Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCECandida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans.

Department of Membrane Transport Institute of Physiology Czech Academy of Sciences Prague Czech Republic

VIB Department of Molecular Microbiology KU Leuven Flanders Belgium; Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Leuven Belgium

VIB Department of Molecular Microbiology KU Leuven Flanders Belgium; Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Leuven Belgium; Department of Membrane Transport Institute of Physiology Czech Academy of Sciences Prague Czech Republic

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Calderone RA, Clancy CJ. 2012. Candida and candidiasis, 2nd ed. ASM Press, Washington, DC.

Mavor AL, Thewes S, Hube B. 2005. Systemic fungal infections caused by Candida species: epidemiology, infection process and virulence attributes. Curr Drug Targets 6:863–874. doi:10.2174/138945005774912735. PubMed DOI

Sobel JD. 2007. Vulvovaginal candidosis. Lancet 369:1961–1971. doi:10.1016/S0140-6736(07)60917-9. PubMed DOI

Cassone A, Cauda R. 2012. Candida and candidiasis in HIV-infected patients: where commensalism, opportunistic behavior and frank pathogenicity lose their borders. AIDS 26:1457–1472. doi:10.1097/QAD.0b013e3283536ba8. PubMed DOI

Pfaller MA, Diekema DJ. 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163. doi:10.1128/CMR.00029-06. PubMed DOI PMC

Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv113. doi:10.1126/scitranslmed.3004404. PubMed DOI

Sandai D, Yin Z, Selway L, Stead D, Walker J, Leach MD, Bohovych I, Ene IV, Kastora S, Budge S, Munro CA, Odds FC, Gow NA, Brown AJ. 2012. The evolutionary rewiring of ubiquitination targets has reprogrammed the regulation of carbon assimilation in the pathogenic yeast Candida albicans. mBio 3:e00495-12. doi:10.1128/mBio.00495-12. PubMed DOI PMC

Brock M. 2009. Fungal metabolism in host niches. Curr Opin Microbiol 12:371–376. doi:10.1016/j.mib.2009.05.004. PubMed DOI

Limjindaporn T, Khalaf RA, Fonzi WA. 2003. Nitrogen metabolism and virulence of Candida albicans require the GATA-type transcriptional activator encoded by GAT1. Mol Microbiol 50:993–1004. doi:10.1046/j.1365-2958.2003.03747.x. PubMed DOI

Fleck CB, Schöbel F, Brock M. 2011. Nutrient acquisition by pathogenic fungi: nutrient availability pathway regulation, and differences in substrate utilization. Int J Med Microbiol 301:400–407. doi:10.1016/j.ijmm.2011.04.007. PubMed DOI

Hruskova-Heidingsfeldova O. 2008. Secreted proteins of Candida albicans. Front Biosci 13:7227–7242. doi:10.2741/3224. PubMed DOI

Naglik JR, Albrecht A, Bader O, Hube B. 2004. Candida albicans proteinases and host/pathogen interactions. Cell Microbiol 6:915–926. doi:10.1111/j.1462-5822.2004.00439.x. PubMed DOI

Jauniaux JC, Grenson M. 1990. GAP1, the general amino acid permease gene of Saccharomyces cerevisiae. Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression. Eur J Biochem 190:39–44. doi:10.1111/j.1432-1033.1990.tb15542.x. PubMed DOI

Magasanik B, Kaiser CA. 2002. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290:1–18. doi:10.1016/S0378-1119(02)00558-9. PubMed DOI

Hein C, André B. 1997. A C-terminal di-leucine motif and nearby sequences are required for NH4+-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae. Mol Microbiol 24:607–616. doi:10.1046/j.1365-2958.1997.3771735.x. PubMed DOI

Ghaddar K, Merhi A, Saliba E, Krammer EM, Prévost M, André B. 2014. Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases. Mol Cell Biol 34:4447–4463. doi:10.1128/MCB.00699-14. PubMed DOI PMC

Hofman-Bang J. 1999. Nitrogen catabolite repression in Saccharomyces cerevisiae. Mol Biotechnol 12:35–73. doi:10.1385/MB:12:1:35. PubMed DOI

Stanbrough M, Magasanik B. 1996. Two transcription factors, Gln3p and Nil1p, use the same GATAAG sites to activate the expression of GAP1 of Saccharomyces cerevisiae. J Bacteriol 178:2465–2468. doi:10.1128/jb.178.8.2465-2468.1996. PubMed DOI PMC

Donaton MC, Holsbeeks I, Lagatie O, Van Zeebroeck G, Crauwels M, Winderickx J, Thevelein JM. 2003. The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae. Mol Microbiol 50:911–929. doi:10.1046/j.1365-2958.2003.03732.x. PubMed DOI

Van Zeebroeck G, Rubio-Texeira M, Schothorst J, Thevelein JM. 2014. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast gap1 amino acid transceptor. Mol Microbiol 93:213–233. doi:10.1111/mmi.12654. PubMed DOI PMC

