genome-wide transcription proﬁling of the early …nina m. agabian1,3,4* departments of cell and...

EUKARYOTIC CELL, Sept. 2005, p. 1562–1573 Vol. 4, No. 91535-9778/05/$08.00�0 doi:10.1128/EC.4.9.1562–1573.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genome-Wide Transcription Profiling of the Early Phase of BiofilmFormation by Candida albicans†

Luis A. Murillo,1 George Newport,1 Chung-Yu Lan,1‡ Stefan Habelitz,2 Jan Dungan,1 andNina M. Agabian1,3,4*

Departments of Cell and Tissue Biology,1 Preventive and Restorative Dental Sciences,2 Microbiology and Immunology,3

and Pharmaceutical Chemistry,4 University of California, San Francisco, California 94143

Received 24 February 2005/Accepted 10 June 2005

The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by mostmicroorganisms to survive in changing environments. Biofilm formation proceeds through distinct develop-mental phases and impacts not only medicine but also industry and evolution. In organisms such as theopportunistic pathogen Candida albicans, the ability to grow as biofilms is also an important mechanism forpersistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medicaldevices. The early stage of C. albicans biofilm is characterized by the adhesion of single cells to the substratum,followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changesin the transcriptome begin within 30 min of contact with the substrate and include expression of genes relatedto sulfur metabolism, in particular MET3, and the equivalent gene homologues of the Ribi regulon inSaccharomyces cerevisiae. Some of these changes are initiated early and maintained throughout the process;others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathwayfor cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production inC. albicans.

Medical advances associated with invasive procedures, ag-gressive immunosuppressive treatments for organ transplanta-tion, and the widespread use of broad-spectrum antibiotics arethe most important causes of nosocomial fungal infection. Theopportunistic pathogen Candida albicans, the predominantspecies recovered from hospitalized patients (39), accounts for10% of hospital-acquired infections; candidiasis is the fourthmost common blood-borne infection with an attributable mor-tality of ca. 38% (14). In immunocompetent individuals, C.albicans occurs as a dimorphic commensal colonizer of muco-sal membranes in the oral cavity, gastrointestinal tract, uro-genital mucosa, and vagina; in immunocompromised patients,however, this organism can become pathogenic, resulting inproliferative growth on mucosal surfaces and systemically. Oneof the features that enhances C. albicans’ pathogenicity and/orinfectivity is its ability to grow tenaciously on the surface ofmedical devices and abiotic surfaces, as well as on mucosa.Mature biofilms are characterized by the production of a thickextracellular matrix and an altered resistance phenotype tocommon antifungal agents (11, 12). The basis of the charac-teristic drug resistance of cells growing in biofilms is a sourceof much speculation, although recent studies suggest multiplefactors involved in this process (5, 53).

Biofilm formation proceeds through characteristic stages

that define the process. It begins with the initial adherence ofsingle cells to the substratum, followed by the formation ofmicrocolonies which grow into a confluent but trabeculatedmonolayer. Once confluence is achieved, the network of yeastcells, pseudohyphae, and hyphae become encased within anextracellular matrix (50). The tenacity of biofilm adherencevaries with cell type, growth conditions, and the properties ofthe abiotic surface. Mutants blocked in either hyphal or yeastmorphology can form biofilms, but these differ in their adhe-sive properties from those produced by wild-type cells (6). Theextracellular matrix secreted by cells growing as adherent bio-films differs from the material secreted by planktonically growncells and from matrices associated with bacterial biofilms (5).

Genome-wide transcription profiling has become an impor-tant and powerful tool in cell biology supporting the genera-tion of testable hypotheses for novel processes not yet charac-terized at the molecular level. To characterize the initiatingevents in biofilm formation, we performed a detailed transcrip-tional analysis of the early stages of biofilm development in C.albicans using an Affymetrix oligonucleotide GeneChip repre-sentative of the entire genome of C. albicans (36, 37). Con-temporaneously, others were performing similar studies by us-ing a PCR-based spotter array composed of 2,002 open readingframes (ORFs), chosen at random from the 6,419 ORFs en-coding proteins of �100 amino acids in the C. albicans haploidORF set (30). In that study (18), gene expression in mature,established biofilms (48 and 72 h) formed in yeast nitrogenbase (YNB) under various conditions of flow, oxygenation, andglucose concentration were characterized; 317 ORFs were ex-pressed independently of hyphal formation, whereas 86 weredependent on hyphal formation (18).

