EUKARYOTIC CELL, Mar. 2008, p. 483–492 Vol. 7, No. 31535-9778/08/$08.00�0 doi:10.1128/EC.00445-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Gene Overexpression/Suppression Analysis of Candidate VirulenceFactors of Candida albicans�

Yue Fu,1,2* Guanpingsheng Luo,1 Brad J. Spellberg,1,2

John E. Edwards, Jr.,1,2 and Ashraf S. Ibrahim1,2

Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance,California 90502,1 and Department of Medicine, David Geffen School of Medicine at University of

California—Los Angeles, Los Angeles, California 900242

Received 7 December 2007/Accepted 18 December 2007

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enablesimultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involvedinsertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy,a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened forvirulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adher-ence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitroresults, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis.In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibilityto neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenouslydisseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitrophenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumu-lation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins.These data indicate that the same gene can increase or decrease candidal virulence in distinct models ofinfection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally,these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidalvirulence factors.

Candida albicans causes hematogenously disseminated andmucosal infections (7, 33). Despite current antifungal therapy,mortality and morbidity are unacceptably high (1, 16, 34).Therefore, new prophylactic and therapeutic strategies are ur-gently needed. The identification of virulence genes that can betargeted to alter pathogenicity is a necessary step in devisingnovel strategies to treat or prevent Candida infections.

Virulence factors in C. albicans include proteins that medi-ate adherence to and invasion of host tissues (43), morpholog-ical change from yeast to hyphae (29, 30), secretion of lyticenzymes (17, 27, 41), maintenance of cell wall integrity (55),and avoidance of the host immune response (39). Many ofthese virulence factors are glycosylphosphatidylinositol (GPI)-anchored proteins, which comprise 88% of all covalently linkedcell wall proteins in C. albicans (23). Examples of GPI-an-chored virulence factors are Phr1p and Phr2p, which mediatecell wall biogenesis and hyphal formation in response tochanges in pH (15); Hwp1p, an epithelial adhesin and biofilmpromoter (32) that is required for virulence during murinehematogenously disseminated candidiaisis (48); Als1p andAls3p, adhesins with broad substrate specificity (13, 22, 43);and Gpi7, an antivirulence factor that reduces candidal resis-

tance to macrophages and virulence in mice (36). NumerousGPI proteins have been identified as virulence factors in C.albicans.

To identify new virulence factors in C. albicans, we used aconditional gene overexpression/suppression approach toscreen a library of genes encoding GPI-anchored proteins forvirulence phenotypes. We used the strong tetracycline-regu-lated (TR) promoter to control gene expression (31, 42). In thepresence of the tetracycline analogue doxycycline (DOX), theexpression of genes controlled by the strong TR promoter issuppressed. In contrast, in the absence of DOX, the gene ispotentially overexpressed. By simultaneously screening forgain and loss of function, we identified a gene, IFF4, that hadcontrasting functions in distinct anatomical contexts, promot-ing epithelial cell adherence but also ameliorating virulenceduring disseminated infection in mice.

(This work was presented in part at the 47th InterscienceConference on Antimicrobial Agents and Chemotherapy, Chi-cago, IL, 17 to 20 September 2007, abstr. B-1445.)

MATERIALS AND METHODS

Strains, media, and plasmids. The C. albicans strains used in this study arelisted in Table 1. All strains were generated from strain THE1, a generous giftfrom Hironobu Nakayama (31). The HIS1-TR promoter cassette plasmid wasconstructed by replacing the URA3 gene in p99CAU1 (kindly provided by H.Nakayama) with a HIS1 fragment (encompassing 543 bp upstream and 355 bpdownstream of its open reading frame [ORF]) amplified from THE1 genomicDNA, using 5�-His1 and 3�-His1 as primers (Table 2). All Candida strains werecultured in YPD medium (1% yeast extract [Difco], 2% Bacto peptone [Difco],2% D-glucose) with or without 20 mg/liter DOX (Sigma-Aldrich, St. Louis, MO)

* Corresponding author. Mailing address: Division of InfectiousDiseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502.Phone: (310) 222-6425. Fax: (310) 782-2016. E-mail: yfu@labiomed.org.

� Published ahead of print on 4 January 2008.

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at 30°C. The growth rates of strains whose virulence levels in animal models werecompared were determined in YPD in the presence or absence of 20 mg/literDOX (38). For strain construction, yeasts were grown in GMM (2% glucose,0.5% ammonium sulfate, and 1� yeast nitrogen base without ammonium sulfateand amino acid) with or without nutrient supplementation. Fluoro-orotic acid(5-FOA) (Sigma-Aldrich; 0.1%) was used for selecting ura3 strains. Media weresolidified with 1.5% Difco agar as necessary.

Construction of a GPI gene overexpression library. Oligonucleotides used inconstruction of the overexpressing/suppressing IFF4 and control strains areshown in Table 2. The library was constructed in THE1, a C. albicans strain thatharbors a codon-optimized TR transactivator. Using a URA3 recyclable markerstrategy (54), one allele of HIS1 in THE1 was disrupted to yield strain THE2.THE2 was used as a control strain for initial GPI gene overexpression libraryscreening. The URA3 marker was eliminated by culturing THE2 on 5-FOAplates to generate THE3. Next, the second HIS1 allele was disrupted in THE3,yielding THE4.

C. albicans THE4 was utilized to generate a GPI gene overexpression libraryusing the strategy outlined in Fig. 1. Briefly, two rounds of PCR were used togenerate fragments C and D. In the first round of PCR, the flanking regions onboth sides of the desired integration site were amplified using primers P1 and P3

to yield fragment A (an upstream sequence of the targeted gene), and primers P2

and P4 were used to yield fragment B (approximately 500 bp from the start of thetargeted gene). Primers P3 and P2 contained a 5� extension (25 bp) that wascomplementary to the 5� HIS1 or 3� TR promoter sequence, respectively (Fig. 1).The second round of PCR utilized primers P1 and PH1 to obtain fragment C,which contained an approximately 500-bp sequence upstream of the ORF of thetargeted gene, as well as a 3�-truncated HIS1. Similarly, fragment D containinga 5�-truncated HIS1, an intact TR promoter, and approximately 500 bp from thestart of the ORF of the targeted gene was generated by using primers PH2 andP4. Therefore, fragments C and D contained flanking sequences to target inte-gration and overlapping, nonfunctional fragments of the HIS1 selection marker.