Kraidlova L, Van Zeebroeck G, Van Dijck P, Sychrová H. 2011. The Candida albicans GAP gene family encodes permeases involved in general and specific aminio-acid uptake and sensing. Eukaryot Cell 10:1219–1229. doi:10.1128/EC.05026-11. PubMed DOI PMC

Kodama Y, Omura F, Takahashi K, Shirahige K, Ashikari T. 2002. Genome-wide expression analysis of genes affected by amino acid sensor Ssy1p in Saccharomyces cerevisiae. Curr Genet 41:63–72. doi:10.1007/s00294-002-0291-1. PubMed DOI

Didion T, Regenberg B, Jørgensen MU, Kielland-Brandt MC, Andersen HA. 1998. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol Microbiol 27:643–650. doi:10.1046/j.1365-2958.1998.00714.x. PubMed DOI

Zaman S, Lippman SI, Zhao X, Broach JR. 2008. How Saccharomyces responds to nutrients. Annu Rev Genet 42:27–81. doi:10.1146/annurev.genet.41.110306.130206. PubMed DOI

Brega E, Zufferey R, Mamoun CB. 2004. Candida albicans Csy1 is a nutrient sensor important for activation of amino acid uptake and hyphal morphogenesis. Eukaryot Cell 3:135–143. doi:10.1128/EC.3.1.135-143.2004. PubMed DOI PMC

Ramachandra S, Linde J, Brock M, Guthke R, Hube B, Brunke S. 2014. Regulatory networks controlling nitrogen sensing and uptake in Candida albicans. PLoS One 9:e92734. doi:10.1371/journal.pone.0092734. PubMed DOI PMC

Inglis DO, Arnaud MB, Binkley J, Shah P, Skrzypek MS, Wymore F, Binkley G, Miyasato SR, Simison M, Sherlock G. 2012. The Candida Genome Database incorporates multiple Candida species: multispecies search and analysis tools with curated gene and protein information for Candida albicans and Candida glabrata. Nucleic Acids Res 40:D667–D674. doi:10.1093/nar/gkr945. PubMed DOI PMC

Rossignol T, Lechat P, Cuomo C, Zeng Q, Moszer I, d’Enfert C. 2008. CandidaDB: a multi-genome database for Candida species and related Saccharomycotina. Nucleic Acids Res 36:D557–D561. doi:10.1093/nar/gkm1010. PubMed DOI PMC

Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, Christie KR, Costanzo MC, Dwight SS, Engel SR, Fisk DG, HIrschman JE, Hitz BC, Karra K, Krieger CJ, Miyasato SR, Nash RS, Park J, Skrzypek MS, Simison M, Weng S, Wong ED. 2012. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res 40:D700–D705. doi:10.1093/nar/gkr1029. PubMed DOI PMC

Rouillon A, Surdin-Kerjan Y, Thomas D. 1999. Transport of sulfonium compounds. Characterization of the S-adenosylmethionine and S-methylmethionine permeases from the yeast Saccharomyces cerevisiae. J Biol Chem 274:28096–28105. doi:10.1074/jbc.274.40.28096. PubMed DOI

Hernday AD, Noble SM, Mitrovich QM, Johnson AD. 2010. Genetics and molecular biology in Candida albicans. Methods Enzymol 470:737–758. doi:10.1016/S0076-6879(10)70031-8. PubMed DOI

Noble SM, Johnson AD. 2005. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4:298–309. doi:10.1128/EC.4.2.298-309.2005. PubMed DOI PMC

Sasse C, Morschhäuser J. 2012. Gene deletion in Candida albicans wild-type strains using the SAT1-flipping strategy. Methods Mol Biol 845:3–17. doi:10.1007/978-1-61779-539-8_1. PubMed DOI

Reuss O, Vik A, Kolter R, Morschhäuser J. 2004. The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127. doi:10.1016/j.gene.2004.06.021. PubMed DOI

Grenson M, Hou C, Crabeel M. 1970. Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J Bacteriol 103:770–777. PubMed PMC

Biswas S, Roy M, Datta A. 2003. N-Acetylglucosamine-inducible CaGAP1 encodes a general amino acid permease which co-ordinates external nitrogen source response and morphogenesis in Candida albicans. Microbiology 149:2597–2608. doi:10.1099/mic.0.26215-0. PubMed DOI

Springael JY, André B. 1998. Nitrogen regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol Biol Cell 9:1253–1263. doi:10.1091/mbc.9.6.1253. PubMed DOI PMC

Maidan MM, Thevelein JM, Van Dijck P. 2005. Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of amino acids and on the G protein coupled receptor Gpr1. Biochem Soc Trans 33:291–293. doi:10.1042/BST0330291. PubMed DOI

Kabir MA, Hussain MA, Ahmad Z. 2012. Candida albicans: a model organism for studying fungal pathogens. ISRN Microbiol 2012:538694. doi:10.5402/2012/538694. PubMed DOI PMC