The analysis reported here begins with individual cell attach-ment and ends when the cells have attained confluence during

* Corresponding author. Mailing address: Department of Cell andTissue Biology, University of California, San Francisco, 521 ParnassusAve., San Francisco, CA 94143-0422. Phone: (415) 476-6845. Fax:(415) 476-0664. E-mail: [email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

‡ Present address: Institute of Molecular and Cell Biology and De-partment of Life Sciences, National Tsing Hua University, Hsin-Chu,Taiwan.

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the first 6 h of C. albicans biofilm formation, an interval in theprocess which differs from later mature stages (11). Our studyalso differs from that previously reported (18) with respect toconditions used for biofilm formation and the number of ORFsassayed, as well as in the methods of data analysis. We findstriking differences in gene expression patterns between non-adherent and adherent cells within 30 min of surface contact;similar and unique patterns of gene expression are also evidentin both biofilm and planktonic cells within these early stages ofbiofilm formation. Our data complement work on gene expres-sion patterns in mature biofilms (18) and indicate that biofilmformation in C. albicans is accompanied by progressivechanges in gene expression. Some of these changes are initi-ated early and maintained throughout the process; others arerestricted to the earliest stages of biofilm formation.

MATERIALS AND METHODS

C. albicans strain and media. The genome sequencing strain SC5314 was usedin these studies (20). Cells are routinely maintained at �80°C and plated on agarcontaining YPD (10 g of yeast extract, 20 g of Bacto peptone, 20 and g ofdextrose/liter) before each experiment.

Establishment of biofilms. A single colony of C. albicans SC5314 was used toinoculate Ham’s F-12 medium (Life Technologies, Gaithersburg, MD) andgrown overnight at 30°C in a water shaker. Cells were centrifuged at 2,100 � gfor 10 min at room temperature and washed twice in fresh F-12 medium at 30°C.Cells at this point are designated as experimental condition time zero (T � 0).For each time point, biofilms were formed in polystyrene petri dishes (100 mmby 15 mm [Fisher Scientific, Pittsburgh, PA]) using 107 cell/ml of inoculum infresh F-12 medium (10-ml final volume per plate). Planktonic cultures wereprepared by using the same inoculum size in 50 ml of F-12 medium in 250-mlpolypropylene flasks, equalizing to the extent possible the surface/volume ratio inbiofilm and planktonic cultures. Both cultures were incubated at 37°C in 5% CO2

with rotation at ca. 50 rpm. After 30 min of incubation, medium from the biofilmplates was aspirated, pooled, and centrifuged, and the number of nonadherentcells was determined in a hemocytometer and used for RNA extraction; rou-tinely, �80% of the cell inoculum was firmly attached to the abiotic surfacewithin 30 min. Biofilm cultures were then thoroughly washed three times inprewarmed medium, and fresh F-12 medium added; plates were incubated for6 h with constant rotation at 37°C and 5% CO2. A total of five time points wereassayed for biofilms and equivalent planktonic cultures: T � 30 min, T � 90 min,T � 150 min, T � 270 min, and T � 390 min. A set of biofilm plates corre-sponding to the five time points described above were stained with phloxine Bdye (Sigma, St. Louis, MO), and propidium iodide and visualized by light mi-croscopy.

AFM. Either cover glass or polystyrene disks (12 mm; Fisher) were submergedin 35-mm wells with 2.5 ml of F-12 medium. Biofilms were formed as describedabove. Each disk was washed twice with F-12 medium and dried at room tem-perature for 1 h before imaging. The disks were glued to a metal disk (12 mm)for magnetic attachment to the piezo-scanner of an atomic force microscope(Digital Instruments, St. Barbara, Calif.). The scan rates were ca. 1 Hz, with scansizes between 15 and 60 �m. All images were flattened manually in x and ydirections by using the glass surface as the flattening reference (Fig. 1). Atomicforce microscopy (AFM) facilitates highly accurate measurements of the topo-graphic height (z axis of image), whereas the width of features is always subjectto a tip-broadening artifact that derives from the shape of the AFM tip (26). Weused height and width measurements of the long axis to more accurately describethe dimensional development of the blastoconidia during biofilm formation. Thewidth measurements were corrected for the tip-broadening artifact by applying atip angle of 35° for DNPS tips as described by Allen et al. (2).