Fragments C and D were cotransformed into strain THE4, and His1� pro-totrophs were selected. A triple-crossover event among fragments C and D andthe chromosome resulted in integration of the HIS1-TR promoter cassette infront of the targeted GPI gene. The desired integration event was confirmed byPCR using a downstream, ORF-specific primer, P5, and a universal upstreamprimer, PH2. Finally, the expression of the gene without DOX or with DOX wasconfirmed by reverse transcription RT-PCR using P2 and P4 as primers.

Construction of strain CAA10-31, in which both IFF4 alleles are controlled bythe TR promoter. The C. albicans strain in which one allele of IFF4 was con-trolled by the TR promoter (strain CAA10) was cultured on plates containing5-FOA, yielding strain CAA10-1. The resulting strain, lacking the URA3 marker,was utilized to construct strain CAA10-31, in which both alleles of IFF4 werecontrolled by the TR promoter, as outlined in Fig. 2. Briefly, a recyclable URA3cassette (54) was used to disrupt the second URA3 allele. Clones were screenedby PCR using primers P6 and P5 to confirm disruption of the second allele (i.e.,the one not controlled by the TR promoter). After CAA10-2 was cultured on

5-FOA plates, clones in which interchromosomal recombination occurred, re-sulting in both alleles being controlled by the TR promoter, were selected,yielding strain CAA10-3. URA3 (from a 3.9-kb NheI-PstI fragment containingthe URA3-IRO1 gene) was inserted into its original locus on the CAA10-3chromosome, resulting in strain CAA10-31. The same URA3-IRO1 fragment wasintegrated into THE3, resulting in THE31, a control strain of CAA10-31.

Confirmation of IFF4 overexpression/suppression. To confirm that IFF4 wasregulated by the TR promoter in strains CAA10 (with one IFF4 allele controlledby the TR promoter) and CAA10-31 (with both IFF4 alleles controlled by the TRpromoter), we performed semiquantitative RT-PCR. After an overnight culture,the strains were grown with DOX (20 �g/ml) and without DOX for 6 h in YPD.Total RNA was extracted by the hot-acidic-phenol method (3). RNA was alsoextracted from strain THE31 grown with DOX and without DOX as a control.To eliminate genomic-DNA contamination, total RNA was treated with RNase-free DNase I (TURBO DNA-free; Ambion, Texas) according to the manufac-turer’s instructions. Lack of genomic-DNA contamination in preparations wasdemonstrated by the absence of a 919-bp band containing the intron of EFB1(50). DNA-free total-RNA samples were cleaned using the RNA Clean-up Kit(Zymo Research, Orange, CA). Next, an RT reaction procedure was performedwith RETROscript (Ambion, Texas). Primers P2 and P4 were used for gene-specific amplification. EFB1 also served as an internal quantitative control, andits specific primers are shown in Table 2.

Effects of overexpression of 12 GPI-anchored proteins on Als1p, anotherGPI-anchored protein. Twelve strains overexpressing RBT1, PGA8, SAP9, PLB3,PLB5, SOD6, HYR1, IFF2, IFF3, IFF4, FGR23, or PGA55 were constructed inthe library and were grown in YPD in the presence or absence of DOX. Over-expression and suppression of these genes encoding GPI-anchored proteins wereconfirmed by RT-PCR. We then used direct immunofluorescence, as we havedescribed previously (13), to quantify the degree of surface expression of Als1pin each of the 12 strains. In brief, the strains were incubated in RPMI 1640medium with glutamine for 1 h at room temperature to induce Als1p expression.Als1p was detected and quantified by incubating intact organisms with a fluo-rescein isothiocyanate-labeled anti-Als1p monoclonal antibody for 1 h. As anegative control, the strains were labeled with a fluorescein isothiocyanate-labeled nonspecific mouse isotype-matched control antibody. Flow cytometrywas then performed to determine the relative expression of Als1p in each straingrown in the presence or absence of DOX using a FACSCaliber (Becton Dick-inson) flow cytometer. The mean fluorescence intensities of 104 events werecalculated using CELLQUEST software.

Adherence assays. Adherence to plastic was tested by growing strains in YPDmedium with or without DOX to early stationary phase in culture tubes (Falcon).The fungal cultures were discarded, and the tubes were gently washed threetimes with tap water. Adherence to plastic was assessed visually. No difference inthe morphologies of overexpressing, suppressing, or wild-type strains was iden-tified.

Adherence of IFF4 overexpression/suppression mutants to the FaDu oralepithelial cell line (ATCC) and endothelial cells was also assessed. FaDu cells

TABLE 1. C. albicans strains in this study

Strain Genotype Source

THE1 ade2::hisG/ade2::hisG ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 H. NakayamaTHE2 ade2::hisG/ade2::hisG HIS1/his1::URA3-dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-

ScHAP4AD-3XHA-ADE2This study

THE3 ade2::hisG/ade2::hisG HIS1/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2

This study

THE31 ade2::hisG/ade2::hisG HIS1/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434::URA3-IRO1 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2

This study

THE4 ade2::hisG/ade2::hisG his1::URA3-dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2

This study

CAA10 ade2::hisG/ade2::hisG his1::URA3-dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 IFF4/HIS1-pTR-IFF4

This study

CAA10-1 ade2::hisG/ade2::hisG his1::dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 IFF4/HIS1-pTR-IFF4

This study

CAA10-2 ade2::hisG/ade2::hisG his1::dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 iff4::URA3-dpl200/HIS1-pTR-IFF4

This study

CAA10-3 ade2::hisG/ade2::hisG his1::dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434 ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 HIS1-pTR-IFF4/HIS1-pTR-IFF4

This study

CAA10-31 ade2::hisG/ade2::hisG his1::dpl200/his1::dpl200 ura3-iro1::imm434/ura3-iro1::imm434::URA3-IRO1ENO1/ENO1-tetR-ScHAP4AD-3XHA-ADE2 HIS1-pTR-IFF4/HIS1-pTR-IFF4

This study

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were maintained in minimal essential medium-Earl’s salts (Irvine Scientific)containing 10% fetal bovine serum, 1 mM pyruvic acid, 2 mM L-glutamine, 0.1mM nonessential amino acids, 100 IU/ml penicillin, and 100 IU/ml streptomycin.The cells were grown to confluence in six-well tissue culture plates (Costar, VanNuys, CA) prior to performance of the adherence assay. Endothelial cells wereobtained from human umbilical veins as we have described previously (13, 21),and second-passage cells were grown to confluence in six-well tissue cultureplates coated with 0.2% gelatin matrix (Collaborative Biomedical Products,Bedford, MA). After the cell monolayers were rinsed twice with prewarmedHanks balanced salt solution (HBSS), blastospores (3 � 102 cells/ml of HBSS)grown in either YPD with DOX or YPD without DOX for 19 h at 25°C wereadded to each well. The plate was incubated in 5% CO2 at 37°C for 30 min, afterwhich the nonadherent organisms were aspirated and the FaDu or endothelialcell monolayers were rinsed twice with 10 ml of HBSS in a standardizedmanner. A 1.5-ml volume of YPD was added to each well and allowed tosolidify. After the plate was incubated at 37°C for 24 h, the number ofadherent organisms was determined by colony counting. Adherence was

expressed as a percentage of the initial inoculum, which was confirmed byquantitative culturing in YPD agar. Each adherence assay was performed intriplicate on three separate occasions.

