Fungal Genetics and Biology xxx (2015) xxx–xxx

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The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans Guobo Guan a,1, Haitao Wang b,1, Weihong Liang a,c,1, Chengjun Cao a,c, Li Tao a, Shamoon Naseem d, James B. Konopka d, Yue Wang b,e, Guanghua Huang a,⇑ a

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Proteos, Singapore 138673, Singapore University of Chinese Academy of Sciences, Beijing 100049, China d Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA e Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 138673, Singapore b c

a r t i c l e

i n f o

Article history: Received 2 October 2014 Revised 12 January 2015 Accepted 17 January 2015 Available online xxxx Keywords: Candida albicans Mcu1 Carbon source utilization Por1 Virulence Filamentation

a b s t r a c t The fungus Candida albicans is both a pathogen and a commensal in humans. The ability to utilize different carbon sources available in diverse host niches is vital for both commensalism and pathogenicity. N-acetylglucosamine (GlcNAc) is an important signaling molecule as well as a carbon source in C. albicans. Here, we report the discovery of a novel gene MCU1 essential for GlcNAc utilization. Mcu1 is located in mitochondria and associated with multiple energy- and metabolism-related proteins including Por1, Atp1, Pet9, and Mdh1. Consistently, inactivating Por1 impaired GlcNAc utilization as well. Deletion of MCU1 also caused defects in utilizing non-fermentable carbon sources and amino acids. Furthermore, MCU1 is required for filamentation in several inducing conditions and virulence in a mouse systemic infection model. We also deleted TGL99 and GUP1, two genes adjacent to MCU1, and found that the gup1/gup1 mutant exhibited mild defects in the utilization of several carbon sources including GlcNAc, maltose, galactose, amino acids, and ethanol. Our results indicate that MCU1 exists in a cluster of genes involved in the metabolism of carbon sources. Given its importance in metabolism and lack of a homolog in humans, Mcu1 could be a potential target for developing antifungal agents. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Candida albicans is a major fungal pathogen of humans, which causes not only cutaneous infections but also life-threatening disease in immunocompromised individuals (Calderone and Fonzi, 2001; Pfaller and Diekema, 2007). This fungus is also a member of the human microbiota and exists in the oral cavity, lower gastrointestinal tract and female genital tract of healthy people (Odds, 2010). To survive and cause infections, C. albicans must adapt to highly diverse environmental niches in the host. Nutrient availability in the host, especially the availability of carbon sources, is totally different from routine growth media used in the laboratory. Therefore, the ability of utilizing different carbon sources is of critical importance for both commensal and pathogenic lifestyles of C. albicans.

⇑ Corresponding author. Fax: +86 10 64806133. 1

E-mail address: [email protected] (G. Huang). These authors contributed equally to this work.

The preferred carbon source, glucose, is often limited in natural niches of C. albicans. N-Acetylglucosamine (GlcNAc) is a component of bacterial and fungal cell walls and the monomeric unit of chitin, which is the second most abundant carbon source in nature (Duo-Chuan, 2006; Kirn et al., 2005; Konopka, 2012). GlcNAc released by commensal bacteria in the mammalian gut is thought to be the major carbon nutrition for C. albicans (Chang et al., 2004; Kirn et al., 2005). Consistent with this hypothesis, genes of the catabolic pathway involved in the utilization of GlcNAc are upregulated in the GUT form (a commensal form) of C. albicans (Pande et al., 2013). The GlcNAc catabolic pathway genes including HXK1 encoding a GlcNAc kinase (Kumar et al., 2000), DAC1 encoding a GlcNAc-6-p deacetylase (Kumar et al., 2000; Yamada-Okabe et al., 2001), and NAG1 encoding a GlcNAc-6-p deaminase (Kumar et al., 2000; Natarajan and Datta, 1993), are clustered in chromosome 6. These three genes are all required for utilization of GlcNAc as a carbon source. The plasma membrane protein Ngt1 is a GlcNAc transporter and required for efficient GlcNAc uptake in C. albicans (Alvarez and Konopka, 2007). Interestingly, the budding yeast Saccharomyces cerevisiae does not contain a