RNA extraction. Before processing the complete set of samples, culture su-pernatants were collected for glucose determination (Glucose [HK] Assay Kit;Sigma, St. Louis, MO) and pH measurement. After each plate was washed twicewith F-12 medium (37°C), cells were scraped from the plate and centrifuged at2,100 � g at room temperature, and RNA was immediately extracted. Planktoniccells were directly harvested by centrifugation as described above and processedconcurrently. After collection by either scraping and/or centrifugation, cells weresuspended in 3 ml of extraction buffer (0.1 M Tris-HCl [pH 7.5], 0.1 M LiCl, and0.02 M dithiothreitol) and transferred to an ice-cold 50-ml tube containing 6 g of

FIG. 1. AFM. All samples were imaged in the contact mode using a DNPS silicon nitride cantilever with a tip radius of ca. 10 nm (Nanosensors);the use of a porous membrane was not required (1). The scan rates were about 1 Hz with scan sizes between 15 and 60 �m. (A) Cell adhesion(T � 30); (B to E) biofilm development (T � 60 to T � 390). Bar: 10 �m (A and B) and 30 �m (C to E).

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acid-washed glass beads, 0.5 ml of 10% sodium dodecyl sulfate, and 5 ml ofphenol-chloroform-isoamyl alcohol (25:24:1). The cells were disrupted by fivecycles of vortexing (3 min each); between cycles the tubes were incubated alter-natively for 3 min at 42°C and then at 0°C. The lysed cells were decanted andcentrifuged at 10,000 � g for 10 min at 4°C and subjected to two additionalphenol-chloroform-isoamyl alcohol extractions before nucleic acid precipitationin 2.5 volumes of ethanol. Samples were stored at �20°C overnight, and RNAprecipitates were collected at 10,000 � g for 10 min. Total RNA was treated withRQ1 DNase (Promega, Madison, WI) for 1 h and cleaned by using an RNeasyColumn (QIAGEN, Valencia, CA) prior to cDNA synthesis (Invitrogen, Carls-bad, CA); RNA samples were processed according to the manufacturer’s instruc-tion for Affymetrix GeneChips according to the protocol for 15 �g of startingmaterial. A total of twelve samples were obtained per experiment (one from timezero, five from biofilm, and five from planktonic and one from the nonadherentcells at 30 min). The experiment was performed in duplicate, and DNA microar-ray analysis was carried out from two independent sets of samples.

DNA microarray design and analysis. A custom high-density oligonucleotideGeneChip manufactured by Affymetrix (Santa Clara, CA) was used; the charac-teristics of these GeneChips and experimental protocols with C. albicans samplesare thoroughly discussed in Lan et al. (36, 37). Gene annotations for the 7,116open reading frames (ORFs) in the microarray and how they were assigned isdescribed elsewhere (http://agabian.ucsf.edu). RNA labeling, target hybridiza-tion, washing, staining, and scanning were performed by using a GeneChipHybridization Oven 640, a GeneChip Fluidics Station 400, and a GeneArrayscanner (Affymetrix) as described previously (36). The technical reproducibilityof the chips is very high due to their density, the random distribution of theprobes within the matrix, and the synthesis of each probe directly on the sub-strate.

