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Isolation of a Candida Albicans Gene, Tightly Linked to URA3, Coding for a Putative Transcription Factor That Suppresses a Saccharomyces Cerevisiae Aft1 Mutation

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Isolation of a Candida Albicans Gene, Tightly Linked to URA3, Coding for a Putative Transcription Factor That Suppresses a Saccharomyces Cerevisiae Aft1 Mutation Yeast Yeast 2001; 18: 301±311. Research Article Isolation of a Candida albicans gene, tightly linked to URA3, coding for a putative transcription factor that suppresses a Saccharomyces cerevisiae aft1 mutation Micaela GoÂmez GarcõÂa1, JoseÂ-Enrique O'Connor2, Lorena Latorre GarcõÂa4, Sami Irar MartõÂnez1, Enrique Herrero3 and Lucas del Castillo Agudo4* 1 Departament de Bioquimica i Biologia Molecular, Facultat de Farmacia, Universitat de ValeÁncia, 46100 Burjassot, Spain 2 Centro de CitometrõÂa, Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat de ValeÁncia, 46010 ValeÁncia, Spain 3 Departament de CieÁncies MeÁdiques BaÁsiques, Facultat de Medicina, Universitat de Lleida, Lleida, Spain 4 Departament de Microbiologia i Ecologia, Facultat de Farmacia, Universitat de ValeÁncia, 46100 Burjassot, Spain *Correspondence to: Abstract L. del Castillo. Agudo, Departament de MicrobiologõÂa i A pathogen such as C. albicans needs an ef®cient mechanism of iron uptake in an iron- EcologõÂa, Facultat de Farmacia, restricted environment such as is the human body. A ferric-reductase activity regulated by Avgda. Vicente AndreÂs EstelleÂs iron and copper, and analogous to that in S. cerevisiae, has been described in C. albicans. s/n, 46100 Burjassot, Spain. We have developed an in-plate protocol for the isolation of clones that complement an aft1 E-mail: [email protected] mutation in S. cerevisiae that makes cells dependent on iron for growth. After transformation of S. cerevisiae aft1 with a C. albicans library, we have selected clones that grow in conditions of iron de®ciency and share an identical plasmid, pIRO1, with a 4500 bp insert containing the URA3 gene and an ORF (IRO1) responsible for the suppression of the iron dependency. IRO1 does not show homology with AFT1 or with other sequences in the databases. Northern analysis demonstrates constitutive expression of IRO1. CAI4, a C. albicans strain isolated as Dura3, also has a deletion of the 3k half of IRO1, and displays in YNB medium similar phenotypic characteristics to S. cerevisiae aft1 mutant strains. Therefore, we consider IRO1 as a gene of C. albicans involved in the utilization of iron. However, in extreme conditions of iron deprivation, CAI4 seems to activate alternative mechanisms of iron uptake that allow a better growth than the wild strain SC5314. Analysis of its predicted protein sequence is in agreement with a role of Received: 9 July 2000 Iro1p as a transcription factor. Copyright # 2000 John Wiley & Sons, Ltd. Accepted: 24 September 2000 Keywords: Candida albicans; iron uptake; transcription factor Introduction dif®cult or almost impossible to obtain when phenotypes are not selected in a simple way. Candida albicans is a polymorphic fungus, its main Consequently, C. albicans is not suitable for genetic morphological forms being yeast and mycelia. In studies. Under these circumstances, molecular recent years, the occurrence of systemic infections approximation can be advantageous (Fonzi and due to C. albicans has been observed more Irwin, 1993) and within this framework we have frequently, as a consequence of increasing immuno- begun to study the molecular components involved de®ciencies of different origins, such as AIDS, anti- in iron uptake by C. albicans. leukaemic treatment or therapeutic immuno Most living beings need iron as a co-factor suppression in transplant patients and others essential for the activity of multiple catalytic (Beck-Sague and Jarvis, 1993; Edwards, 1990; Neil proteins. This element is not easily available in the and Ampel, 1996; Sobel and Vazquez, 1990). host, which sequesters this element as a defence C. albicans belongs to the imperfect fungi, lacking mechanism. Because of this, pathogenic microor- a known sexual cycle and having a diploid or ganisms are dependent on extremely effective aneuploid genetic constitution, and so mutants are systems of iron uptake (Kontoghiorghes and Copyright # 2000 John Wiley & Sons, Ltd. 302 M. G. GarcõÂa et al. Weinberg, 1995; Weinberg, 1998) and these systems the host complement system, based in a C. albicans represent important virulence factors. For this complement receptor-like molecule, has been reason, the mechanisms of iron uptake in different reported (Moors et al., 1992). C. albicans possesses bacterial pathogens are currently being studied ferric-reductase activity, regulated by iron and (Guerinot, 1994). The bacterial mechanisms differ copper (Morrissey et al., 1996), that would be from eukaryotic systems by the presence of side- analogous to the FRE1-andFRE2-associated rophores, as in Escherichia coli (Bagg and Neilands, activities in S. cerevisiae. The latter have been 1987), or by the ability to acquire iron by capturing shown to play an important role in iron transport mammalian proteins, as in Neisseria (Genco and in this species (Dancis et al., 1990; Georgatsou and Desai, 1996). Alexandraki, 1994; Stearman