http://dx.doi.org/10.1016/j.fgb.2015.01.006 1087-1845/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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homolog of Ngt1 possibly due to its adaptive evolution to a different environmental habitat, where GlcNAc is not a primary carbon source (Alvarez and Konopka, 2007). It is also well known that GlcNAc acts as a signal molecule in the regulation of phenotypic transitions in C. albicans (Huang, 2012; Konopka, 2012). GlcNAcinduced filamentation is independent on its metabolism but dependent on the Ras1-cAMP-PKA signaling pathway (Biswas et al., 2007; Cassone et al., 1985; Huang et al., 2010; Naseem et al., 2011). Chitin is a major component of fungal cell wall. Mammals and plants cannot synthesize chitin, although they do secrete chitinases that degrade fungal chitin (Duo-Chuan, 2006). Lysine motifs (LysMs) have been known as chitin-binding modules (Buist et al., 2008; de Jonge and Thomma, 2009). To avoid triggering host immunity, plant pathogenic fungi secrete LysM domain-containing proteins, also known as LysM effectors, to sequester chitin oligosaccharides, the degradation products of fungal cell wall during infections. For example, the effector protein Avr4 of the tomato fungal pathogen Cladosporium fulvum shields chitin on the cell wall and prevents degradation by the host chitinases (van den Burg et al., 2004, 2003). C. albicans adopts a different strategy to prevent pathogen-specific response in the host. Its cell wall is coated with an outer layer of mannoproteins covalently linked to the inner beta-glucan layer, which can be recognized by the immune system (Wheeler et al., 2008). We wondered whether there are any LysM effector proteins in C. albicans, which could play a similar role in preventing chitininduced host immunity. In the present study, we identify a pseudogene (psorf19.4984) encoding a putative LysM domain-containing protein in C. albicans by performing a BLAST search against the Candida Genome Database (CGD, Assembly 22, http://www.candidagenome.org) (Muzzey et al., 2013). While deletion of the putative open reading frame (ORF) region of psorf19.4984 has no obvious effect on cell growth, deletion of its promoter region (a part of the intergenic region between MCU1 or orf19.4983 and psorf19.4984) leads to serious defect in utilization of GlcNAc. We, therefore, identify MCU1 (Multiple Carbon source Utilizer 1, orf19.4983) as an essential gene for utilizing GlcNAc, amino acids, and non-fermentable sugars as carbon sources. The mcu1/mcu1 null mutant also shows filamentous growth defect and attenuated virulence in a mouse systemic infection model. 2. Materials and methods 2.1. Strains and culture conditions The strains used in this study are listed in Table S1. Lee’s glucose (Huang et al., 2010) and YPD medium (20 g/L glucose, 20 g/L peptone, 10 g/L yeast extract) were used for routine growth of C. albicans. Lee’s glucose, Lee’s GlcNAc (Huang et al., 2010), Lee’s glucose + GlcNAc (with 6.25 g/L glucose and 6.25 g/L GlcNAc), and Spider medium, supplemented with 5 lg/ml phloxine B were used for filamentous growth. Spot growth assay media: yeast nitrogen base (YNB with (NH4)2SO4) agar containing 2% GlcNAc, 2% glucose, 2% maltose, 2% galactose, 2% sucrose, 2% fructose, 1% galactose plus 1% GlcNAc, 1 mM arginine, 7 amino acids (1 mM of alanine or A, arginine or R, glutamine or Q, glutamic acid or E, asparagine or N, proline or P, and serine or S), 2% mannitol, 2% sodium citrate, 2% sodium pyruvate, 4% acetate, 6% ethanol, or 4% glycerol. 2.2. Construction of plasmids The primers used are listed in Table S2. A fragment containing the open reading frame (ORF) and 326 bp of the 30 -untranslated

