Candida Albicans Hom6 Is a Homoserine Dehydrogenase

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ORIGINAL ARTICLE Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion Pei-Wen Tsai a, Chu-Yang Chien a, Ying-Chieh Yeh a, Luh Tung b, Hsueh-Fen Chen a, Tien-Hsien Chang b, Chung-Yu Lan a,c,* a Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan b Genomics Research Center, Academia Sinica, Nankang, Taipei, Taiwan c Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan

Received 21 October 2015; received in revised form 22 February 2016; accepted 9 March 2016 Available online ---

KEYWORDS Abstract Background/Purpose: Candida albicans is a common fungal pathogen in humans. In Candida albicans; healthy individuals, C. albicans represents a harmless commensal organism, but infections can cell adhesion; be life threatening in immunocompromised patients. The complete genome sequence of C. al- Hom6; bicans is extremely useful for identifying genes that may be potential drug targets and impor- homoserine tant for pathogenic virulence. However, there are still many uncharacterized genes in the dehydrogenase; Candida genome database. In this study, we investigated C. albicans Hom6, the functions of protein synthesis which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome proﬁling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Copyright ª 2016, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/).

* Corresponding author. Institute of Molecular and Cellular Biology, National Tsing Hua University, Number 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. E-mail address: [email protected] (C.-Y. Lan). http://dx.doi.org/10.1016/j.jmii.2016.03.001 1684-1182/Copyright ª 2016, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Materials and methods