et al., 1996). In this Although a clear relationship between iron work, we employ an S. cerevisiae Daft1 mutant to uptake and virulence has not been established, isolate and characterize C. albicans genes involved different observations attest to the importance of in iron transport. iron uptake in C. albicans virulence (Fratti et al., 1998). It is known that salivary lactoferrin is one of the factors that reduce the infective capacity of this fungus in the oral cavity (Palma et al., 1992; Xu Materials and methods et al., 1999). Human serum inhibits the growth of C. albicans, fundamentally due to the presence of Strains, libraries and probes transferrins; proteins such as lactoferrin and ovo- S. cerevisiae strains: CML126 (MATa leu2-3,112 transferrin have similar effects to serum. On the ura3-52 trp1-1 his4 can1R aft1-D5::URA3)and other hand, haemoglobin addition suppresses the CML128 (MATa leu2-3,112 ura3-52 trp1-1 his4 effect of serum and these proteins (Radke et al., can1R) were described in Casas et al., (1997). 1994; Watanabe et al., 1997). These data suggest Dmac1, and Dfre1/Dfre2 strains were kindly pro- that C. albicans must compete for iron with these vided by Dr Despina Alexandraki (Foundation iron-complexing proteins and, therefore, iron- for Research and Technology ± Hellas, Institute of sequestering mechanisms must be considered as Molecular Biology and Biotechnology). C. albicans: virulence factors. Furthermore, the adherence to ATCC2655 (ATCC) and SC5314 (Gillum et al., epithelial cells and formation of germ tube are also 1984) were wild-type clinical isolates. CAI4 regulated by iron availability. A change in the (Dura3::limm434/Dura3::limm434) was obtained electrophoretic pattern of cell wall proteins, as well from SC5314 by Fonzi and Irwin (1993). C. albicans as in antigenic determinants, has been reported in genomic library (in YRp7 vector; Struhl et al.,1976) response to iron concentration (Abe et al., 1985; has been described previously (Nieto et al., 1993). Paul et al., 1989; Sweet and Douglas, 1991). C. albicans URA3 probe has also been described The importance of iron for the development of previously (del Castillo Agudo et al., 1993). IRO1 life, together with the potential toxicity of an excess probe was obtained by labelling a HincII±HincII of this element, gave rise to the development of pIRO1 fragment or by PCR ampli®cation with complex mechanisms that regulate the uptake of speci®c primers; the URA3 and IRO1 probes over- iron from media. In S. cerevisiae, highly speci®c lap and contain the EcoRV site of the IRO1 open mechanisms of iron uptake and transport have been reading frame (ORF) (Figure 1). described (for reviews, see Eide, 1998; Guerinot, 1994; Stearman et al., 1996). These mechanisms are regulated at the transcriptional level by the product Enzymes and chemicals of AFT1 in response to iron (Yamaguchi-Iwai et al., 1995, 1996; Casas et al., 1997). In C. albicans the DNA restriction endonucleases and T4 DNA ligase mechanisms of iron uptake are not well character- were supplied by Boehringer-Mannheim, Pharmacia ized, although this fungus probably uses a system of or Promega. Taq DNA polymerase was purchased iron uptake through siderophores (Holzberg and from Pharmacia. Ferrozine, aak-dipyridil (DPD), Artis, 1983; Ismail and Lupan, 1986; Ismail and ferrichrome and ¯uorochromes rhodamine 123 Bedell, 1986; Minnick et al., 1991). A mechanism (RH123) and dihydroethidine (HE), were purchased of iron acquisition from blood cells which exploits from Sigma (St. Louis, MO, USA). Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2001; 18: 301±311. URA3-linked C. albicans ORF that suppresses S. cerevisiae iron dependency 303 Birnboim and Doly (1979). DNA fragments were isolated from agarose gels by the freeze±squeeze method of Tautz and Renz (1983). Plasmids were isolated from yeast according to Rose et al. (1990). Probes for Southern analysis were labelled by the digoxygenin method of Boehringer-Mannheim, either with random primer extension or PCR ampli®cation, following the instructions provided by the manufacturer. Southern blot analysis was carried out as described by Nieto et al. (1993) and Northern blots as described in Casas et al. (1997), except that transfer was carried out by capillarity and RNA crosslinks were made by incubation for 30 min at 120uC. Cell transformation E. coli transformations were carried out according to Inoue et al. (1990). Yeast cells were transformed Figure 1. Comparative maps of C. albicans DNA inserts and using the lithium acetate method of Ito et al. (1983), ¯anking regions (dark areas in plasmids pIRO1 and pAL2). with modi®cations described by Rose et al. (1990). Spaces between vertical markers=1kb Media and growth conditions Selection of Daft1 wild-type transformant clones C. albicans and S. cerevisiae were grown in YPD rich medium (2% peptone, 2% glucose, 1% Yeast S. cerevisiae CML126 carrying Daft1 mutation is Extract). SD minimal medium, was prepared by unable to grow in iron-de®cient media when adding the appropriate amino acids to 0.67% Yeast glycerol/ethanol were used as carbon sources. In Nitrogen Base without amino acids (Difco) and these conditions, CML128 (AFT1) strain is able supplemented with 2% glucose or 3% glycerol+2% to grow.
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