region (UTR) of MCU1 was amplified by PCR from genomic DNA of SC5314. The PCR product was digested with SalI and BglII and subcloned into the plasmid pNIM1 to replace the GFP fragment, generating the ectopic expression plasmid pNIM-MCU1. A fragment containing only the ORF region of MCU1 was amplified by PCR from genomic DNA of SC5314. The PCR product was digested with SalI and subcloned into the plasmid pNIM1, generating the MCU1-GFP fusion expression plasmid pNIM-MCU1-GFP (TETp-MCU1-GFP-SAT1). To construct the MCU1-reconstituted plasmid pSFS2a-MCU1pMCU1, a fragment of the 30 -UTR of MCU1 and a fragment containing the MCU1 ORF plus 479 bp of the promoter region were sequentially inserted into the SacII/SacI and ApaI/XhoI sites of the plasmid pSFS2A (Reuss et al., 2004). The GFP coding sequence of the plasmid pGFPutr (Wang et al., 2012) was replaced with the 6  Myc sequence at the XhoI/ClaI site, generating the plasmid pMYCutr. To construct the Myc-tagged ATP1 and POR1 expression plasmids, a 30 -fragment of ATP1 or POR1 was amplified from the genomic DNA of C. albicans by PCR. The PCR products were digested with KpnI and XhoI and subcloned into pMYCutr, generating the Myc-tagged ATP1 and POR1 expression plasmids. These constructs were linearized at the unique HpaI (ATP1) or HindIII site (POR1) before used for C. albicans transformation. 2.3. Construction of C. albicans mutant strains To generate the ps19.4984 / (a) mutant, the primers 49845DR and 4984-3DR were used to amplify the HIS1 and ARG4 selectable markers from the plasmids pGEM-HIS1 and pRS-ARG4DSpeI (Wilson et al., 1999), respectively. PCR products of the ARG4 marker were used for deletion of the first copy of ps19.4984, generating the 19.4984 +/ heterozygous mutant. PCR products of the HIS1 marker were used for deletion of the second copy of ps19.4984, generating the homozygous null mutant 19.4984 / (a). To construct the 19.4984 / (b) mutant, the second copy of the putative ORF region of ps19.4984 was deleted with PCR products of the HIS1 marker in the 19.4984 +/ heterozygous mutant. The primers 4984-5DRb and 4984-3DR were used for PCR. The MCU1 gene was disrupted by using the fusion PCR assay (Noble and Johnson, 2005). To delete the first copy of MCU1, the strain BWP17 was transformed with fusion PCR products of the ARG4 gene flanked by the MCU1 gene 50 - and 30 -fragments. The plasmid pRS-ARG4DSpeI was used as a PCR template. The second copy of MCU1 was deleted with fusion PCR products of the HIS1 marker flanked by the MCU1 gene 50 - and 30 -fragments. The plasmid pGEM-HIS1 was used as a PCR template (Wilson et al., 1999). The primers used are listed in Table S2. The MCU1 reconstituted strain (mcu1/mcu1+MCU1p-MCU1) was constructed by transforming the mcu1/mcu1 mutant with the ApaI/ SacI digested pSFS2a-MCU1p-MCU1 plasmid. Fusion PCR assays were used to generate the gup1/gup1, tgl99/ tgl99 and por1/por1 mutants in the wild type strain SN152 (Noble and Johnson, 2005). Briefly, the HIS1 and CmLEU2 markers flanked by GUP1 gene, TGL99 gene or POR1 gene 50 - and 30 -fragments were amplified with fusion PCR assays. The plasmids pGEM-HIS1 and pSN40 were used as the PCR templates. Primers used for PCR are listed in Table S2. PCR products of the HIS1 and CmLEU2 markers were sequentially transformed into SN152, generating the mutants. 2.4. Protein extraction and Western blotting Lysis buffer: 50 mM Tris (pH 7.5), 1 mM EDTA, 1% NP-40, 150 mM KCl, EDTA-free protease inhibitor mix (Roche), and phosphatase inhibitor cocktail (2 mM NaF, 4 mM sodium orthovanadate, 0.2 mM Na4P2O7, 2.3 mM sodium molybdate and

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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0.2 mM b-glycerol phosphate). Cells were suspended in the lysis buffer and cell lysates were prepared using the bead-beating assay. Protein concentration of the lysate was determined using bicinchoninic acid (BCA) protein assay (Galen). After SDS–PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis), proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was immersed in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% non-fat dry milk for 1 h at room temperature, followed by incubation in TBST with 1% milk containing the primary antibody and secondary antibody conjugated with hydrogen peroxidase (HRP) consecutively for 1 h each. The target protein was visualized by using a chemiluminescence (ECL) system. Anti-Myc antibodies were purchased from Santa Cruz (USA). Anti-GFP antibodies were purchased from Roche (USA). 2.5. Immunoprecipitation (IP) and Co-IP Generally, 500 ll of the cell pellet was suspended in 1 volume of lysis buffer containing 50 mM Tris (pH 7.5), 1 mM EDTA, 1% NP-40, 150 mM KCl, and EDTA-free protease inhibitor mix (Roche). Cells were lysed using Tomy Microsmash by five rounds of beadbeating at 4 °C (50-s beating at 5, 500 rpm plus a 2-min cooling process on ice for each round). The supernatant of cell lysates was collected after centrifugation at 13,200g for 30 min at 4 °C. Antibody-conjugated beads (GFP-Trap) (30–40 ll; ChromoTekGermany) were washed twice with 500 ll lysis buffer and mixed with the cell lysate for incubation at 4 °C for 3 h. The beads were washed three times with the lysis buffer and then boiled for 5 min in 1  loading buffer. Proteins in loading buffer were separated by SDS–PAGE and probed by Western blotting using appropriate antibodies. 2.6. Analysis of Mcu1-associated proteins To identify Mcu1-associated proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), cells of the MCU1GFP fusion strain in 1 L of exponential phase cultures were collected and re-suspended in 12 ml of lysis buffer before beadbeating assays. The cell lysates were incubated with 200 ll of aGFP-coupled beads (GFP-Trapl; ChromoTek-Germany) at 4 °C for 6 h. The beads were washed 4 times with 1 ml of the lysis buffer. Proteins were eluted by 2% TFA (v/v) and the dry samples were used for LC-MS/MS assays. Cells of the strain transformed with the plasmid pNIM1 were used as the control. 2.7. Microscopy Cells collected from liquid cultures or from nutrient agar plates as indicated in the main text were used for morphological analysis. Differential interference contrast (DIC) optics was used for standard cell morphology detection. Cells used for subcellular localization analysis were grown in liquid Lee’s glucose medium for 16 h in the absence or presence of 40 lg/ml doxycycline. Mitochondrial staining assays with the dye Mito-Tracker Red CMX Ros (Molecular Probes, Invitrogen Inc.) were performed according to the manufacturer’s protocol. 2.8. Virulence experiments All animal experiments were performed according to the guidelines approved by the Animal Care and Use Committee of the Institute of Microbiology, Chinese Academy of Sciences. The present study was approved by the Committee. Systemic infection of mice was performed according to our previous studies with slight modifications (Du et al., 2012). Briefly, female BALB/c mice aged 4–5 weeks were used for systemic infections. Ten mice were used

