JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.13.4090–4093.2001

July 2001, p. 4090–4093 Vol. 183, No. 13

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

NOTES

Efg1, a Morphogenetic Regulator in Candida albicans, Is aSequence-Specific DNA Binding Protein

PING LENG,† PHILIP R. LEE,‡ HONG WU,§ AND ALISTAIR J. P. BROWN*

Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences,Foresterhill, Aberdeen AB25 2ZD, United Kingdom

Received 2 March 2001/Accepted 10 April 2001

Efg1 is essential for hyphal development in the human pathogen Candida albicans under most conditions.Efg1 is related to basic helix-loop-helix regulators, and therefore most workers presume that Efg1 is atranscription factor. Here we confirm that Efg1 is a DNA binding protein that can interact specifically with theE box.

Candida albicans is the major fungal pathogen of humans(19). This fungus causes frequent and recurrent oral and vag-inal infections and systemic infections in severely immunocom-promised patients. A number of factors are thought to pro-mote establishment and progression of C. albicans infections,including yeast hypha morphogenesis (5, 16, 20). Several sig-

nalling pathways appear to activate hyphal development, andone of these pathways is defined by the factor Efg1 (9, 26).Efg1 probably lies on a Ras-cAMP-protein kinase A signallingpathway (5, 9, 10, 24). Mutations that inactivate Efg1 blockhyphal development under most experimental conditions invitro and in vivo (16, 26). For example, C. albicans efg1/efg1mutants are unable to form hyphae following serum stimula-tion or pH induction in vitro or in the kidneys of infectedanimals. However, efg1/efg1 mutants still form hyphae whenthey are embedded in agar and in some infection models (6,21). Nevertheless, Efg1 is considered a major morphogeneticregulator in C. albicans (9).

Ernst and coworkers pointed out that Efg1 contains a basichelix-loop-helix (bHLH) domain with significant sequence sim-ilarity to the APSES family of proteins (9, 26). Members of thisfamily include morphogenetic regulators from other fungi,such as Sok2 and Phd1 from Saccharomyces cerevisiae, Asm1from Neurospora crassa, and StuA from Aspergillus nidulans.This bHLH domain, which is characteristic of some transcrip-tion factors, is thought to promote dimerization and DNAbinding (18, 22). Therefore, it is generally presumed that Efg1is a transcription factor, and most models of morphogenetic

FIG. 1. Efg1p is required for HYR1 and ALS8 activation duringserum-induced morphogenesis. We performed a Northern analysiswith isogenic C. albicans strains grown for 2 h in YPD containing 10%fetal calf serum at 37°C; the strains used were wild-type strain SC5314(WT) (11), cph1/cph1 strain JKC19 (15), efg1/efg1 strain HLC52 (16),and cph1/cph1 efg1/efg1 strain HLC54 (54). ALS8 and HYR1 blots wereexposed for 8 h and 4 days, respectively. Data from one of two replicateexperiments are shown.

* Corresponding author. Mailing address: Department of Molecularand Cell Biology, University of Aberdeen, Institute of Medical Sci-ences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. Phone:44-1224-273183. Fax: 44-1224-273144. E-mail: al.brown@abdn.ac.uk.

† Present address: Division of Cellular & Molecular Biology, On-tario Cancer Institute, University of Toronto, Toronto, Ontario M5G2M9, Canada.

‡ Present address: Laboratory of Developmental Neurobiology,NICHD, National Institutes of Health, Bethesda, MD 20892-4480.

§ Present address: Eye Research Institute of Canada, University ofToronto, Toronto, Ontario M5T 2S8, Canada.

FIG. 2. Hypha-specific promoters contain E boxes. The positions ofE boxes (59-CANNTG-39) (stars) are shown in the 59 regions of theALS8, HYR1 ALS3, HWP1, ECE1 and RBT4 promoters (http://www-sequence.stanford.edu/group/candida). The scale is relative to the firstnucleotide of each coding region.

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regulation are based on this presumption (5, 9, 26). However,this remains to be confirmed.

Hypha-specific genes carry E-boxes in their promoters. Anumber of hypha-specific genes have been identified in C.albicans. Several of these genes, such as HWP1, HYR1, ALS3,and ALS8, encode hyphal wall proteins (1, 12, 14, 25). Thefunctions of other genes, such as ECE1 and RBT4, remainobscure (2–4). Activation of some hypha-specific genes de-pends upon Efg1 but not upon Cph1 (3, 23). Cph1 is a Ste12-

like transcription factor which lies on the MAP kinase signal-ling pathway that regulates C. albicans morphogenesis (13, 15).We tested whether ALS8 and HYR1 are regulated by Efg1 andCph1 by using an isogenic set of C. albicans strains (Fig. 1).Both mRNAs were induced following serum stimulation inwild-type and cph1/cph1 cells, but they were not induced inefg1/efg1 cells or an efg1/efg1 cph1/cph1 double mutant. Hence,as expected (3, 23), activation of ALS8 and HYR1 was depen-dent upon Efg1 but not upon Cph1 under these conditions.

bHLH transcription factors related to Efg1 are known tobind to the E box (consensus sequence, 59-CANNTG-39 [18,22]). Hence, we studied whether the promoter regions of hy-pha-specific genes contain E boxes by using regulatory se-quence analysis tools (27). The ALS3, ALS8, ECE1, HYR1,HWP1, and RBT4 promoters all contain multiple E boxes (Fig.2), which is consistent with the idea that Efg1 interacts with theE box to regulate expression of these promoters.