DNA microarray data analysis. GeneChips were scanned, and the resultingimage files were used to calculate, normalize, and compare the hybridizationintensity data by using the Microarray Suite 5.0 software (Affymetrix). Thefluorescence signals of each array were normalized by global scaling with a targetintensity of 500. The statistical algorithm within this software was used for theabsolute analysis of each individual microarray. The single array analysis mea-sures a relative level of expression of a transcript (signal) and determines thedetection call indicating whether a transcript is detected (present [P]) or notdetected (absent [A]). Absolute analysis of each microarray was followed bycomparison analysis between two separate arrays; T � 0 was selected as thebaseline for comparison. This estimates the magnitude of fold change (signal logratio) and the direction of the change (increase, decrease, and no change) in theexpression of a transcript across the experimental time course for either biofilmor planktonic cultures. The data derived from each experiment was exported toMicrosoft Excel, where the “signal log ratios” are converted to fold changes. Forcomparative purposes four variables were considered: (i) values with a no-changecall, (ii) an absent signal with an increase- or decrease-change call, (iii) foldchanges of �2, and (iv) intensity values of �100. If any of these criteria werepositive in both planktonic and biofilm data sets, the probe set was excluded fromanalysis. The final results thus obtained consist of three different data sets: oneset is described as the common gene set (CGS); genes in this set are upregulatedor downregulated in both planktonic and biofilm cultures with respect to T � 0.The second and third data sets contain the ORFs that are up- or downregulateddifferentially in either biofilm or planktonic cultures. The complete data set isavailable online (http://agabian.ucsf.edu).

Reverse transcriptase PCR (RT-PCR). Reactions were carried out by usingSuperScript One Step RT-PCR with Platinum Taq (Invitrogen) and a PTC-200Peltier Thermal Cycler (MJ Research, Waltham, MA). A total of 1 ng of DNase-treated total RNA was used as a template in each reaction. Primers for each geneare included 5�-GAACACAAATGTCATGAAAC-3� and 5�-CAAGACTGAGGCACCAGC-3� for YWP1, 5�-GGTCACTTATTTACCTGATG and 5�-TGTGATTCTACGTTCACCC-3� for MET3, and 5�-ACACCTGAGTTGACTCCA-3�and 5�-ACACCTGAGTTGACTCCA-3� for ORF19.6824. The reactions wereperformed with 1 cycle of 50°C for 30 min and 94°C for 2 min; 35 cycles of 94°Cfor 15 s, 50°C for 30 s, and 72°C for 2 min; and 1 cycle at 72°C for 10 min. PCRproducts were visualized in 1% agarose gels and in some instances qualitativelycorrelated with signals obtained from DNA microarray intensity data.

RESULTS AND DISCUSSION

In vitro biofilm model for C. albicans. The surfaces to whichC. albicans adheres range from host mucosal membranes andskin to abiotic substrates used in medical devices. Coupled withC. albicans’ ability to alter morphology and adapt to a range of

environmental conditions, biofilm formation facilitates thetransfer to and rapid proliferation of the fungus in the manyanatomic sites that, in the immunocompromised host, supportits growth. To approximate in vivo conditions, we used F-12medium, 5% CO2, and 37°C; F-12 medium approximates hu-man physiological conditions regarding fluid constituents, os-molarity, glucose concentration, and pH stability. Each ofthese factors, as well as the shearing force applied to the petridish, critically influenced the rate of formation and the adhe-sive strength of these biofilms (15). These conditions are dis-tinct from those used in a number of other reported biofilmstudies (11, 18). The kinetics of glucose consumption wereessentially identical in both planktonic and biofilm cultures,with more than half of the glucose being consumed within 4 h(initial concentration of 10 mM) and entirely consumed after6 h (data not shown). The pH of the medium was stable overthe 6 h of the experiment, ranging between 7.6 and 7.8. Ap-proximately 80% of the initial cell inoculum adhered to thepetri dish within the 30-min attachment period.

AFM was used to visualize biofilm development. The AFMimages shown in Fig. 1 were obtained from glass substrates butare representative of biofilm formation on polystyrene as well(data not shown). Image quality was enhanced when glass diskswere used due to the reduced surface roughness of the glassversus polystyrene. AFM images of biofilms provided the op-portunity to measure cellular parameters during the earlyphases of biofilm formation. Hyphal structure showed an ap-proximately cylindrical shape, and the height measurementdirectly translates into a measurement of the diameter of thehyphae. In contrast, the blastoconidia is an ellipsoid structurewith its long axis usually perpendicular to the hyphal axis. At T� 30, C. albicans blastoconidia were on average 1.4 �m highand 2.2 �m wide, and germ tube development (length � 3 �m)was evident. At the density of “seeding” of the biofilms used inthese experiments, germ tubes attained lengths up to 10 �m byT � 90. At T � 90, the beginnings of segmentation wereevident; hyphae were generally straight and without evidenceof branching or budding and the mother cells have increased toca. 2.2 and 4.7 �m in height and width, respectively. By T �150 a more intricate network was formed where most hyphaerange in length from 20 to 30 �m and form contacts either withneighboring hyphae or mother cells (57) and branching is ini-tiated. The biofilm increased in density with time, and theAFM images at T � 270 show a highly connected network ofoverlapping hyphae. At this time the pores or potential waterchannels in the network are reduced to �10 �m in diameter.At T � 390, the pore size is further reduced to an average of1 �m; this pore size is maintained during continued biofilmdevelopment and retained in mature biofilms (data notshown).