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for injection of each strain. 1  106 cells in 200 ll of 1  PBS were injected into each mouse via the tail vein. For fungal burden assays, 1  106 cells of each strain were injected into a mouse via tail vein. Three mice were used for each strain. Mice were sacrificed at 24 h after injection. Kidneys and spleens were homogenated, diluted in 1  PBS and then plated on YPD medium containing 100 lg/ml ampicillin and 50 lg/ml kanamycin for two days of growth at 37 °C for CFU assays. 3. Results 3.1. Identification of a putative LysM domain-containing protein in C. abicans We set out to identify LysMs-domain containing proteins, which may bind chitin or GlcNAc and play a role in their metabolism in C. albicans. A BLAST search against the CGD database (http://www.candidagenome.org) with the amino acid sequences of several previously identified LysM domain-containing proteins (Buist et al., 2008) was performed. The putative protein p19.4984, encoded by the ‘‘orf19.4984’’ gene in C. albicans, was found to carry a LysM domain. Moreover, this putative protein contains several other conserved domains or motifs, including a chitin-binding domain (Cht_BD), a glycosyl hydrolase domain (Glyco_18), and a putative signal peptide sequence (Fig. 1A). The sequence feature indicates that p19.4984 could be involved in the utilization of chitin or GlcNAc as a carbon source. We further found that two metabolism-related genes TGL99 (orf19.4982, with a predicted role in lipid metabolism) and GUP1 (orf19.4985, with a predicted role in glycerol uptake) are clustered in the same locus in Chromosome 6 (Fig. 1B). Functional descriptions and protein sequence features of p19.4982, p19.4983, p19.4984, and p19.4985 are shown in Fig. 1C. Interestingly, p19.4982, p19.4983, and p19.4985, but not p19.4984, are conserved in the related species Candida dubliniensis and Candida tropicalis (Fig. S1). 3.2. Mcu1 can rescue the defect of the psorf19.4984D/D mutant in utilizing GlcNAc Genomic DNA of the orf19.4984 gene was amplified with PCR and subcloned into the pGEM-T vector (Promega, Inc) for sequencing. We found that one allele of this gene has a nonsense mutation (a TGA stop codon) at nucleotides 40–42 and the other allele carries a frameshift mutation at nucelotide 60. Therefore, both alleles of this gene cannot make a functional protein. Recently released assembly 22 of the SC5314 genome sequence also identified this pseudogene (CGD, Assembly 22, http://www.candidagenome.org) (Muzzey et al., 2013). The orf19.4984 also encodes a pseudogene in the clinical strain WO-1 (Butler et al., 2009). Hereafter, the pseudogene orf19.4984 is re-named as psorf19.4984. Since the putative protein p19.4984 was predicted to play a role in utilizing chitin or GlcNAc, we wondered whether the locus of this pseudogene still has some function. As expected, deletion of one allele of psorf19.4984 (ORF+214 bp of promoter region + 48 bp of 30 -UTR) had no obvious effect on cell growth on either Lee’s glucose or Lee’s GlcNAc medium (Fig. 2B). The same region of the second copy psorf19.4984 was subsequently deleted, generating the homozygous deletion mutant (a). Surprisingly, this mutant demonstrated serious growth defect on Lee’s GlcNAc medium (Fig. 2B). The intergenic region between psorf19.4984 and MCU1 (orf19.4983) is only 363 bp. We predicted that this intergenic sequence could be a dual promoter for these two genes/pseudogenes. Deletion of 214 bp of this region in mutant (a) might have affected the expression of MCU1. To test this idea, we generated a different mutant (b), in which only the ORF plus 48 bp of

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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Fig. 1. Identification of a pseudogene (ps19.4984) encoding a putative LysM domain containing protein in C. albicans. (A) Domain architecture of the orf19.4984 protein according to its putative protein sequence. (B) Four genes involved in the utilization of different carbon sources are clustered on the chromosome (Chr_1). orf19.4984 was identified as a pseudogene by DNA sequencing. (C) Functional descriptions and conserved domains/motifs of the proteins encoded by the four metabolism-related genes.

