of normal glycosylation. Thus, the high Ca2+ levels maintained by Spf1 are critical for protein glycosylation in the ER. Moreover, ␤-galactosidase assays revealed a significant decrease in the expression level of SEC61 in the mutant, which may be involved in defects of ER functions. These data demonstrate that Spf1 is indispensible for ER functions, especially protein glycosylation. In addition, the Golgi-localized Pmr1, a Golgi-localized P-type Ca2+ /Mn2+ ATPases, functions in supplying the Golgi with both Ca2+ and Mn2+ and is also required for maintenance of calcium homeostasis and glycosylation (Bates et al., 2005). Thus, Spf1 and Pmr1 are not functionally redundant P-type ATPases. This may be due to

the different functions between the ER and the Golgi in C. albicans physiological processes. Recently, the cell wall has been a focus of attention, mainly attributed to its important role in survival and pathogenesis of C. albicans. A number of reports have shown that maintenance of CWI, including synthesis, delivery, and organization of cell wall components, is controlled by complicated mechanisms (Chaffin, 2008; Scrimale et al., 2009). There is evidence for a link between ER functions and CWI in S. cerevisiae. Unfolded protein response (UPR) pathway, initiated by ER or cell wall stress, is crucial for protein quality control and consequent proper cell wall construction. Uncompensated ER stress caused by disruption of the UPR-related gene IRE1 highly affects CWI (Scrimale et al., 2009). Moreover, Nglycosylation, mainly mediated by the ER-localized proteins Stt3 and Kre5, is required for the synthesis and organization of ␤-(1,6)glucan in the cell wall (Chavan et al., 2003; Yan and Lennarz, 2002). In this study, the spf1/ mutant displays severe CWI phenotypes, including hypersensitivity to cell wall stresses, delayed reconstruction of cell wall, and abnormal cell wall composition. It seems likely that these cell wall consequences in C. albicans are also associated with malfunction of ER. Disruption of SPF1 may cause uncompensated Ca2+ depletion of ER, disturbing such ER functions as protein folding, glycosylation and quality control. Consequently, abundant misfolded, non-glycosylated cell wall proteins are released from ER and incorporated into the carbohydrate network of the cell wall, resulting in the CWI defect. This is emphasized by the fact that soluble proteins abnormally increased in the mutant cell wall. Hence, we demonstrate that ER Ca2+ homeostasis is required for maintenance of CWI in C. albicans. In addition, since many cell wall proteins play important roles in morphogenesis (Chaffin, 2008), the defects in morphogenesis, including hyphal development and biofilm formation (Yu et al., 2012), are supposed to be associated with this damage of CWI in the mutant. The CWI pathway is a well-known signaling cascade that responds to cell wall damage and activates the transcription of CWI genes (Levin, 2005; Bruno et al., 2006). Here, we further assess activation of this pathway in the spf1/ mutant. Expectedly, in this mutant, ECM331 is up-regulated with no drug treatment, and PGA13 and DFG5 are also up-regulated with the treatment of tunicamycin or FK506. However, some of these genes are downregulated in this mutant under the drug treatments. These results may reflect that the regulation of CWI gene expression is not only regulated by the traditional CWI pathway, but also by other mechanisms, such as the calcieneurin signaling pathway. In addition, we demonstrated that the mutant showed abnormal expression of the ER protein SEC61, which is also related to calcineurin signaling. RT-PCR analysis further reveals that the expression of those genes related to morphogenesis, such as HWP1, ECE1 and ALS3, is significantly decreased in the mutant under hyphal- and biofilminducing conditions. It reveals a link between malfunction of the ER and the regulation of expression of morphogenesis-related genes. Collectively, these data indicate that the disruption of SPF1 may provoke a broad transcription response. Genome-wide analysis of gene expression profiles is required to evaluate detailed mechanisms in response to disruption of SPF1 and consequent defects in ER functions. Fungal cell adhesion, an interaction between fungal cells and host surfaces, is conferred by abundant adhesins, a class of specialized cell wall-surface proteins (Verstrepen and Klis, 2006). In C. albicans, several protein families of adhesins, such as Als family and Hwp family, are crucial for this interaction (Butler et al., 2009), and Sfl1 is one of the main transcription factors that negatively regulates flocculation and filamentous growth (Bauer and Wendland, 2007). In this study, the spf1/ mutant displays decreased flocculation and adhesion to polystyrene surface and BEC. When the expression level of SFL1 and adhesin-encoding genes in the mutant

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Fig. 7. Flocculation (A) and adhesion (B–E) analysis of the wild-type (WT), spf1/ and reconstituted (spf1/+SPF1) strains. (A) Flocculation analysis. Cells were incubated with shaking in RPMI1640 medium, vortexed, and photographed. (B and D) Adhesion to polystyrene surface. Cells in RPMI1640 medium were incubated on polystyrene plates at 37 ◦ C for 4 h, washed, and stained with crystal violet. Crystal violet was then extracted with 10% acetic acid and A595 of the extract was determined. (C and E) Adhesion to BEC. C. albicans cells and BEC were mixed and incubated at 37 ◦ C for 4 h. BEC were then fixed, stained with eosin and photographed. The BEC adhered with C. albicans cells were further determined. Percentage of adhered BECs was calculated as the number of adhered BEC × 100%/the number of total BEC. Values represent mean + SD of five independent assays. *Statistically significant differences between spf1/ (or spf1/+SPF1) and WT (unpaired student t-test). P < 0.01.

was examined under flocculation- and adherence-inducing conditions, no difference relative to the control strains was observed (our unpublished data). Therefore, the phenotypes of flocculation and adhesion in the mutant, are primarily attributed to the disturbance of protein modification and quality control in the ER, and this disturbance leads to altered cell wall composition, especially a decrease of functional adhesins. In conclusion, we provide evidence that Spf1, as a P-type ATPase, contributes to essential ER functions, such as protein glycosylation and quality control, and thus supports synthesis and organization of cell wall components required for CWI. We also revealed that ER functions maintained by Spf1 are involved in regulation of gene expression related to CWI and morphogenesis. Hence, these results indicate that Ca2+ homeostasis in the ER is crucial for CWI, which

may be closely associated with morphogenesis, cell adhesion, and virulence of C. albicans.

Acknowledgments We thank Dana Davis (University of Minnesota, USA), Steven Bates (University of Exeter, UK), and Linghuo Jiang (Jiangnan University, China) for generously providing strains and plasmids. We also thank reviewers for critical reading and helpful suggestions. This work was supported by National Natural Science Foundation of China (grant numbers 81171541 and 31070126) and Natural Science Foundation of Tianjin (13JCYBJC20700).

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