Candida albicans commensally colonizes skin and mucosal Strains and growth conditions surfaces; however, this commensalism can be disrupted when the immune system is compromised, at which time C. C. albicans strains are listed in Supplementary Table S1. albicans can cause superficial, subcutaneous, and even life- Cells were grown in yeast-extract peptone dextrose (YPD) 1,2 threatening invasive infections. To date, only a small medium (1% yeast extract, 2% peptone, and 2% glucose) or number of antifungals are available, and these drugs target synthetic complete (SC) medium (0.67% yeast nitrogen base 2,3 only a few molecular pathways. Moreover, the persistent with ammonium sulfate, 2% glucose, 0.079% complete use of these drugs has led to drug-resistant C. albicans in supplement mixture of amino acids; MP Biochemicals, 3 clinical settings. Thus, it is critically important to char- Solon, OH, USA). For amino acid-depletion experiments, we acterize different aspects of C. albicans physiology to used synthetic minimal (SM) medium (0.67% yeast nitrogen identify alternative drug targets that can be used to base with ammonium sulfate and 2% glucose) or SCThr (SM develop new antifungal therapies. with 0.069% complete supplement mixture lacking threo- Metabolic pathways are important targets for developing nine; Sunrise Science Products Inc., San Diego, CA, USA). All 4 new antifungals. The sulfur assimilatory (SA) pathways, for reagents were purchased from Sigma-Aldrich (St. Louis, MO, example, are significantly different in humans and fungi. USA) unless otherwise indicated. The yeast Saccharomyces cerevisiae chemically reduces and incorporates inorganic sulfates to synthesize methio- Gene deletion and reintegration nine de novo, whereas humans are unable to utilize inorganic sulfates.4 Additionally, the sequential reactions that To delete C. albicans HOM6, the SAT1-flipper method was convert methionine to homocysteine, cystathionine, and 13 used. Briefly, the 5ʹ- and 3ʹ-flanking regions of HOM6 were cysteine are reversible in most fungi, but in humans, amplified from the SC5314 genome with the primer pairs methionine is an essential amino acid that must be ob- 4 0 CaHOM6-up-F-ApaI/CaHOM6-up-R-XhoI and CaHOM6-dn-F- tained from the diet. Moreover, Adenosine-5 -triphosphate SacII/CaHOM6-dn-R-SacI, respectively. All the primers used (ATP) sulfurylase (Met3), which reduces sulfur for subse- in this study are listed in Supplementary Table S2. These quent incorporation into metabolites, is essential for flanking sequences were independently cloned into plasmid infection by the pathogenic yeast Cryptococcus neofor- pSFS2A to generate pSFS2A-CaHOM6.13 For HOM6 reinte- mans.5 The aspartate metabolic pathway is related to the gration, the 5ʹ-flanking region plus full-length HOM6 were SA pathway and is another potential antifungal target, as amplified with the primer pair CaHOM6-up-F-ApaI/CaHOM6- humans lack enzymes involved in this pathway.6 In S. cer- R2-XhoI. This DNA fragment and the 3ʹ-flanking region of evisiae, aspartate is initially converted to beta-aspartate HOM6 were independently cloned into pSFS2A to generate semialdehyde. This reaction is sequentially catalyzed by pSFS2A-CaHOM6-6. The HOM6 deletion and reintegration aspartate kinase (Hom3) and aspartate semialdehyde de- cassettes were excised by ApaI/SacI digestion from pSFS2A- hydrogenase (Hom2). Beta-aspartate semialdehyde is CaHOM6 and pSFS2A-CaHOM6-6, respectively, and trans- further converted to homoserine by homoserine dehydro- 13 formed into the C. albicans SC5314 strain as described. genase (Hom6). Importantly, homoserine is the intermedi- 7 The HOM6-heterozygous mutant was used for the second ate used to synthesize methionine and threonine. In S. round of cassette integration/excision to generate the cerevisiae, homoserine transacetylase (Met2) converts HOM6-homozygous mutant. To generate the HOM6 reinte- homoserine to O-acetylhomoserine, which is then con- grated strain, the HOM6-homozygous mutant was subjected verted to homocysteine and methionine. Homoserine can to two rounds of integration/excision of the linear HOM6 be also converted to threonine by homoserine kinase (Thr1) reintegrated cassette. and threonine synthase (Thr4),8 and threonine can be used to synthesize isoleucine. Although several studies have Southern blot and reverse transcription polymerase been conducted in S. cerevisiae, the aspartate pathway in pathogenic yeast has not been well characterized. C. chain reaction (RT-PCR) albicans THR1-deleted mutants are serum sensitive, and this phenotype can be suppressed by adding threonine to Genomic DNA and RNA isolation, Southern blot, and RT-PCR 14 the growth medium, suggesting that serum sensitivity re- were performed as described. For RT-PCR assays, the sults from the accumulation of homoserine.9 Additionally, primer pairs HOM6-forward/HOM6-reverse and ACT1- both C. albicans THR1 and C. neoformans MET2 are forward/ACT1-reverse were used for HOM6 and ACT1, required for fungal virulence.9,10 Expression of C. albicans respectively. Hom6 can be induced by amino acid starvation and macrophage phagocytosis.11,12 The function of Hom6 in C. Assay for homoserine dehydrogenase activity albicans, however, is mostly unknown. In this study, we identified functions for C. albicans Cells grown in SM medium were resuspended in 200 mL lysis Hom6. Since mammals lack Hom6, our results provide buffer [100mM Tris-HCl (pH 9.0), 0.6M sorbitol, 1mM phe- important information for developing new antifungal nylmethylsulfonyl fluoride, and 10% glycerol] containing an agents directed toward this enzyme and/or its biosynthetic equal volume of acid-washed glass beads. The mixture was pathway. vortexed 10 times (45 s each) and kept on ice for 30 seconds