30 -UTR region of the second copy of psorf19.4984 was deleted (Fig. 2A). We found that the mutant (b) showed no growth defect on Lee’s GlcNAc medium. These results suggest that the pseudogene psorf19.4984 does not play a role in the utilization of GlcNAc, while the expression level of MCU1 could be important in this process. To prove this hypothesis, we then generated a regulatable TETpcontrolled MCU1-expression plasmid (pNIM-MCU1) (Park and Morschhauser, 2005). As shown in Fig. 2C, ectopic expression of MCU1 in the WT strain had no obvious effect on colony growth. However, ectopic expression of MCU1 could rescue the growth defect of the mutant (a) of psorf19.4984 on Lee’s GlcNAc medium under both non-inducing (without doxycycline) and inducing condition (with 100 lg/ml doxycycline). These results suggest that (i) deletion of 214 bp of the intergenic region affected the expression of MCU1, (ii) Mcu1 plays a critical role in utilizing GlcNAc, and (iii) the endogenous expression level of MCU1 could be very low. The low expression level of MCU1 was confirmed by testing the fluorescence signal of a GFP-tagged-Mcu1 (data not shown). 3.3. Mcu1 is essential for the utilization of GlcNAc as a carbon source We next deleted the MCU1 gene in a WT strain of C. albicans (BWP17). As shown in Fig. 3, deletion of one copy of MCU1 did not affect colony growth on Lee’s glucose and Lee’s GlcNAc media at both 25 and 37 °C. However, deleting both copies of MCU1 completely blocked cell growth on Lee’s GlcNAc medium and reduced the growth rate on Lee’s glucose medium at both 25 and 37 °C. To verify that the growth defect was due to the inactivation of MCU1, a copy of MCU1 was re-integrated into the original locus

of the mutant. The re-constituted strain grew as normally as the WT control (Fig. 3). These results suggest that MCU1 is an essential gene for the utilization of GlcNAc as a sole carbon source. 3.4. Mcu1 is located in mitochondria and associated with a number of mitochondrial proteins involved in respiratory metabolism Mcu1 contains a transmembrane domain suggesting that it could be a membrane protein (Fig. 1). To examine the subcellular localization of Mcu1 in C. albicans, we constructed a TETp-controlled MCU1-GFP fusion expression strain. The localization of the Mcu1GFP fusion protein showed a punctate pattern as indicated by the green fluorescence (Fig. 4), suggesting that Mcu1 is located in mitochondria. To further confirm the subcellular localization of Mcu1, we stained mitochondria with the dye Mitotracker (Molecular Probes, Invitrogen Inc.). Fluorescence microscopy revealed colocalization of Mcu1-GFP and the Mitotracker in a punctate pattern, which is in contrast to the diffuse cytoplasmic localization of GFP in cells of the TETp-GFP control strain (Fig. 4). Homologs of Mcu1 were not found in S. cerevisiae and higher eukaryotic organisms. Functional analysis of Mcu1 in other fungi has never been reported. To explore its roles and underlying molecular mechanisms in C. albicans, we immunoprecipitated Mcu1-GFP for mass spectrometry (LC-MS/MS) analysis of Mcu1associated proteins. We identified 32 candidates, among which most are metabolism-related proteins including a number of putative or previously reported mitochondrial proteins (e.g. Atp1, Atp2, Por1, Pet9, Mdh1, Ilv5, Aco1, Tom70, and Osm2) (Table 1). To verify the LC-MS/MS results, we performed coimmunoprecipitation (Co-IP) and Western blot assays and found

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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Fig. 2. Role of the pseudogene (ps19.4984) locus in GlcNAc utilization. (A) Strategy of generating the two mutants (a, b) of ps19.4984. Mutant (a) a region containing the putative ORF and a part of the promoter sequence (214 bp) was deleted in both chromosome alleles. Mutant (b) a region containing the putative ORF and a part of the promoter sequence (214 bp) was deleted in one allele, and a region containing only the putative ORF sequence was deleted in the other allele. (B) Mutant (a), but not (b), of ps19.4984 showed growth defect on Lee’s GlcNAc medium. Scale bar, 1 cm. C. Ectopic expression of MCU1, could rescue the defect of mutant (a) of ps19.4984 on GlcNAc utilization.

that two representative mitochondrial proteins Atp1 and Por1 were physically associated with Mcu1 (Fig. 5). ATP1 encodes the ATP synthase a subunit, while POR1 encodes a mitochondrial outer membrane porin. These mitochondrial proteins are involved in energy and carbon metabolism.

