The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants.
References
[1]
Romani L, Bistoni F, Puccetti P (2003) Adaptation of Candida albicans to the host environment: the role of morphogenesis in virulence and survival in mammalian hosts. Curr Opin Microbiol 6: 338–343.
[2]
Hube B (2004) From commensal to pathogen: stage- and tissue-specific gene expression of Candida albicans. Curr Opin Microbiol 7: 336–341.
[3]
Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2: 73–85.
[4]
Akins RA (2005) An update on antifungal targets and mechanisms of resistance in Candida albicans. Med Mycol 43: 285–318.
[5]
Ghannoum MA, Rice LB (1999) Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12: 501–517.
[6]
White T, Marr K, Bowden R (1998) Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11: 382–402.
[7]
Sanglard D, Coste A, Ferrari S (2009) Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation. FEMS Yeast Res 9: 1029–1050.
[8]
Chandra J, Kuhn D, Mukherjee P, Hoyer L, McCormick T, et al. (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bact 183: 5385–5394.
[9]
Ramage G, Bachmann S, Patterson T, Wickes B, Lopez-Ribot J (2002) Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms. J Antimicrob Chemother 49: 973–980.
[10]
Sellam A, Tebbji F, Nantel A (2009) Role of Ndt80p in sterol metabolism regulation and azole resistance in Candida albicans. Eukaryot Cell 8: 1174–1183.
[11]
Netea MG, Maródi L (2010) Innate immune mechanisms for recognition and uptake of Candida species. Trends in Immunology 31: 346–353.
[12]
Bourgeois C, Majer O, Frohner I, Tierney L, Kuchler K (2010) Fungal attacks on mammalian hosts: pathogen elimination requires sensing and tasting. Curr Opin Microbiol 13: 401–408.
[13]
van de Veerdonk F, Kullberg B, Netea M (2010) Pathogenesis of invasive candidiasis. Curr Opin Crit Care 16: 453–459.
[14]
van de Veerdonk F, Netea M, Joosten L, Van der Meer J, Kullberg B (2010) Novel strategies for the prevention and treatment of Candida infections: the potential of immunotherapy. FEMS Microbiol Rev 34: 1063–1075.
[15]
Davis DA (2009) How human pathogenic fungi sense and adapt to pH: the link to virulence. Curr Opin Microbiol 12: 365–370.
[16]
Almeida R, Wilson D, Hube B (2009) Candida albicans iron acquisition within the host. FEMS Yeast Res 9: 1000–1012.
[17]
Klis F, Sosinska G, de Groot P, Brul S (2009) Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence. FEMS Yeast Res 9: 1013–1028.
[18]
Brown AJP, Odds FC, Gow NAR (2007) Infection-related gene expression in Candida albicans. Curr Opin Microbiol 10: 307–313.
[19]
Hube B (2006) Infection-associated genes of Candida albicans. Future Microbiol 1: 209–218.
[20]
Román E, Arana DM, Nombela C, Alonso-Monge R, Pla J (2007) MAP kinase pathways as regulators of fungal virulence. Trends Microbiol 15: 181–190.
[21]
Thewes S, Kretschmar M, Park H, Schaller M, Filler SG, et al. (2007) In vivo and ex vivo comparative transcriptional profiling of invasive and non-invasive Candida albicans isolates identifies genes associated with tissue invasion. Mol Microbiol 63: 1606–1628.
[22]
Homann OR, Dea J, Noble SM, Johnson AD (2009) A Phenotypic Profile of the Candida albicans Regulatory Network. PLoS Genet 5: e1000783.
[23]
Becker JM, Kauffman SJ, Hauser M, Huang L, Lin M, et al. (2010) Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model. Proc Natl Acad Sci USA 107: 22044–22049.
[24]
Noble SM, French S, Kohn LA, Chen V, Johnson AD (2010) Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nature Genetics. pp. 1–11.
[25]
Boon J, Lambert T, Sisson A, Davis A, Smith B (2003) Facilitated phosphatidylserine (PS) flip-flop and thrombin activation using a synthetic PS scramblase. JAmChemSoc 125: 8195–8201.
[26]
Davis D, Edwards J, Mitchell A, Ibrahim A (2000) Candida albicans RIM101 pH response pathway is required for host-pathogen interactions. Infect Immun 68: 5953–5959.