Please cite this article in press as: Tsai P-W, et al., Candida albicans Hom6 is a homoserine dehydrogenase involved in protein synthesis and cell adhesion, Journal of Microbiology, Immunology and Infection (2016), http://dx.doi.org/10.1016/j.jmii.2016.03.001 + MODEL Hom6 in C. albicans 3 between each vortex. The protein extract was collected by analyzed at 254 nm using an UA-6 UV/VIS detector (Tele- centrifugation and quantified using a bicinchoninic acid dyne Isco, Inc.). (BCA) protein assay kit (Thermo Scientific Pierce, Rockford, Ribosomes were dissociated from polysomes as IL, USA). described with modifications.16,17 Early exponential-phase Homoserine dehydrogenase activity was measured as cells (OD600 Z 1.0e1.5) were mixed with crushed ice and described.15 Whole-cell extracts (25 mg) were mixed with a centrifuged. The cell pellets were washed twice with 5 mL solution [100mM Tris-HCl (pH 9.0), 2mM NADP, and 50mM L- ribosome-dissociation buffer [1mM Tris-HCl (pH 7.5), 50mM homoserine] and incubated at 30C for 20 minutes. The KCl, and 0.1mM ethylenediaminetetraacetic acid], resus- production of NADPH was measured by reading the absor- pended in 500 mL ribosome-dissociation buffer, and lysed by bance at 340 nm (OD340). A mixture that lacked L-homo- vortexing (as described above). Whole-cell extracts serine was used as a control, and a mixture that lacked (OD260 z 10) were loaded onto a 5% to 21% sucrose gradient protein extract was used as a blank. Homoserine dehydro- and centrifuged (35,000 rpm for 5 h at 4C). The ribosome- genase activity was calculated as: [(OD340 with homoserine dissociation profile was analyzed at 254 nm using a UA-6 e OD340 without homoserine)/6.22] 1000 (nmol NADP/ UV/VIS detector (Teledyne Isco, Inc.). To quantify the mg/min). The value 6.22 represents the absorption coeffi- polysome profile, the area of integration for each peak was cient of NADPH. defined as the area enclosed by the basal lines and the boundary lines using Unicorn 4.00 software (GE Healthcare Amino acid analysis Life Sciences, Taipei, Taiwan). The boundary line was defined by the two points at which the slope of the trace line exhibited the biggest difference with that of the Cells grown in SM medium were centrifuged, washed, and adjacent point. resuspended in 200 mL water containing 1mM phenyl- methylsulfonyl fluoride. The suspension was mixed with glass beads and vortexed 10 times (45 s each), with Adhesion of C. albicans to polystyrene 30 seconds on ice between each vortex. After centrifugation, the protein extract was assayed using the BCA protein To assess cell adhesion to polystyrene, exponential-phase assay kit. cells were suspended in Roswell Park Memorial Institute To quantify intracellular amino acids, a Pico-Tag work (RPMI)-1640 medium at a concentration of 6 107 cells/ station and a 2796 bioseparations module (Waters Corpora- mL. Next, 250 mL of cell suspension was loaded into each tion, Milford, MA, USA) were used. Briefly, protein samples well of a 24-well plate and incubated at 37C for 30 mi- were freeze dried, resuspended in ethanol:water:triethyl- nutes with shaking (100 rpm). Cells were then washed amine [2:2:1 (v/v)], and then freeze dried again. The samples twice with phosphate-buffered saline (PBS) and analyzed were then incubated with derivatization reagent [phenyl- for the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H- isothiocyanate in ethanol:water:triethylamine, 1:7:1:1 (v/ tetrazolium-5-carboxanilide (XTT) reduction.18 Absor- v)] for 20 minutes at room temperature and then dried. Dil- bance at 490 nm was determined using a microplate uents [0.07% Na2HPO4 (pH 7.4) and 5% acetonitrile] were reader, and data were normalized to the wild-type added, and the samples were assayed. Levels of intracellular control. amino acids were normalized to the number of cells. Statistical analysis Polysome profiling Differences between groups were statistically analyzed Cells grown in YPD (OD600 Z 1.0e1.5) were transferred to using the two-tailed Student t test. Differences were SC or SCThr medium for an additional 2 hours. Immedi- considered significant at p < 0.05. ately before harvesting cells, 2 mL of 10 mg/mL cycloheximide and 1/3 volume (w67 mL) of crushed ice were added to the cultures. Cells were collected, washed twice with Results 5 mL ice-cold lysis buffer [10mM Tris-HCl (pH 7.5), 100mM C. albicans NaCl, 30mM MgCl2, 100 mg/mL cycloheximide, 200 mg/mL Hom6 is a homoserine dehydrogenase heparin, and 0.2% diethylpyrocarbonate (DEPC)], and resuspended in 500 mL ice-cold lysis buffer. The cells were In the Candida Genome Database (http://candidagenome. mixed with glass beads and lysed by vortexing six times org), orf19.2951 is named HOM6, but its function is mostly (30 s each), with 30 seconds on ice between each vortex. uncharacterized. In this work, we first aligned/compared Cell lysates were collected by centrifugations (6500g for its amino acid sequence between C. albicans and S. cer- 5 min at 4C, followed by centrifugation again at 9200g for evisiae using ClustalW2 (http://www.ebi.ac.uk/Tools/ 10 min at 4C) and stored at 80C. Sucrose was dissolved msa/clustalw2/), which revealed 64% amino acid similar- in a solution containing 50mM Tris-acetate (pH 7.0), 50mM ity and 79% identity (Fig. 1). Additionally, searching the C. NH4Cl, 12mM MaCl2, 1mM DTT, and 0.1% DEPC, and a 7% to albicans sequence against the Prosite database (http:// 50% sucrose density gradient was prepared using a Foxy Jr. prosite.expasy.org/) indicated that residues 199e221 fraction collector with a Tris pump (Teledyne Isco Inc., represent the homoserine dehydrogenase signature. Lincoln, NE, USA). Cell extracts (OD260 Z 1020) were To functionally characterize C. albicans Hom6, we loaded onto the sucrose gradient, centrifuged with a generated HOM6-deleted (HOM6/hom6D and hom6D/ Beckman SW-41 rotor (21,000g for 3.5 h at 4C), and hom6D) and HOM6-reintegrated (hom6D/hom6D::HOM6