3.5. Mcu1 is essential for the utilization of amino acids and nonfermentable carbon sources Based on the subcellular localization and CoIP data, we predicted that Mcu1 might also play a role in the utilization of other

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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Fig. 3. Mcu1 is essential for GlcNAc utilization in C. albicans. (A) The WT (BWP17), MCU1/mcu1, mcu1/mcu1 mutants and the MCU1 re-constituted strain were grown on Lee’s glucose or Lee’s GlcNAc medium at 25 °C for 6 days. (B) The WT (BWP17), MCU1/mcu1, mcu1/mcu1 mutants and the MCU1 re-constituted strain were grown on Lee’s glucose or GlcNAc medium at 37 °C for 4 days. Scale bar, 1 cm.

carbon sources besides GlcNAc. As shown in Fig. 6, the mcu1/mcu1 mutant could grow on the media with glucose, maltose, galactose, sucrose, fructose, or mannitol as the carbon source, but could not use amino acids (arginine or the mixture of seven amino acids including A, R, Q, E, N, P, and S) and non-fermentable carbohydrates (e.g. acetate, ethanol, glycerol, citrate, and pyruvate) as carbon sources (Fig. 6). Of note, the mcu1/mcu1 mutant grew much slower than the WT in the glucose, sucrose, and galactose media (Figs. 6 and S2). These results indicate that the mcu1/mcu1 mutant has a defect in respiratory metabolism and can probably acquire energy (or ATPs) only through fermentative metabolism. 3.6. Deletion of MCU1 impairs filamentation and virulence Because Mcu1 is important in the utilization of different carbon sources, we predicted that it could also be important in the regulation of filamentation. As shown in Figs. S3, S4, and 6, deletion of

MCU1 notably attenuated the ability of filamentous growth on a number of media. The mcu1/mcu1 mutant formed relatively smooth colonies, while the WT, MCU1/mcu1 mutant, and the MCU1-reconstituted strain formed wrinkled and filamentous colonies on Lee’s glucose, Lee’s GlcNAc, Lee’s glucose + GlcNAc, and Spider medium. Consistent with our earlier finding, the mcu1/mcu1 mutant could not grow on Lee’s GlcNAc medium. We next examined cellular morphologies of some representative colonies. While most WT cells exhibited filamentous growth, only a small percentage of mcu1/mcu1 cells underwent filamentation on all media tested (Figs. S3 and S4). And the filaments of WT, MCU1/mcu1 and the reconstituted strains were much longer than those of the mcu1/mcu1 mutant (Figs. S3 and S4). The abilities of utilizing different carbon sources and filamentation are related to virulence in C. albicans. We thus performed the virulence assay using a mouse systemic infection model. All the mice died within 13 days post infection when infected with

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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Fig. 4. Mitochondrial localization of Mcu1 in C. albicans. The strains of mcu1/mcu1+pNIM1 (TETp-GFP) and mcu1/mcu1+pNIM-MCU1 (TETp-MCU1-GFP) were grown in liquid Lee’s glucose medium at 25 °C for 16 h. Doxycycline was added to the medium for induction of the TET promoter. The dye mitoTracker was used to stain mitochodria.

Table 1 Mcu1-associated proteins identified by Co-IP and LC-MS/MS assays. Gene name

ORF

Protein Description

Sequence coverage (%)

Unique peptides detected

emPAI

MCU1 ATP1 ATP2 PGK1 POR1 PET9 MDH1 ADH1 CIT1 HSP12 RPL12 QCR2 HSP60 HSP90 GPM1 FDH1 IDP2 SSC1 ILV5 PGI1 CCP1 RPS4A ACO1 KGD1 TOM70

19.4983 19.6854 19.5653 19.3651 19.8644 19.930 19.7481 19.3997 19.4393 19.4216 19.1635 19.2644 19.717 19.6515 19.8522 19.638 19.3733 19.1896 19.7733 19.3888 9.7868 19.5341 19.6385 19.4021 19.4983

48 23.4 19.7 52.5 19.1 23.6 20.6 17.3 18.1 21.4 18.2 7.8 15.4 4.8 14.1 5.5 13.3 7.9 6.8 5.1 3.6 13.1 5.8 1.1 1.6

18 11 7 6 4 8 6 7 6 3 2 2 6 3 3 2 3 3 2 2 1 2 4 1 1

1.35 0.68 0.39 0.50 0.40 0.91 0.42 0.59 0.41 0.39 0.40 0.08 0.16 0.12 0.12 0.08 0.14 0.09 0.15 0.05 0.08 0.12 0.04 0.03 0.04