Rao KH, Ghosh S, Natarajan K, Datta A. 2013. N-Acetylglucosamine kinase, HXK1 is involved in morphogenetic transition and metabolic gene expression in Candida albicans. PLoS One 8:e53638. doi:10.1371/journal.pone.0053638. PubMed DOI PMC

Lee KL, Buckley HR, Campbell CC. 1975. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13:148–153. doi:10.1080/00362177585190271. PubMed DOI

Liu H, Köhler J, Fink GR. 1994. Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266:1723–1726. doi:10.1126/science.7992058. PubMed DOI

Dunkel N, Hertlein T, Franz R, Reuß O, Sasse C, Schäfer T, Ohlsen K, Morschhäuser J. 2013. Roles of different peptide transporters in nutrient acquisition in Candida albicans. Eukaryot Cell 12:520–528. doi:10.1128/EC.00008-13. PubMed DOI PMC

Martínez P, Ljungdahl PO. 2004. An ER packaging chaperone determines the amino acid uptake capacity and virulence of Candida albicans. Mol Microbiol 51:371–384. doi:10.1046/j.1365-2958.2003.03845.x. PubMed DOI

Sychrová H, Chevallier MR. 1993. Transport properties of a C. albicans amino-acid permease whose putative gene was cloned and expressed in S. cerevisiae. Curr Genet 24:487–490. doi:10.1007/BF00351710. PubMed DOI

McNemar MD, Gorman JA, Buckley HR. 2001. Isolation of a gene encoding a putative polyamine transporter from Candida albicans, GPT1. Yeast 18:555–561. doi:10.1002/yea.697. PubMed DOI

Van Zeebroeck G, Bonini BM, Versele M, Thevelein JM. 2009. Transport and signaling via the amino acid binding site of the yeast gap1 amino acid transceptor. Nat Chem Biol 5:45–52. doi:10.1038/nchembio.132. PubMed DOI

Serneels J, Tournu H, Van Dijck P. 2012. Tight control of trehalose content is required for efficient heat-induced cell elongation in Candida albicans. J Biol Chem 287:36873–36882. doi:10.1074/jbc.M112.402651. PubMed DOI PMC

Martínez P, Ljungdahl PO. 2005. Divergence of Stp1 and Stp2 transcription factors in Candida albicans places virulence factors required for proper nutrient acquisition under amino acid control. Mol Cell Biol 25:9435–9446. doi:10.1128/MCB.25.21.9435-9446.2005. PubMed DOI PMC

Morschhäuser J. 2011. Nitrogen regulation of morphogenesis and protease secretion in Candida albicans. Int J Med Microbiol 301:390–394. doi:10.1016/j.ijmm.2011.04.005. PubMed DOI

Dabas N, Morschhäuser J. 2008. A transcription factor regulatory cascade controls secreted aspartic protease expression in Candida albicans. Mol Microbiol 69:586–602. doi:10.1111/j.1365-2958.2008.06297.x. PubMed DOI

Law MJ, Ciccaglione K. 2015. Fine-tuning of histone H3 Lys4 methylation during pseudohyphal differentiation by the CDK submodule of RNA polymerase II. Genetics 199:435–453. doi:10.1534/genetics.114.172841. PubMed DOI PMC

Parsek MR, Val DL, Hanzelka BL, Cronan JE Jr., Greenberg EP. 1999. Acyl homoserine-lactone quorum-sensing signal generation. Proc Natl Acad Sci U S A 96:4360–4365. doi:10.1073/pnas.96.8.4360. PubMed DOI PMC

Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. PubMed

Gimeno CJ, Ljungdahl PO, Styles CA, Fink GR. 1992. Unipolar cell divisions in the yeast Saccharomyces cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68:1077–1090. doi:10.1016/0092-8674(92)90079-R. PubMed DOI

Maresová L, Sychrová H. 2007. Applications of a microplate reader in yeast physiology research. Biotechniques 43:667–672. doi:10.2144/000112620. PubMed DOI

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3. PubMed DOI

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔCT) method. Methods 25:402–408. doi:10.1006/meth.2001.1262. PubMed DOI

Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. doi:10.1038/nprot.2008.73. PubMed DOI

Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ. 1997. Yeast-enhanced green fluorescent protein (yEGFP) a reporter of gene expression in Candida albicans. Microbiology 143:303–311. doi:10.1099/00221287-143-2-303. PubMed DOI

Gerami-Nejad M, Berman J, Gale CA. 2001. Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans. Yeast 18:859–864. doi:10.1002/yea.738. PubMed DOI

Gietz RD, Schiestl RH, Willems AR, Woods RA. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360. doi:10.1002/yea.320110408. PubMed DOI

Gillum AM, Tsay EYH, Kirsch DR. 1984. Isolation of the Candida albicans gene for orotidine-5′-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198:179–182. doi:10.1007/BF00328721. PubMed DOI