Endothelial cell damage assay. The ability of IFF4 overexpression/suppressionto modulate C. albicans-induced endothelial cell injury was determined with thechromium (51Cr) release assay in 96-well tissue culture plates as describedpreviously (52).

Neutrophil-mediated killing assay. In vitro human neutrophil-mediated killingwas quantified by a modification of our previously described method (46).Briefly, 4 � 104 purified neutrophils were cocultured with 2 � 104 C. albicanscells (2:1 ratio) for 1 h at 37°C in RPMI plus 10% pooled human serum (Sigma-Aldrich). The cultures were sonicated to kill residual neutrophils, serially diluted,overlaid with YPD, and incubated overnight at 37°C. CFU were counted andcompared to the number of CFU plated from C. albicans cultures withoutneutrophils to assess killing of C. albicans.

Murine models. The effect of IFF4 overexpression/suppression on the viru-lence of C. albicans was assessed in the murine models of vaginal candidiasis (47)

TABLE 2. Oligonucleotides used in this study

Oligonucleotide Sequence

Oligonucleotides used for a wild-type HIS1gene cloning and HIS1-pTR cassette

5�-His1...........................................................................................CGGGATCCAGAATGTGCCGTTGTGTTTG3�-His1...........................................................................................CGGGATCCGTACCAGGTGAACTGTTTAATTG

Oligonucleotides used for generating his1 null mutantrequired for GPI gene overexpressionlibrary construction

HISKN-1A....................................................................................AAGGGCAGATTATACGAAAAATGCTGTAACTTATTGAGTGGTGCCGATATACAGTTTAGAGTTTTCCCAGTCACGACGTT

HISKN-1B ....................................................................................AGATCTAATAGATTAGATATAGCACTTTCTACAAACTTGCCAATTGCATTAATCTTCTTGGTTTTCCCAGTCACGACGTT

HISKN-2A....................................................................................TTAAAGGTGTGTACATCAAGGTGGTAGATAAAGATGGTATAAGACAGATTGAGTCAAATTTGTGGAATTGTGAGCGGATA

HISKN-2B ....................................................................................TCTACAATTTGATATCTCGAGTACCAATATATCGGTTGCACCAGCTTTCTTCAATTCGTCTGTGGAATTGTGAGCGGATA

HisConfirm1.................................................................................CCAGACCGTTTGTTATTTGCTGHisConfirm2.................................................................................GTCAAGGAATTACAAACGAGAATGC5-detect .........................................................................................GTTTTCCCAGTCACGACGTTGTAAAACGAC3-detect .........................................................................................TGTGGAATTGTGAGCGGATAACAATTTCAC

Oligonucleotides used for making andconfirming IFF4 conditionaloverexpression/suppression strain

P1 ...................................................................................................ACCACCATATTGCGCTTTTCTAP2 ...................................................................................................ACAGCTTTATCTCAGAAAAACTAGTTCCGTTTTCTTTCACGTCAACP3 ...................................................................................................CGACAAACACAACGGCACATTCTGGCTTTAACGATTTGCAAAATTTAGP4 ...................................................................................................ACCACTGTTGGTAATAGATCCPH1.................................................................................................GTCGTCGCTGTGTTTGTCPH2.................................................................................................CGTTGGAGAAGGTAATTGTGAP5 ...................................................................................................AGTGGAATCACCATTAGCAGAP6 ...................................................................................................CTAAATTTTGCAAATCGTTAAAGCCIFF4KN1.......................................................................................GCACCAATCTCATCAAGTGAGTTTATTAGTTTGGAATCGTGTTTTCCC

AGTCACGACGTTIFF4KN2.......................................................................................GGATGTTTCAAACGGTAATTCGCTCCAACAAGAGGGTTCATGTGGAA

TTGTGAGCGGATAIFF4CONF1.................................................................................CACTTTAGCTAAAGTATCATCTACTGIFF4CONF2.................................................................................TTCGGAATAGGATGGTTTGAC

Oligonucleotides used for detecting IFF4in vivo expression

IFF4 specific 1 .............................................................................CTGCACCAATCTCATCAAGTGAIFF4 specific 2 .............................................................................GATCCACCAGCACTTGAGGAEFB1a ...........................................................................................ATTGAACGAATTCTTGGCTGACEFB1b...........................................................................................CATCTTCTTCAACAGCAGCTTG

Oligonucleotides used for confirming URA3-IRO1in its original locus

URA3 Conf1.................................................................................TGCTGGTTGGAATGCTTATTTGURA3 Conf2.................................................................................TGCAAATTCTGCTACTGGAGTT

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and hematogenously disseminated candidiasis. For the vaginal model, BALB/cmice (Charles River) were treated with estradiol valerate (30 �g subcutaneously)in peanut oil (both from Sigma-Aldrich) on day �3 relative to infection to inducepseudoestrus. On the day of infection, mice were sedated by intraperitonealadministration of 100 mg/kg of body weight of ketamine. The sedated mice wereinfected intravaginally with 106 blastospores of C. albicans in 10 �l of endotoxin-free phosphate-buffered saline. On day 1 postinfection, the vaginas and approx-imately 1 centimeter of each uterine horn were dissected en bloc, homogenized,and quantitatively cultured.

For survival studies, male BALB/C mice were infected with 5 � 105 blasto-spores of C. albicans through the tail vein (21). The mice were monitored threetimes daily, and moribund mice were euthanized. A second group of mice wereinfected and sacrificed 5 days postinfection to determine the tissue fungal bur-den. Kidneys, brain, and liver were removed, homogenized, and quantitativelycultured on YPD containing 50 �g/ml chloramphenicol. Values were expressedas log CFU per gram of tissue.

The mice were given food and water ad libitum throughout the course of theexperiment. Mice infected with IFF4 conditional overexpression/suppression orcontrol strains were given water with or without DOX (2 mg/ml) dissolved in 5%sucrose solution throughout the period of the experiment starting from day �2relative to infection (42). All procedures involving mice were approved by theinstitutional animal use and care committee, following the National Institutes ofHealth guidelines for animal housing and care.