GRE3 HTB1 RPS24 – TAL1 RPL15A OSM2

19.4317 19.6925 19.5466 19.5565 19.930 19.493 19.5005

Hypothetical protein Mcu1 ATP alpha synthase chain, mitochondrial precursor F1 beta subunit of F1F0 ATPase complex Glyceraldehyde-3-phosphate dehydrogenase Mitochondrial outer membrane porin, Por1 Potential mitochondrial inner membrane ATP/ADP translocator Likely mitochondrial malate dehydrogenase Alcohol dehydrogenase Citrate synthase Putative heat shock protein Ribosomal protein L12, 60S ribosomal subunit Potential ubiquinol cyt-c reductase core protein 2 Heat shock protein 60 Heat shock protein 90 Putative tetrameric phosphoglycerate mutase Potential NAD-formate dehydrogenase Isocitrate dehydrogenase Heat shock protein, SSC1 Likely mitochondrial ketol-acid reductoisomerase Glucose-6-phosphate isomerase Cytochrome-c peroxidase N terminus Likely cytosolic ribosomal protein S4 Likely mitochondrial aconitate hydratase Putative 2-oxoglutarate dehydrogenase Protein channel activity and role in protein import into mitochondrial inner membrane Putative D-xylose reductase Histone H2b Likely cytosolic ribosomal protein S24 Putative 3-hydroxyisobutyrate dehydrogenase Transaldolase Putative ribosomal protein Putative mitochondrial fumarate reductase

5.7 9.6 18.5 3.1 8.4 4.9 1.5

1 1 2 1 2 1 1

0.07 0.32 0.21 0.08 0.09 0.13 0.04

Brief protein descriptions were adapted from the CGD database (http://www.candidagenome.org). Sequence coverage (%), percentage of reads mapping to each potential Mcu1-associated protein. emPAI, exponentially modified protein abundance index. Cells in exponential phase were used for Co-IP and LC-MS/MS assays. Stains (BWP17GFP and BWP17NGFP) were used for Co-IP assays. Proteins only identified in the BWP17NGFP Co-IP assay, but not in the BWP17GFP control, are presented as potential Mcu1associated proteins.

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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3.7. Roles of Por1, Tgl99, and Gup1 in the utilization of different carbon sources Por1 is an Mcu1-associated porin protein, which could probably function in material transportation between cytoplasm and mitochondria in C. albicans. Deletion of POR1 notably affected cell growth on the medium containing GlcNAc as the sole carbon source (Fig. 8). The TGL99 and GUP1 genes are located closely to MCU1 on the same chromosome (Fig. 1) and both are predicted to be involved in metabolism. We therefore tested whether these two genes are also involved in the utilization of different carbon sources. As shown in Fig. 8, deletion of TGL99 did not affect cell growth on any of the media tested. Deletion of GUP1 slightly reduced cell growth rates on media with GlcNAc, maltose, galactose, amino acids, or ethanol as the sole carbon source (Fig. 8). Fig. 5. Mcu1 is associated with mitochondrial proteins Atp1 and Por1. The C-terminus of Atp1 (lane 1 and 2) or Por1 (lane 3 and 4) was tagged with 13  Myc. Lane 1 and 3, strains were transformed with the plasmid pNIM1 (TETp-GFP). Lane 2 and 4, strains were transformed with the plasmid pNIM1-MCU1 (TETp-MCU1-GFP). To turn on the TET promoter, doxycycline (20 lg/ml) was added to the medium. Cell lysate was incubated with GFP antibody. Cell lysate and the co-precipitated proteins were used for Western blot assays (WB) with Myc antibody.

the WT, MCU1/mcu1, or MCU1-reconstituted strain. However, no mice died within 20 days post infection when infected with the mcu1/mcu1 mutant (Fig. 7A). These results were verified by fungal burden assays (Fig. 7B and C). Therefore, deletion of MCU1 has a profound effect on virulence in C. albicans.

4. Discussion The ability to utilize different carbon nutrition is critical for microbial organisms to be successful in evolution. The carbon sources in different natural environments are extremely diverse. To adapt to human or mammalian host niches, C. albicans must have the ability to utilize different carbon sources other than the preferred glucose. As a commensal microbe in the human gut and genital tract, where glucose is scarce, C. albicans may need to explore other carbon sources. GlcNAc is a component of the mammalian intestinal mucus and cell wall peptidoglycan of commensal bacteria. GlcNAc and other non-glucose carbon sources are

Fig. 6. Effect of deletion of MCU1 on utilization of different carbon sources in C. albicans. The WT (BWP17), MCU1/mcu1, mcu1/mcu1 mutants and the re-constituted strain were adjusted to 5  105 cells/ml, and then 10-fold serial dilutions of cells were spotted onto different medium plates. Cells were cultured at 37 °C for 4 d. YNB, yeast nitrogen base containing 5 grams/L of (NH4)2SO4. YNB+7 AAs, medium containing YNB and seven amino acids (A, R, Q, E, N, P, and S).

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

G. Guan et al. / Fungal Genetics and Biology xxx (2015) xxx–xxx

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Fig. 7. Mcu1 is required for virulence in a mouse systemic infection model. (A) Survival rates (%). 1  106 cells in 200 ll PBS of the WT (BWP17AHU), MCU1/mcu1, mcu1/mcu1 mutants or the re-constituted strain were injected into each mouse via lateral tail veins. Ten mice were used for each strain. (B) Kidney fungal burden. (C) Spleen fungal burden. Error bars indicate standard deviations (SD).