Synthetic Efg1 binds to an E box. To test whether Efg1 caninteract with an E box, we made two forms of synthetic Efg1.First, Efg1 was made with the TNT coupled in vitro transcrip-tion-translation system (Promega, Southampton, United King-dom) by using pGEM-EFGI, which contains the PCR-ampli-fied and resequenced EFG1 open reading frame in pGEM11Z(Promega). Second, a maltose binding protein (MBP)-Efg1fusion was made in Escherichia coli by using pMAL-EFG1(17), which contains the EFG1 open reading frame cloned intopMAL-c (New England BioLabs, Beverly, Mass.) (8). The lev-els of expression of MBP-Efg1 were low, apparently becausethis protein was toxic to E. coli. Nevertheless, the in vitro Efg1and MBP-Efg1 were the expected sizes (Fig. 3) given the pre-dicted molecular mass of Efg1, 60 kDa (26).

FIG. 3. Production of synthetic Efg1s. (A) In vitro transcriptionand translation of EFG1: sodium dodecyl sulfate-polyacrylamide gelelectrophoresis gel containing [35S]-labelled reaction product. Lane M,markers; lane 1, control reaction with empty pGEM11Z vector; lane 2,reaction with pGEM-EFG1. (B) Synthesis of a MBP-Efg1 fusion pro-tein in E. coli: Coomassie blue-stained sodium dodecyl sulfate-polyac-rylamide gel electrophoresis gel containing soluble protein extracts.Lane M, size markers; lane 1, pMAL-c and no IPTG (isopropyl-b-D-thiogalactopyranoside); lane 2, pMAL-c with IPTG; lane 3, pMAL-EFG1 with IPTG. The positions of MBP (open arrow) and MBP-Efg1(solid arrow) are indicated on the right.

FIG. 4. Synthetic Efg1s interact specifically with the E box in vitro. (A) Gel shift assays in which an E box-containing fragment from the ALS8promoter (2196 to 2106) was used. (B) Gel shift assay in which an E box-containing oligonucleotide was used. The extracts used included a controlin vitro transcription and translation extract lacking EFG1 (iv), an analogous extract containing EFG1 (iv Efg1p), a control E. coli extractcontaining the empty MBP expression plasmid (MBP), and an analogous extract containing the MBP-EFG1 fusion plasmid (MBP-Efg1p) (Fig. 3).The probes and competitors used were an ALS8 promoter fragment (F) (2196 to 2106), an E box-containing oligonucleotide (E) (59-AGAGATGCATTTGCTAGAGATGCATTTGCTAGACTT), and a control oligonucleotide lacking E boxes (C) (59-AGAGATGTGCCGATTAGAGATGTGCCGATTAGACTT).

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Both synthetic Efg1s were used in gel shift experiments (7)to test whether they interacted specifically with E box-contain-ing DNA fragments. Both Efg1s formed complexes with a90-bp region of the ALS8 promoter that contains an E box(Fig. 4A) and an E box-containing oligonucleotide (Fig. 4B).These complexes were competed out by E box-containingDNA molecules but not by a control oligonucleotide lacking anE box. Furthermore, the complexes were not formed by controlextracts lacking Efg1. Therefore, Efg1 is a sequence-specificDNA binding protein capable of binding the E box.

A hypha-specific promoter forms an Efg1-dependent com-plex. We then tested whether a hypha-specific promoter is ableto form Efg1-dependent complexes with C. albicans cell ex-tracts. A 90-bp ALS8 promoter fragment, which contained anE box, formed sequence-specific complexes with wild-type ex-tracts (Fig. 5A). This fragment formed similar complexes withcell extracts prepared from yeast or hyphal cells (data notshown). Formation of the largest of these complexes was in-hibited by inactivation of Efg1 but not by inactivation of Cph1.Therefore, the E box-containing region of the ALS8 promoterformed an Efg1-dependent complex.

The specificity of this interaction was examined further bytesting whether other fragments of the ALS8 promoter, whichlacked an E box, were capable of forming this Efg1-dependentcomplex. The following fragments were examined by usingextracts prepared from wild-type and efg1/efg1 cells: 2696 to

2597, 2596 to 2495, 2496 to 2388, 2412 to 2281, and 2306to 2197. None of these ALS8 promoter fragments generatedthe Efg1-dependent complex (data not shown), unlike the Ebox region (2196 to 2106) (Fig. 5). The observation that theEfg1-dependent complex can form with cell extracts from yeastcells (Fig. 5A) and the observation that synthetic forms of Efg1can interact directly with the E box (Fig. 4) suggest that thisDNA binding activity is not dependent on activation of Efg1 bya morphogenetic stimulus.

Ernst (9) has pointed out that bHLH proteins can regulategenes that do not contain an E box. Also, we did not identifyan E box in the 59 region of RBT1, which is regulated by Efg1(3). It is conceivable that RBT1 is regulated indirectly by Efg1or that Efg1 can bind sequences other than E boxes. Never-theless, our data confirm that Efg1 is a sequence-specific DNAbinding protein that is capable of binding the E box and thatmost hypha-specific genes contain E boxes in their promoters.This is consistent with the idea that Efg1 interacts directly withhypha-specific promoters in C. albicans to activate transcrip-tion of the promoters during hyphal development, and this hasimportant implications for regulation of cellular morphogene-sis in this human pathogen.

We thank Gerald Fink and Joachim Ernst for providing strains, LoisHoyer for providing information about the ALS3 promoter, and SusanBudge for excellent technical assistance. We are grateful to the Stan-ford DNA Sequencing and Technology Center for access to theirC. albicans genome sequence data (http://www-sequence.stanford.edu/group/candida), which were generated with the support of the NIDRand the Burroughs Wellcome Fund.

P.L. was supported by the UK Biotechnology and Biological Sci-ences Research Council (grants 1/CEL4563 and 97/B1/P/03008).P.R.L. and A.J.P.B. were supported by The Wellcome Trust (grants041399, 055015, and 063204).

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