Figure 1. Amino acid sequence alignment of Candida albicans and Saccharomyces cerevisiae Hom6p. The alignment was performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Black shading indicates identities, and grey shading indicates similarities. and hom6D::HOM6/hom6D::HOM6) strains. The successful proliferation in different liquid media. In amino aciderich strain constructions were veriﬁed by Southern blot and RT- YPD medium, there was no signiﬁcant differences in cell PCR (Fig. 2A). To determine whether C. albicans Hom6 is a proliferation among the strains tested (data not shown). homoserine dehydrogenase, enzymatic activity was However, Hom6-homozygous mutants exhibited a growth measured using L-homoserine as a substrate and NADP as a delay when starved for amino acids (Fig. 3). These results cofactor. Almost no homoserine dehydrogenase activity was suggested that Hom6 plays a role in cellular responses to detected for HOM6-homozygous mutants as compared with amino acid starvation, perhaps by helping to synthesize the wild-type variant (Fig. 2B). When both HOM6 alleles certain amino acids. were reintegrated, homoserine dehydrogenase activity was To test whether C. albicans Hom6 is involved in amino largely restored (Fig. 2B). acid biosynthesis, a spot assay was performed. HOM6-homozygous mutants exhibited a severe growth defect in e C. albicans Hom6p is involved in the response to amino acid starved SM medium as compared with that e amino acid starvation growth observed in amino acid rich YPD medium (Fig. 4). HOM6 reintegration rescued the growth defect observed in the HOM6-homozygous mutants (Fig. 4). Moreover, supple- Amino acid starvation-induced Hom6 expression11 raises a menting SM agar with methionine did not restore wild-type possibility that C. albicans Hom6 is involved in cellular levels of growth to HOM6-homozygous mutants, whereas response to amino acid starvation. We analyzed cell

Figure 2. Hom6p possesses homoserine dehydrogenase activity in Candida albicans. (A) Upper panel shows a Southern blot of the HOM6 locus for the indicated strains. The 4-kb fragment contains intact HOM6, whereas a 2.8-kb fragment indicates the HOM6 deletion. Lower panel shows the detection of HOM6 RNA by RT-PCR in the indicated strains. Two independent clones of each strain were used for the assays. ACT1 was used as the RT-PCR control; and (B) homoserine dehydrogenase activity was measured for each strain. HOM6-homozygous mutants had very little activity as compared with wild-type cells. The activity was rescued in the HOM6- reconstituted strain. Results are the mean SD of three independent assays. **p < 0.01. HSD Z homoserine dehydrogenase; RT- PCR Z reverse transcription polymerase chain reaction; SD Z standard deviation.

Figure 3. HOM6-homozygous mutants show a severe growth delay when starved for amino acids. Cells were grown in SM medium, and samples were collected at different times. Results are the mean SD of three independent assays. SD Z standard deviation; SM Z synthetic minimal medium. threonine supplementation partially restored this growth alone. These results suggested that C. albicans Hom6 par- phenotype (Fig. 4). Importantly, HOM6-homozygous mu- ticipates in the biosynthesis of methionine and threonine. tants grew at wild-type levels when both threonine and To further investigate the role of Hom6 in amino acid methionine were added to SM or homoserine was added biosynthesis, levels of intracellular amino acids were

Figure 4. Candida albicans Hom6 affects methionine and threonine synthesis. HOM6-homozygous mutants did not grow when starved for amino acids (SM), but growth was recovered by adding threonine plus methionine, or homoserine alone. Cells in the early exponential phase of growth (3 107 cells/mL) were serially diluted with PBS and then spotted on agar plates as indicated. PBS Z phosphate-buffered saline; SM Z synthetic minimal medium.