In vivo expression of IFF4. IFF4 expression by wild-type C. albicans SC5314was examined in the models of vaginal or hematogenous disseminated candidi-asis. BALB/c mice were infected intravaginally or intravenously as describedabove. Twenty-four hours later, the mice were sacrificed and the vaginas orkidneys were collected as described above, cut into small pieces, and put into ared tube (Q-BIOgene) with a 1/4-inch ceramic bead (Q-BIOgene). Next, thesamples were frozen in liquid nitrogen in the presence of 1 ml Tri reagent(Ambion) and homogenized twice with Fastprep FP120 (Bio 101 Thermo Elec-tro corporation) at speed 4 for 25 seconds. Total-RNA samples were extractedaccording to the manufacturer’s instructions. cDNA synthesis and RT-PCR werecarried out as outlined above, except that IFF4-specific primers 1 and 2 wereused to amplify IFF4-specific bands and primers EFB1a and EFB1b were used toamplify the housekeeping gene, EFB1 (Table 2). The PCR conditions were asfollows: denaturing at 94°C for 2 min and amplification for 40 cycles at 94°C for1 min, 52°C for 1 min, and 72°C for 2 min.

Statistical analysis. Adherence and neutrophil-mediated killing were com-pared by the Mann-Whitney U test for unpaired comparisons. The nonparamet-ric log rank test was utilized to determine differences in the survival times of themice. Tissue fungal burdens among different groups were compared by the Steel

test for nonparametric multiple comparisons (35) or the Mann-Whitney U testfor unpaired comparisons, as appropriate. P values of �0.05 were consideredsignificant.

RESULTS

Iff4p mediates C. albicans adherence to plastic. Using asplit-marker, triple-crossover strategy (Fig. 1), we made anincomplete GPI gene overexpression library that included thefollowing members: RBT1, PGA8, SAP9, PLB5, SOD6, PLB7,HYR1, IFF2, IFF3, IFF4, FGR23, SOD5, ORF19.6420, ORF19.893, ORF19.4404, ORF19.13035, ORF19.3738, ORF19.3740,ORF19.6336, ORF19.2475, ORF19.5141, ORF19.5303, ORF19.5760, ORF19.2759, and ORF19.6321. The gene products werebelieved to be cell surface localized. The constructed strains hadone allele of the targeted gene controlled by the TR promoter.Overexpression of these genes in the absence of DOX wasconfirmed by RT-PCR, comparing RNA samples extractedfrom mutants grown in the presence or absence of DOX (datanot shown).

To determine if overexpression of a GPI-anchored proteinaffected the expression of other GPI proteins, we investigatedthe expression of Als1p (a known GPI-anchored protein) in 12randomly picked strains, each of which overexpressed a differ-ent GPI-anchored protein. Als1p surface accumulation wasquantified by direct immunofluorescence and flow cytometry(13). No effect on Als1p surface accumulation was detectedamong the 12 strains tested (Fig. 2).

To screen the library for an adherence function, we grew theoverexpression strains in plastic tubes containing YPD with orwithout DOX. One clone, overexpressing the IFF4 gene, dem-onstrated enhanced adherence to plastic (Fig. 3). This clonewas selected for further study. The other library strains showedno enhanced adherence to plastic.

FIG. 1. Diagram detailing our split-marker strategy to insert the TR promoter in front of the gene encoding putative GPI-anchored pro-teins.

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IFF4 overexpression/suppression modulates the adherenceof C. albicans to epithelial cells. To completely control theexpression of IFF4, we placed the second allele of IFF4 underthe control of the TR promoter by interchromosomal recom-bination (Fig. 4). The generated strain (CAA10-31) had bothIFF4 alleles controlled by the TR promoter. To avoid artifactsrelated to the chromosomal locus of the URA3 marker (4, 6, 8,26, 49, 51), we also restored URA3-IRO1 to its original locus.After confirming the abrogation of IFF4 expression with DOXand IFF4 overexpression without DOX compared to the con-

trol (THE31) (Fig. 5A), we compared the abilities of thesestrains to adhere to human epithelial cells. Overexpression ofIFF4 enhanced adherence to epithelial cells by more than 68%compared to IFF4 suppression (IFF4 without DOX), whereassuppression of IFF4 decreased adherence by 22% compared tothe control strain grown with DOX (Fig. 5B).

Overexpression of IFF4 increased the tissue fungal burdenduring vaginal candidiasis. To determine the in vivo signifi-cance of IFF4-mediated adherence to epithelial cells, we usedour murine model of candidal vaginitis (47). Since adherence isan early event during mucosal infection, we determined thevaginal fungal burden at 24 h in mice infected with the IFF4-conditional expressing strain (CAA10-31) or control C. albi-cans (THE31) grown without DOX or with DOX. Mice in-fected with C. albicans overexpressing IFF4 (without DOX)had a significantly higher vaginal fungal burden than thoseinfected with IFF4-suppressed C. albicans (with DOX) orthose infected with the control strain (Fig. 6A). There was nodifference in vaginal fungal burdens between mice infectedwith C. albicans CAA10-31 grown under IFF4 suppressionconditions and those infected with the control strain, THE31.

We also sought to confirm that the IFF4 gene was expressed

FIG. 2. Overexpression of GPI-anchored proteins does not affect the expression of Als1p, a known GPI-anchored protein. Twelve overex-pression strains and one control strain were screened by flow cytometry. A representative strain is shown; all other strains demonstrated equivalentcharacteristics. The strains were grown under conditions in which Als1p was either known not to be expressed or known to be expressed (13).Overexpression of the GPI protein had no effect on Als1p surface accumulation (no difference between the results without DOX [�DOX] andwith DOX [�DOX]).

FIG. 3. Overexpression of IFF4 induces adherence of C. albicans toculture plastic tubes. The control strain used was C. albicans THE2.

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during murine vaginitis. Both wild-type C. albicans and IFF4-overexpressing C. albicans (the positive control) expressedIFF4 at 24 h during murine vaginitis, while no band was am-plified from uninfected mice (negative control) (Fig. 6B). The

constitutively expressed candidal EFB1 gene served as a quan-titative control, as well as a control for lack of genomic con-tamination of C. albicans (50) (Fig. 6B).