Fig. 8. Roles of Tgl99, Gup1 and Por1 in utilization of different carbon sources in C. albicans. The WT (BWP17AHU), tgl99/tgl99, gup1/gup1 and por1/por1 mutants were adjusted to 5  105 cells/ml, and then 10-fold serial dilutions of cells were spotted onto different media. Cells were cultured at 37 °C for 4 d. YNB, yeast nitrogen base containing 5 g/L of (NH4)2SO4. YNB+7 AAs, medium containing YNB and seven amino acids (A, R, Q, E, N, P, and S).

abundant in the mammalian gut (Chang et al., 2004). In the present study, we have discovered that the mitochondrial protein Mcu1 plays a critical role in the utilization of GlcNAc, amino acids, and non-fermentable carbohydrates as carbon sources in C. albicans.

Mcu1 is also involved in the regulation of filamentation and virulence possibly via metabolism processes. In C. albicans, the putative protein p19.4984 carries a LysM domain and several other conserved domains related to chitin

Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

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hydrolysis (Fig. 1). We searched the genomic sequence of Candida tropicalis, another human fungal pathogen closely related to C. albicans, and found that there are at least three genes encoding LysM domain-containing proteins (CTRT_00875, CTRG_05676, CTRT_04177). No orthologs of these genes were found in C. albicans. One would wonder why C. tropicalis contains so many LysM domain-containing protein encoding genes, while the single one (psorf19.4984) in C. albicans became inactivated. One possible explanation is the different natural habitats of these two species. Although both of them are human fungal pathogens, C. albicans is normally associated with humans or warm-blooded animals as a commensal, while C. tropicalis is not only a commensal of humans, but is also widespread in the environment such as oceans and soil. The ability of binding and hydrolyzing environmental chitin may be important for C. tropicalis to acquire carbon sources. C. albicans may not need the ability of binding chitin through LysM domaincontaining proteins any more after it became a commensal of humans, which do not produce chitin. So, genes encoding these proteins were lost or are undergoing degeneration like psorf19.4984. Genes with similar biological functions or involved in different reactions of the same biological process are often located in same regions of a chromosome. The MCU1 gene is located immediately adjacent to the psorf19.4984 pseudogene on chromosome 6. Mcu1 has a putative transmembrane motif and a coiled-coil motif, indicating that it may form a dimer to function as a mitochondrial protein. The biological roles of homologs of Mcu1 in other species remain unclear. Our data demonstrate that, together with a number of other mitochondrial proteins, Mcu1 plays a critical role in the regulation of carbon and energy metabolisms. For example, the Mcu1-associated protein Por1 is also involved in the utilization of a number of carbon sources (Fig. 8). Deletion of POR1 decreased the growth rate of C. albicans on some culture conditions, although this growth defect was much weaker than that of the mcu1/mcu1 mutant. A possible explanation for this difference could be that many Mcu1-associated mitochondrial proteins are involved in the utilization of different carbon sources. However, the exact molecular mechanism of Mcu1 in these processes remains unclear. We propose that Mcu1 and metabolism-related proteins could form a functional complex. In the absence of Mcu1, the complex becomes unstable or unable to bind its substrate molecules. We also noted that several heat shock proteins (Hsps) were identified in the Co-IP assay of Mcu1. Given their general role in facilitating protein folding, the interaction between Mcu1 and the Hsps could be non-specific. The MCU1-related genes TGL99 and GUP1 are also involved in carbon source metabolism, suggesting that functionrelated genes have been clustered during the process of evolution. By searching the NCBI protein database, we found that Mcu1 homologs seem to exist only in the species of the Candida clade and several other fungi (such as Kuraishia capsulate and Yarrowia lipolytica). The biological roles of this protein in pathogenic Candida species could be involved in adaptation to the unique host environment. Therefore, it makes sense that Mcu1 plays a role in the regulation of filamentation, a major virulence factor. Given the importance of Mcu1 in metabolism and the lack of a homolog in humans, it could be a good candidate for potential antifungal targets. Acknowledgments This work was supported by the grants (31170086, 31370175, and 81322026) from the Chinese National Natural Science Foundation (to G.H.) and ‘‘100 Talent Program’’ grant from the Chinese Academy of Sciences (to G.H.). Y.W. was supported by the Agency for Science, Technology and Research, Singapore.

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Please cite this article in press as: Guan, G., et al. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans. Fungal Genet. Biol. (2015), http://dx.doi.org/10.1016/j.fgb.2015.01.006

The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans.

The fungus Candida albicans is both a pathogen and a commensal in humans. The ability to utilize different carbon sources available in diverse host ni...
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