Table 1 Intracellular concentrations of threonine, methionine, isoleucine, and glycine (nmol/109 cells) for cells in amino acid-starvation conditions. 1 h in SM medium 2 h in SM medium Wild type hom6D/ hom6D::HOM6/ Wild type hom6D/ hom6D::HOM6/ hom6D hom6D::HOM6 hom6D hom6D::HOM6 Thr 17.84 0.49 ND 20.13 0.54 21.41 3.65 ND 21.37 1.23 Met 4.65 0.13 4.30 0.46 5.98 0.03 5.01 0.84 4.21 0.04 5.82 0.62 Ile 9.86 0.07 3.35 0.39 10.14 0.49 9.24 1.01 4.11 0.37 10.13 0.47 Gly 38.86 1.39 23.86 3.68 45.53 0.40 45.33 5.03 23.45 0.95 51.85 2.17 Gly Z glycine; Ile Z isoleucine; Met Z methionine; ND Z not detected; SM Z synthetic minimal medium.; Thr Z threonine.

measured after 1 hour or 2 hours of growth in SM medium. C. albicans Hom6 plays a role in C. albicans Intracellular threonine was not detected in HOM6-homo- adhesion zygous mutants (Table 1), and levels of isoleucine and glycine, which are synthesized from threonine, were Using a Hom6-green ﬂuorescent protein (GFP) construct, reduced, while levels of methionine were slightly lower in the fusion protein was found in both the cytosolic and cell- HOM6-homozygous mutants (Table 1). Intracellular levels of wall fractions (Supplementary Fig. S1). Therefore, we other amino acids were also affected in HOM6-homozygous tested whether Hom6 could affect cell adhesion. HOM6- mutants (Supplementary Table S3). Thus, Hom6 affected homozygous mutants adhered poorly to polystyrene as both amino acid biosynthesis via the aspartate pathway and compared with other strains (Fig. 6), indicating a role for the size of the intracellular amino acid pool. Hom6 in C. albicans adhesion.

C. albicans Hom6 affects translation Discussion

Low levels of intracellular amino acids can downregulate Here, we showed that Hom6 may participate in the biosyn- translation. To determine the effects of HOM6 on trans- thesis of threonine and methionine through the aspartate lation, we assessed cell susceptibility to translation in- pathway. Wild-type and HOM6-homozygous mutant strains hibitors. HOM6-homozygous mutants were hypersensitive grew at similar rates in amino aciderich YPD medium (data to hygromycin B and cycloheximide as compared with wild- not shown); however, in amino acidedepleted SM medium, type, HOM6-heterozygous, and HOM6-reintegrated strains HOM6-homozygous mutants exhibited slow growth (Fig. 3). (Fig. 5A). Supplementing the SM medium with both methionine and To further investigate whether C. albicans Hom6p af- threonine rescued the growth of HOM6-homozygous mutants fects translation, we performed polysome profiling.19 (Fig. 4). Addition of threonine alone to the SM medium Wild-type and HOM6-homozygous mutant strains showed partially rescued the growth phenotype, whereas addition very similar polysome profiles in threonine-rich SC me- of methionine alone did not (Fig. 4). One explanation is that dium (Fig. 5B). In SC medium lacking threonine (SCThr), methionine may be synthesized by the aspartate pathway however, polysome levels were lower in HOM6-homozy- and alternative routes, such as the SA pathway and through gous mutants as compared with wild-type cells (Fig. 5B). cysteine metabolism.4,20,21 This hypothesis is partially sup- Simultaneously, the levels of 40S and 60S ribosomal sub- ported by our analysis of intracellular amino acids. After units increased in HOM6-homozygous mutants as amino acid starvation, similar levels of methionine were compared with wild-type variants (Fig. 5B). These results detected in wild-type, HOM6-homozygous, and HOM6-rein- suggested that HOM6 deletion affected translation and tegrated strains. In contrast, threonine was undetectable in led to the accumulation of free ribosomes, as indicated by HOM6-homozygous mutants (Table 1). Similarly, isoleucine the increases in 40S and 60S subunits. Alternatively, the and glycine can be generated from threonine or through the increase in free ribosomal 40S and 60S subunits may citraconate pathway (isoleucine)22 and serine metabolism represent an indirect effect of HOM6 deletion in the event (glycine).20 Moreover, methionine synthesis involves biofilm that HOM6 affects the synthesis of other ribosomal formation in C. albicans.23 Deletion of ECM17, which en- subunits. codes a sulfite reductase involved in methionine/cysteine To differentiate between these possibilities, total levels biosynthesis, reduces cell adhesion and biofilm formation in of 40S and 60S subunits were determined by measuring both C. albicans.23,24 Our results highlighted complex in- available free ribosomes and subunits obtained from the teractions between different pathways of amino acid dissociation of existing polysomes. Total amounts of 40S biosynthesis and possible links between host amino acid and 60S subunits did not differ between HOM6-homozygous levels and C. albicans infection. mutants and wild-type variants (Fig. 5C), suggesting that HOM6-homozygous mutants were hypersensitive to increases in free 40S and 60S subunits resulted from a hygromycin B and cycloheximide (Fig. 5A). A previous study translational defect (i.e., they were released from reported a correlation between hygromycin B sensitivity in polysomes). S. cerevisiae and defects in protein glycosylation.25