IFF4 overexpression increases neutrophil killing of C. albi-cans. To define potential interactions of IFF4 with host cellsencountered during hematogenously disseminated infection,we investigated the conditional expression strain’s ability tobind to or damage endothelial cells and its susceptibility toneutrophil-mediated killing. Overexpression/suppression ofIFF4 did not have any effect on C. albicans adherence to ordamage of endothelial cells (data not shown). However, C.albicans overexpressing IFF4 (without DOX) was more sus-ceptible to neutrophil-mediated killing in vitro than suppressedcells (with DOX) or the control strain (Fig. 7A).

Overexpression of IFF4 reduces the severity of hematog-enously disseminated candidaisis in normal, but not neutro-penic, mice. To determine the in vivo significance of the en-hanced neutrophil-mediated killing of IFF4-overexpressing C.albicans, we tested the contribution of IFF4 to C. albicansvirulence during hematogenously disseminated candidiasis inimmunocompetent and neutropenic mice. All C. albicansstrains tested for virulence showed similar growth rates invitro, with P values of �0.05 (doubling times were as follows:THE31 without DOX, 1.38 h; THE31 with DOX, 1.34 h;CAA10-31 without DOX, 1.45 h; and CAA10-31 with DOX,1.35 h). In the immunocompetent-mouse model, overexpres-sion of IFF4 (without DOX) significantly reduced the virulenceof C. albicans CAA10-31 compared to mice infected with thesame strain grown under IFF4 suppression conditions (withDOX) or those infected with the control strain, THE31 (Fig.7B). In addition to enhanced survival, mice infected with theIFF4 overexpression strain had more than a 10-fold reductionin the kidney fungal burden compared to mice infected withthe same strain grown under IFF4 suppression conditions (withDOX) or mice infected with the control strain. THE31 (Fig.7C). In contrast, there was no difference in survival between

FIG. 4. Strategy to generate a homozygous mutant in which both alleles of the desired gene were controlled by the TR promoter.

FIG. 5. Overexpression of IFF4 results in enhanced adherence ofC. albicans to FaDu epithelial cells. (A) RT-PCR results for IFF4demonstrating overexpression of the gene without DOX medium andlack of expression with DOX medium. The P2 and P4 primers (Table2) were used to amplify IFF4. The EFB1 fragment was coamplified andserved as a control. Lack of genomic-DNA (gDNA) contamination incDNA preparations was demonstrated by the absence of a 919-bp bandcontaining the intron of EFB1. THE31 was the control strain, andCAA10-31 was the strain overexpressing IFF4. (B) Adherence ofstrains THE31 and CAA10-31 grown without DOX (overexpressioncondition) or with DOX (suppression condition) to FaDu epithelialcells. The data are displayed as the median � the interquartile. *, P 0.01 compared to control plus DOX; **, P � 0.002 versus all others.

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neutropenic mice infected with C. albicans overexpressingIFF4 (without DOX) and those infected with the same straingrown under IFF4-suppressing conditions (with DOX) orthose infected with the control strain (Fig. 7D).

Because overexpression of IFF4 decreased the virulence ofC. albicans in mice infected via the tail vein, we hypothesizedthat this gene is likely not expressed in the wild-type strainduring hematogenously disseminated candidiasis. Consistentwith this hypothesis, we could not detect expression of IFF4 byRT-PCR in kidneys harvested from mice 24 h after intravenousinfection with wild-type C. albicans (Fig. 8).

DISCUSSION

We used a conditional gene overexpression/suppression ap-proach to identify a candidal virulence factor, Iff4p. The IFF4gene was found to promote epithelial adherence and mucosalinfection in mice. Of note, a recent publication showed that thegerm tubes of a C. albicans iff4 null mutant generated by genedisruption had decreased adherence to plastic surfaces (24).

When we suppressed IFF4 expression in our URA3 repairedstrains, we also found decreased adherence to epithelial cellsusing blastospores. However, the reduced epithelial adherenceduring suppression of IFF4 expression did not translate into avirulence phenotype in vivo, likely due to functional redun-dancy with other adhesins. It is also possible that our failure todetect a loss-of-function phenotype in vivo was due to low andundetectable IFF4 expression under suppression conditions(with DOX).

IFF4 belongs to the IFF gene family, which contains 12members (IFF1 to IFF11 and HYR1) (37). The proteins have aconserved domain that does not display any significant homol-ogy to proteins with known functions (5). Most of the IFFfamily members encode proteins that exhibit the characteristicstructure of GPI-anchored cell wall proteins, with the excep-tion of Iff10p and Iff11p, which do not have any signal sequencefor GPI anchor linkages (37). In addition, IFF7 appears to belocated in the plasma membrane rather than in the cell wall(5). The function of this gene family is largely unknown, withthe exception of IFF11, which encodes a secreted protein thatis required for cell wall structure and virulence (5). In thisstudy, we found that IFF4 promotes adherence to plastic andepithelial cells, but not endothelial cells. This epithelial adher-ence function was specific to IFF4, since overexpression/sup-pression of two other members of the IFF family, namely, IFF2(HYR3) and IFF3, did not alter adherence (data not shown).The lack of altered adherence in these strains of IFF2 and IFF3suggests that the adherence function of IFF4 is not localized inthe conserved region.

Overexpression of IFF4 also increased the susceptibility ofC. albicans to neutrophil-mediated killing in vitro. Susceptibil-ity to neutrophil-mediated killing correlated with diminishedvirulence of C. albicans overexpressing IFF4 in the hematoge-nous model of infection using immunocompetent mice. Thevirulence of C. albicans overexpressing IFF4 was restored inneutropenic mice, emphasizing that the diminished virulencein normal mice was due to enhanced clearance of the yeast byneutrophils. Not surprisingly, wild-type C. albicans did notexpress the antivirulence factor, IFF4, during hematogenousseeding of the kidney. However, since forced expression of thegene caused a marked decrement in severity of infection, ac-tivation of IFF4 expression by small molecules is a potentiallynovel treatment for disseminated candidiasis.

The virulence factors of C. albicans are complex, and asdemonstrated by transcriptional-profiling studies, different vir-ulence factors may be operative in the same organism at dif-ferent times, depending upon the anatomical context (2, 11, 12,14). Our data therefore underscore the importance of testingC. albicans virulence factors that directly participate in hostinteraction in multiple models at different time points, reflect-ing the diversity of anatomical contexts that represent differentin vivo niches of infection.