[27]
Ikeda F, Wakai Y, Matsumoto S, Maki K, Watabe E, et al. (2000) Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis. Antimicrob Agents Chemother 44: 614–618.
[28]
Moye-Rowley WS (2002) Transcription factors regulating the response to oxidative stress in yeast. Antioxid Redox Signal 4: 123–140.
[29]
Felk A, Kretschmar M, Albrecht A, Schaller M, Beinhauer S, et al. (2002) Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs. Infect Immun 70: 3689–3700.
[30]
Lachke S, Srikantha T, Soll D (2003) The regulation of EFG 1 in white-opaque switching in Candida albicans involves overlapping promoters. Mol Microbiol 48: 523–536.
[31]
Staib P, Kretschmar M, Nichterlein T, Hof H, Morschhauser J (2002) Transcriptional regulators Cph1p and Efg1p mediate activation of the Candida albicans virulence gene SAP5 during infection. Infect Immun 70: 921–927.
[32]
Kiesewetter DO, Finn RD, Rice KC, Monn JA (1990) Synthesis of 11C-labeled (+-)-5-methyl-10,11-dihydro-5H-dibenzo[a？,d]cyclohepten-5,10-imine [(+-)-[11C]MK801]. Int J Rad Appl Instrum A 41: 139–142.
[33]
Johnston D, Eberle K, Sturtevant J, Palmer G (2009) Role for endosomal and vacuolar GTPases in Candida albicans pathogenesis. Infect Immun 77: 2343–2355.
[34]
Fonzi W, Irwin M (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134: 717–728.
[35]
Wilson R, Davis D, Mitchell A (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bact 181: 1868–1874.
[36]
Sanglard D, Ischer F, Monod M, Bille J (1996) Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 40: 2300–2305.
[37]
Dennison P, Ramsdale M, Manson C, Brown A (2005) Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system. Fungal Genet Biol 42: 737–748.
[38]
Liu O, Chun C, Chow E, Chen C, Madhani H, et al. (2008) Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135: 174–188.
[39]
Moyrand F, Lafontaine I, Fontaine T, Janbon G (2008) UGE1 and UGE2 regulate the UDP-glucose/UDP-galactose equilibrium in Cryptococcus neoformans. Eukaryot Cell 7: 2069–2077.
[40]
Bruno VM, Kalachikov S, Subaran R, Nobile CJ, Kyratsous C, et al. (2006) Control of the C. albicans cell wall damage response by transcriptional regulator Cas5. PLoS Pathog 2: e21.
[41]
Nobile C, Andes D, Nett J, Smith F, Yue F, et al. (2006) Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLoS Pathog 2: e63.
[42]
Nobile C, Mitchell A (2005) Regulation of cell-surface genes and biofilm formation by the C. albicans transcription factor Bcr1p. Curr Biol 15: 1150–1155.
[43]
MacCallum DM, Odds FC (2005) Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice. Mycoses 48: 151–161.
[44]
Moyrand F, Fontaine T, Janbon G (2007) Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. Molecular Microbiology 64: 771–781.
[45]
Bader T, Bodendorfer B, Schr？ppel K, Morschh？user J (2003) Calcineurin is essential for virulence in Candida albicans. Infect Immun 71: 5344–5354.
[46]
Blankenship J, Wormley F, Boyce M, Schell W, Filler S, et al. (2003) Calcineurin is essential for Candida albicans survival in serum and virulence. Eukaryot Cell 2: 422–430.
[47]
Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003) Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48: 959–976.
[48]
Coste AT, Karababa M, Ischer F, Bille J, Sanglard D (2004) TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 3: 1639–1652.
[49]
Coste AT, Ramsdale M, Ischer F, Sanglard D (2008) Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae. Microbiology 154: 1491–1501.
[50]
Talibi D, Raymond M (1999) Isolation of a putative Candida albicans transcriptional regulator involved in pleiotropic drug resistance by functional complementation of a pdr1 pdr3 mutation in Saccharomyces cerevisiae. J Bact 181: 231–240.
[51]
Hoot S, Oliver B, White T (2008) Candida albicans UPC2 is transcriptionally induced in response to antifungal drugs and anaerobicity through Upc2p-dependent and -independent mechanisms. Microbiology 154: 2748–2756.