Figure 5. HOM6 affects Candida albicans translation. (A) HOM6-homozygous mutants were hypersensitive to hygromycin B and cycloheximide. Cells in the early exponential phase of growth were serially diluted with PBS as indicated and then spotted onto YPD plates with or without 150 mg/mL hygromycin B or 2 mg/mL cycloheximide; (B) HOM6 deletion affected polysome levels. Wild-type or HOM6-homozygous mutant cells in the exponential phase of growth were transferred into SC (top panel) or SCThr (bottom panel) medium and incubated for 2 hours. The polysome proﬁle shows ribosomal subunits (40S and 60S), monosomes (80S), and polysomes from different fractions of the sucrose gradient. Representative plots from two (upper) or three (lower) independent experiments with similar results are shown. The area under each peak (mean SD) is shown. *p < 0.05; and (C) Total levels of 40S and 60S ribosomal subunits (including those derived from dissociated polysomes) were similar between wild-type and mutant cells in SCThr medium. A representative plot from three independent experiments with similar results is shown. The area under each peak (mean SD) is indicated. PBS Z phosphate-buffered saline; SC Z synthetic complete medium; SCThr Z synthetic complete medium lacking threonine; SD Z standard deviation; YPD Z yeast-extract peptone dextrose.

Notably, glycosylation can modulate the structure and infection.31,32 Here, C. albicans adhesion to polystyrene function of cell-surface proteins in yeast. Interestingly, C. was studied in amino acid-containing RPMI medium, albicans Hom6-GFP fusion protein localized to both the resulting in reduced adhesion of HOM6-deleted strains cytosolic and cell-wall fractions. The localization of C. (Fig. 6). Since wild-type and HOM6-deleted strains grew at albicans Hom6 within the cell-wall fraction was also iden- similar rates in amino acid-rich YPD medium, the reduced tified by several proteomic studies.26,27 Moreover, S. cer- adhesion observed in the mutant strain may not be due to a evisiae Hom6 localizes to the cytosol and the nucleus.28 growth defect. Threonine is the key residue for O-linked Proteins that localize to multiple cellular compartments mannosylation,33 and threonine deficiencies likely reduce commonly perform more than one function. For example, levels of mannoproteins, which are thought to be important C. albicans Adh1 protein localizes to the cell surface of for C. albicans adhesion and virulence.34 Because Hom6 is yeast-form cells and to the cytosol of hyphal cells.29,30 Adh1 involved in threonine and methionine biosynthesis, the is an alcohol dehydrogenase involved in biofilm formation possible mechanisms by which Hom6 affects O-linked and possible adhesion to the extracellular matrix during mannosylation and adhesion require further study.

(Academia Sinica; AS-99-TP-B20 and AS-103-TP-B12), and Academia Sinica. LT was supported by an Academia Sinica postdoctoral fellowship.

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