The advantage of our conditional-expression system is theability to test both gain and loss of function in the same back-ground strain simultaneously. In Saccharomyces cerevisiae,gene phenotypes and pathway mapping can be achieved bysystematic gene overexpression (10, 45). However, in C. albi-cans, virulence gene function analysis using a similar approachis still in its infancy, despite the fact that gene dosage effectsare well documented in the organism (9, 53). Multiple lines of

FIG. 6. IFF4 overexpression increases the tissue fungal burden inthe mouse vagina. (A) Mice (n 8 per group) infected with C. albicansoverexpressing IFF4 had an increased vaginal fungal burden 24 hpostinfection compared to the same strain gown with DOX or to acontrol strain. The data are displayed as the median � the interquar-tile. �, P � 0.037 compared to IFF4 plus DOX or the control straincultured without DOX or with DOX. (B) RT-PCR results demonstrat-ing expression of IFF4 in the mouse vagina infected with wild-type C.albicans. RT-PCR of RNA samples extracted from a mouse vaginainfected with C. albicans overexpressing IFF4 was included as a posi-tive control, whereas RNA extracted from uninfected mice was in-cluded as a negative control. The EFB1 fragment was coamplified andserved as a loading control. Furthermore, lack of genomic DNA (g-DNA) contamination in cDNA preparations was demonstrated by theabsence of a 919-bp band containing the intron of EFB1.

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research indicate that overexpression of a gene results in adetectable, authentic phenotype (13, 18–20, 25, 28, 40, 44). Ourapproach of conditional gene overexpression/suppression cir-cumvents the need to construct a gene-complemented strain to

verify virulence phenotypes, as is required in the gene disrup-tion approach. Furthermore, insertion of the TR promoter intoone allele is well suited to C. albicans, a diploid organism,because this genetic maneuver maintains the expression of anyessential gene(s) even under suppression conditions (withDOX), allowing identification of the virulence function(s) ofan essential gene. In contrast, gene disruption cannot be usedto study the functions of essential genes.

One concern with using an overexpression strategy is so-called “capacity utilization.” For example, overexpression of aparticular GPI-anchored protein could potentially result inlimited space on the cell surface and restrict the expression ofother GPI-anchored proteins. Alternatively, overexpression ofa GPI-anchored protein could possibly alter the expressionof other proteins because of limited enzyme activity to add GPIanchors. We found no evidence of a capacity utilization prob-lem, as introduction of 12 randomly selected GPI genes, in-cluding IFF4, did not alter the surface accumulation of Als1p(another GPI-anchored protein).

In summary, we have described a conditional overexpres-sion/suppression approach that can be broadly used to identifynew genes encoding virulence-related phenotypes in C. albi-cans. This approach allows simultaneous evaluation of gain

FIG. 7. IFF4 overexpression increases neutrophil killing of C. albicans and reduces the severity of hematogenously disseminated candidiasis. (A) Invitro neutrophil-mediated killing of C. albicans strain THE31 or CAA10-31 grown under overexpression or suppression conditions. The data are displayedas the median � the interquartile. ‡, P � 0.001 compared to IFF4 plus DOX. (B) Survival of mice (n 8 per group) infected with a control strain orC. albicans IFF4 grown under overexpression or suppression conditions. �, P � 0.002 compared to mice infected with C. albicans IFF4 with DOX or thecontrol strain. The experiment is representative of two studies with similar findings. (C) Burden of C. albicans in kidneys of immunocompetent mice (n 8 per group) infected with C. albicans IFF4 or a control strain grown under overexpressing (�DOX) or suppressing (�DOX) conditions. Kidneys wereharvested 5 days postinfection. The data are displayed as the median � the interquartile. �, P � 0.025 versus no expression of IFF4 or the control strain.(D) In the absence of neutrophils, the virulence of IFF4-overexpressing C. albicans was restored. Shown is the survival of neutropenic mice (n 8 pergroup) infected with a control strain or C. albicans IFF4 grown under overexpression or suppression conditions.

FIG. 8. RT-PCR of RNA samples extracted from mouse kidneysinfected with wild-type C. albicans for 24 h. RNA extracted fromuninfected mice was included as a negative control, whereas RNAextracted from wild-type C. albicans SC5314 in vitro was included as apositive control. The EFB1 fragment was coamplified and served as aloading control. Lack of genomic-DNA contamination in cDNA prep-arations was demonstrated by the absence of a 919-bp band containingthe intron of EFB1.

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and loss of function. Using this new approach, we identifiedIFF4, a gene encoding a GPI-anchored protein, as an epithelialcell adhesin gene that modestly exacerbated mucosal infectionin vivo. We also identified IFF4 as encoding an antivirulencefactor during hematogenously disseminated infection. Thesedata underscore the need to study virulence genes in multipleanatomical contexts to discern the full potential range of phe-notypes and raise the possibility of targeting forced expressionof IFF4 as a treatment for disseminated infection.

ACKNOWLEDGMENTS

We thank Hironobu Nakayama for providing the TR expressionsystem. Research described in the article was conducted at the re-search facilities of the Los Angeles Biomedical Research Institute atHarbor-UCLA Medical Center.

This study was supported by Public Health Service grant R21AI066010 and American Heart Association Western State Affiliategrant 0665041Y to Y.F. and Public Health Service grants R01 AI19990and AI063382 to J.E.E. A.S.I. is supported by Public Health Servicegrants R01 AI063503 and R21 AI064716. B.J.S. is supported by PublicHealth Service grants K08 AI060641 and R01 AI072052 and AmericanHeart Beginning Grant-in-Aid 0665154Y.

REFERENCES

1. Alonso-Valle, H., O. Acha, J. D. Garcia-Palomo, C. Farinas-Alvarez, C.Fernandez-Mazarrasa, and M. C. Farinas. 2003. Candidemia in a tertiarycare hospital: epidemiology and factors influencing mortality. Eur. J. Clin.Microbiol. Infect. Dis. 22:254–257.

2. Andes, D., A. Lepak, A. Pitula, K. Marchillo, and J. Clark. 2005. A simpleapproach for estimating gene expression in Candida albicans directly from asystemic infection site. J. Infect. Dis. 192:893–900.

3. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith, and K. Struhl. 1994. Current protocols in molecular biology. JohnWiley & Sons, Inc., New York, NY.

4. Bain, J. M., C. Stubberfield, and N. A. Gow. 2001. Ura-status-dependentadhesion of Candida albicans mutants. FEMS Microbiol. Lett. 204:323–328.

5. Bates, S., J. M. de la Rosa, D. M. MacCallum, A. J. Brown, N. A. Gow, andF. C. Odds. 2007. Candida albicans Iff11, a secreted protein required for cellwall structure and virulence. Infect. Immun. 75:2922–2928.