[52]
Hoot S, Brown R, Oliver B, White T (2010) The UPC2 promoter in Candida albicans contains two cis-acting elements that bind directly to Upc2p, resulting in transcriptional autoregulation. Eukaryot Cell 9: 1354–1362.
[53]
Silver PM, Oliver BG, White TC (2004) Role of Candida albicans transcription factor Upc2p in drug resistance and sterol metabolism. Eukaryot Cell 3: 1391–1397.
[54]
Heilmann C, Schneider S, Barker K, Rogers P, Morschhauser J (2010) An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans. Antimicrob Agents Chemother 54: 353–359.
[55]
Chamilos G, Nobile CJ, Bruno VM, Lewis RE, Mitchell AP, et al. (2009) Candida albicans Cas5, a regulator of cell wall integrity, is required for virulence in murine and toll mutant fly models. J Infect Dis 200: 152–157.
[56]
Brown V, Sexton J, Johnston M (2006) A glucose sensor in Candida albicans. Eukaryot Cell 5: 1726–1737.
[57]
Uppuluri P, Chaffin W (2007) Defining Candida albicans stationary phase by cellular and DNA replication, gene expression and regulation. Mol Microbiol 64: 1572–1586.
[58]
Lan C, Rodarte G, Murillo L, Jones T, Davis R, et al. (2004) Regulatory networks affected by iron availability in Candida albicans. Mol Microbiol 53: 1451–1469.
[59]
Sudbery P, Gow N, Berman J (2004) The distinct morphogenic states of Candida albicans. Trends Microbiol 12: 317–324.
[60]
Chen C, Pande K, French SD, Tuch BB, Noble SM (2011) An Iron Homeostasis Regulatory Circuit with Reciprocal Roles in Candida albicans Commensalism and Pathogenesis. Cell Host Microbes 10: 118–135.
[61]
Liu TT, Znaidi S, Barker KS, Xu L, Homayouni R, et al. (2007) Genome-Wide Expression and Location Analyses of the Candida albicans Tac1p Regulon. Eukaryot Cell 6: 2122–2138.
[62]
Brun S, Aubry C, Lima O, Filmon R, Bergès T, et al. (2003) Relationships between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob Agents Chemother 47: 847–853.
[63]
Lupetti A, Paulusma-Annema A, Welling MM, Dogterom-Ballering H, Brouwer CPJM, et al. (2003) Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species. Antimicrobial Agents Chemother 47: 262–267.
[64]
Brun S, Dalle F, Saulnier P, Renier G, Bonnin A, et al. (2005) Biological consequences of petite mutations in Candida glabrata. J Antimicrob Chemother 56: 307–314.
[65]
Zucchi PC, Davis TR, Kumamoto CA (2010) A Candida albicans cell wall-linked protein promotes invasive filamentation into semi-solid medium. Mol Microbiol 76: 733–748.
[66]
Mitchell AP (1998) Dimorphism and virulence in Candida albicans. Curr Opin Microbiol 1: 687–692.
[67]
Sexton JA, Brown V, Johnston M (2007) Regulation of sugar transport and metabolism by the Candida albicans Rgt1 transcriptional repressor. Yeast 24: 847–860.
[68]
Chiranand W, McLeod I, Zhou H, Lynn J, Vega L, et al. (2008) CTA4 transcription factor mediates induction of nitrosative stress response in Candida albicans. Eukaryot Cell 7: 268–278.
[69]
Oh J, Fung E, Schlecht U, Davis RW, Giaever G, et al. (2010) Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection. PLoS Pathog 6:
[70]
MacPherson S, Akache B, Weber S, De Deken X, Raymond M, et al. (2005) Candida albicans zinc cluster protein Upc2p confers resistance to antifungal drugs and is an activator of ergosterol biosynthetic genes. Antimicrob Agents Chemother 49: 1745–1752.
[71]
Ondeyka J, Harris G, Zink D, Basilio A, Vicente F, et al. (2009) Isolation, structure elucidation, and biological activity of virgineone from Lachnum Wirgineum using the genome-wide Candida albicans fitness test. J Nat Prod 72: 136–141.