6. Brand, A., D. M. MacCallum, A. J. Brown, N. A. Gow, and F. C. Odds. 2004.Ectopic expression of URA3 can influence the virulence phenotypes andproteome of Candida albicans but can be overcome by targeted reintegrationof URA3 at the RPS10 locus. Eukaryot. Cell 3:900–909.

7. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candidaalbicans. Trends Microbiol. 9:327–335.

8. Cheng, S., M. H. Nguyen, Z. Zhang, H. Jia, M. Handfield, and C. J. Clancy.2003. Evaluation of the roles of four Candida albicans genes in virulence byusing gene disruption strains that express URA3 from the native locus.Infect. Immun. 71:6101–6103.

9. De Backer, M. D., P. T. Magee, and J. Pla. 2000. Recent developments inmolecular genetics of Candida albicans. Annu. Rev. Microbiol. 54:463–498.

10. Forsburg, S. L. 2001. The art and design of genetic screens: yeast. Nat. Rev.Genet. 2:659–668.

11. Fradin, C., P. De Groot, D. MacCallum, M. Schaller, F. Klis, F. C. Odds, andB. Hube. 2005. Granulocytes govern the transcriptional response, morphol-ogy and proliferation of Candida albicans in human blood. Mol. Microbiol.56:397–415.

12. Fradin, C., M. Kretschmar, T. Nichterlein, C. Gaillardin, C. d’Enfert, and B.Hube. 2003. Stage-specific gene expression of Candida albicans in humanblood. Mol. Microbiol. 47:1523–1543.

13. Fu, Y., A. S. Ibrahim, D. C. Sheppard, Y.-C. Chen, S. W. French, J. E. Cutler,S. G. Filler, and J. E. J. Edwards. 2002. Candida albicans Als1p: an adhesinthat is a downstream effector of the EFG1 filamentation pathway. Mol.Microbiol. 44:61–72.

14. Garaizar, J., S. Brena, J. Bikandi, A. Rementeria, and J. Ponton. 2006. Useof DNA microarray technology and gene expression profiles to investigatethe pathogenesis, cell biology, antifungal susceptibility and diagnosis of Can-dida albicans. FEMS Yeast Res. 6:987–998.

15. Ghannoum, M. A., B. Spellberg, S. M. Saporito-Irwin, and W. A. Fonzi. 1995.Reduced virulence of Candida albicans PHR1 mutants. Infect. Immun. 63:4528–4530.

16. Gudlaugsson, O., S. Gillespie, K. Lee, J. Vande Berg, J. Hu, S. Messer, L.Herwaldt, M. Pfaller, and D. Diekema. 2003. Attributable mortality of nos-ocomial candidemia, revisited. Clin. Infect. Dis. 37:1172–1177.

17. Hube, B., D. Sanglard, F. C. Odds, D. Hess, M. Monod, W. Schafer, A. J.Brown, and N. A. Gow. 1997. Disruption of each of the secreted aspartyl

proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuatesvirulence. Infect. Immun. 65:3529–3538.

18. Huh, W. K., S. T. Kim, H. Kim, G. Jeong, and S. O. Kang. 2001. Deficiencyof D-erythroascorbic acid attenuates hyphal growth and virulence of Candidaalbicans. Infect. Immun. 69:3939–3946.

19. Hwang, C. S., Y. U. Baek, H. S. Yim, and S. O. Kang. 2003. Protective rolesof mitochondrial manganese-containing superoxide dismutase against vari-ous stresses in Candida albicans. Yeast 20:929–941.

20. Hwang, C. S., J. H. Oh, W. K. Huh, H. S. Yim, and S. O. Kang. 2003. Ssn6,an important factor of morphological conversion and virulence in Candidaalbicans. Mol. Microbiol. 47:1029–1043.

21. Ibrahim, A. S., F. Mirbod, S. G. Filler, Y. Banno, G. T. Cole, Y. Kitajima,J. E. Edwards, Jr., Y. Nozawa, and M. A. Ghannoum. 1995. Evidence im-plicating phospholipase as a virulence factor of Candida albicans. Infect.Immun. 63:1993–1998.

22. Kamai, Y., M. Kubota, T. Hosokawa, T. Fukuoka, and S. G. Filler. 2002.Contribution of Candida albicans ALS1 to the pathogenesis of experimentaloropharyngeal candidiasis. Infect. Immun. 70:5256–5258.

23. Kapteyn, J. C., L. L. Hoyer, J. E. Hecht, W. H. Muller, A. Andel, A. J.Verkleij, M. Makarow, H. Van Den Ende, and F. M. Klis. 2000. The cell wallarchitecture of Candida albicans wild-type cells and cell wall-defective mu-tants. Mol. Microbiol. 35:601–611.

24. Kempf, M., V. Apaire-Marchais, P. Saulnier, P. Licznar, C. Lefrancois, R.Robert, and J. Cottin. 2007. Disruption of Candida albicans IFF4 geneinvolves modifications of the cell electrical surface properties. Colloids Surf.B 58:250–255.

25. Kohler, G. A., T. C. White, and N. Agabian. 1997. Overexpression of a clonedIMP dehydrogenase gene of Candida albicans confers resistance to thespecific inhibitor mycophenolic acid. J. Bacteriol. 179:2331–2338.

26. Lay, J., L. K. Henry, J. Clifford, Y. Koltin, C. E. Bulawa, and J. M. Becker.1998. Altered expression of selectable marker URA3 in gene-disrupted Can-dida albicans strains complicates interpretation of virulence studies. Infect.Immun. 66:5301–5306.

27. Leidich, S. D., A. S. Ibrahim, Y. Fu, A. Koul, C. Jessup, J. Vitullo, W. Fonzi,F. Mirbod, S. Nakashima, Y. Nozawa, and M. A. Ghannoum. 1998. Cloningand disruption of caPLB1, a phospholipase B gene involved in the pathoge-nicity of Candida albicans. J. Biol. Chem. 273:26078–26086.

28. Leng, P., P. E. Sudbery, and A. J. Brown. 2000. Rad6p represses yeast-hyphamorphogenesis in the human fungal pathogen Candida albicans. Mol. Mi-crobiol. 35:1264–1275.

29. Lo, H. J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, andG. R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell90:939–949.

30. Mitchell, A. P. 1998. Dimorphism and virulence in Candida albicans. Curr.Opin. Microbiol. 1:687–692.

31. Nakayama, H., T. Mio, S. Nagahashi, M. Kokado, M. Arisawa, and Y. Aoki.2000. Tetracycline-regulatable system to tightly control gene expression inthe pathogenic fungus Candida albicans. Infect. Immun. 68:6712–6719.

32. Nobile, C. J., J. E. Nett, D. R. Andes, and A. P. Mitchell. 2006. Function ofCandida albicans adhesin Hwp1 in biofilm formation. Eukaryot. Cell 5:1604–1610.

33. Odds, F. C. 1988. Candida and candidiasis: a review and bibliography. Bal-liere Tindall, London, United Kingdom.

34. Pappas, P. G., J. H. Rex, J. Lee, R. J. Hamill, R. A. Larsen, W. Powderly,C. A. Kauffman, N. Hyslop, J. E. Mangino, S. Chapman, H. W. Horowitz,J. E. Edwards, and W. E. Dismukes. 2003. A prospective observational studyof candidemia: epidemiology, therapy, and influences on mortality in hospi-talized adult and pediatric patients. Clin. Infect. Dis. 37:634–643.

35. Rhyne, A. L., and R. G. Steel. 1967. A multiple comparisons sign test: allpairs of treatments. Biometrics 23:539–549.

36. Richard, M., S. Ibata-Ombetta, F. Dromer, F. Bordon-Pallier, T. Jouault,and C. Gaillardin. 2002. Complete glycosylphosphatidylinositol anchors arerequired in Candida albicans for full morphogenesis, virulence and resistanceto macrophages. Mol. Microbiol. 44:841–853.

37. Richard, M. L., and A. Plaine. 2007. Comprehensive analysis of glycosylphos-phatidylinositol-anchored proteins in Candida albicans. Eukaryot. Cell6:119–133.

38. Rieg, G., Y. Fu, A. S. Ibrahim, X. Zhou, S. G. Filler, and J. E. Edwards, Jr.1999. Unanticipated heterogeneity in growth rate and virulence among Can-dida albicans AAF1 null mutants. Infect. Immun. 67:3193–3198.

39. Romani, L., F. Bistoni, and P. Puccetti. 2003. Adaptation of Candida albi-cans to the host environment: the role of morphogenesis in virulence andsurvival in mammalian hosts. Curr. Opin. Microbiol. 6:338–343.

40. Rottmann, M., S. Dieter, H. Brunner, and S. Rupp. 2003. A screen inSaccharomyces cerevisiae identified CaMCM1, an essential gene in Candidaalbicans crucial for morphogenesis. Mol. Microbiol. 47:943–959.

41. Sanglard, D., B. Hube, M. Monod, F. C. Odds, and N. A. Gow. 1997. A tripledeletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 ofCandida albicans causes attenuated virulence. Infect. Immun. 65:3539–3546.

42. Saville, S. P., A. L. Lazzell, C. Monteagudo, and J. L. Lopez-Ribot. 2003.Engineered control of cell morphology in vivo reveals distinct roles for yeast

VOL. 7, 2008 C. ALBICANS GENE OVEREXPRESSION/SUPPRESSION ANALYSIS 491

on October 16, 2020 by guest

http://ec.asm.org/

Dow

nloaded from

and filamentous forms of Candida albicans during infection. Eukaryot. Cell2:1053–1060.

43. Sheppard, D. C., M. R. Yeaman, W. H. Welch, Q. T. Phan, Y. Fu, A. S.Ibrahim, S. G. Filler, M. Zhang, A. J. Waring, and J. E. Edwards, Jr. 2004.Functional and structural diversity in the Als protein family of Candidaalbicans. J. Biol. Chem. 279:30480–30489.

44. Sonneborn, A., D. P. Bockmuhl, M. Gerads, K. Kurpanek, D. Sanglard, andJ. F. Ernst. 2000. Protein kinase A encoded by TPK2 regulates dimorphismof Candida albicans. Mol. Microbiol. 35:386–396.

45. Sopko, R., D. Huang, N. Preston, G. Chua, B. Papp, K. Kafadar, M. Snyder,S. G. Oliver, M. Cyert, T. R. Hughes, C. Boone, and B. Andrews. 2006.Mapping pathways and phenotypes by systematic gene overexpression. Mol.Cell 21:319–330.

46. Spellberg, B. J., M. Collins, S. W. French, J. E. Edwards, Jr., Y. Fu, and A. S.Ibrahim. 2005. A phagocytic cell line markedly improves survival of infectedneutropenic mice. J. Leukoc. Biol. 78:338–344.

47. Spellberg, B. J., A. S. Ibrahim, V. Avanesian, Y. Fu, C. Myers, Q. T. Phan,S. G. Filler, M. R. Yeaman, and J. E. J. Edwards. 2006. Efficacy of theanti-Candida rAls3p-N or rAls1p-N vaccines against disseminated and mu-cosal candidiasis. J. Infect. Dis. 194:256–260.

48. Staab, J. F., S. D. Bradway, P. L. Fidel, and P. Sundstrom. 1999. Adhesiveand mammalian transglutaminase substrate properties of Candida albicansHwp1. Science 283:1535–1538.

49. Staab, J. F., and P. Sundstrom. 2003. URA3 as a selectable marker for

disruption and virulence assessment of Candida albicans genes. Trends Mi-crobiol. 11:69–73.

50. Stehr, F., A. Felk, A. Gacser, M. Kretschmar, B. Mahnss, K. Neuber, B.Hube, and W. Schafer. 2004. Expression analysis of the Candida albicanslipase gene family during experimental infections and in patient samples.FEMS Yeast Res. 4:401–408.

51. Sundstrom, P., J. E. Cutler, and J. F. Staab. 2002. Reevaluation of the roleof HWP1 in systemic candidiasis by use of Candida albicans strains withselectable marker URA3 targeted to the ENO1 locus. Infect. Immun. 70:3281–3283.

52. Toenjes, K. A., S. M. Munsee, A. S. Ibrahim, R. Jeffrey, J. E. Edwards, Jr.,and D. I. Johnson. 2005. Small-molecule inhibitors of the budded-to-hyphal-form transition in the pathogenic yeast Candida albicans. Antimicrob AgentsChemother. 49:963–972.

53. Uhl, M. A., M. Biery, N. Craig, and A. D. Johnson. 2003. Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the dip-loid human fungal pathogen C. albicans. EMBO J. 22:2668–2678.

54. Wilson, R. B., D. Davis, B. M. Enloe, and A. P. Mitchell. 2000. A recyclableCandida albicans URA3 cassette for PCR product-directed gene disruptions.Yeast 16:65–70.

55. Zaragoza, O., C. de Virgilio, J. Ponton, and C. Gancedo. 2002. Disruption inCandida albicans of the TPS2 gene encoding trehalose-6-phosphate phos-phatase affects cell integrity and decreases infectivity. Microbiology 148:1281–1290.

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