After the initial attachment period at T � 30 in biofilmdevelopment we observed what seemed to be a population ofsignificantly larger and more elliptically shaped yeast formcells, reminiscent of the opaque phenotype of the WO-1 strain(56). Phloxine B, a stain that reflects alterations in cell mem-brane permeability, is used to differentiate white (W) andopaque (O) cells, as well as living and dead colonies in variousyeasts (58). Staining of biofilms with Phloxine B produced adifferent pattern over the course of biofilm development: ca.30% of the cells stained pink at T � 30 and T � 90 (Fig. 2),

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suggesting heterogeneity in the population of yeast cells at-tached to the substratum. However, there was no preferentialstaining of large versus small cells. At later times (T � 270 andT � 390), the proportion of stained cells decreased until onlythe tips of the hyphae took up stain (Fig. 2). Propidium iodide,which only stains cells with disrupted cell membranes, did notstain any of the cells throughout the experiment (data notshown), indicating that all cells within the biofilm remainedviable for the 6 h of the study. Moreover, during the course ofthe experiment, biofilms grew as homogeneous hyphal cultureswhile the planktonic cells became progressively more hetero-

geneous mixtures of hypha-pseudohypha-yeast forms (see be-low).

RT-PCR. Three differentially expressed ORFs, two biofilm(MET3, and ORF19.6824) and one planktonic (YWP1; cyclicalexpression pattern) were assayed by RT-PCR in addition toDNA microarray analysis. Comparisons of the RT-PCR prod-ucts with DNA microarray signal values are shown in Fig. 3.MET3 transcripts are detected in both biofilm and planktoniccultures; however, at equal concentrations of total RNA andsimilar cycling programs, the amounts of product were sub-stantially different in each case and consistent with the patternobtained from DNA microarray analysis. YWP1 gene expres-sion in planktonic cells is cyclical, both by DNA microarrayanalysis and RT-PCR, reflecting the initial transition fromyeast to hyphal cells and the rapid reappearance of yeast formsin the culture. Conversely, YWP1 gene expression decreases inbiofilm cultures and is essentially undetectable through T �390, both by RT-PCR and DNA microarray analysis, reflectingthe hyphal nature of biofilms at this stage of developmentunder our conditions. ORF19.6824, another gene upregulatedin biofilm similarly confirms that the expression patterns areequivalent in both types of analysis.

Gene expression data. We assayed genome-wide changes ingene expression during the earliest stages of biofilm formation,beginning with the attachment of individual cells to the sub-stratum through the formation of a contiguous cellular layer onthe abiotic surface. The transcription data derived from theseexperiments was compared in several ways. “Vertical” compar-isons were made during the time course (i.e., 1 h biofilm versus1 h planktonic), similar to comparisons made in the study ofgene expression in mature biofilms (18). We then went on andcompared each vertical comparison longitudinally across theentire time course of the experiment. In these comparisons,genes that are equivalently induced as a function of timeand/or morphogenesis in both biofilm and planktonic culturesare not scored since their values cancel each other; ORFswhich display variable (i.e., cyclic) patterns of expression dur-ing the experimental time course were more difficult to com-pare across cultures, especially because the adherent earlystage biofilms are essentially completely hyphal, whereas theplanktonic cultures are heterogeneous with respect to morpho-logic types. In the “horizontal” comparison we analyzedchanges in gene expression as a function of time within eitherbiofilm or planktonic cultures, respectively, using the T � 0samples as a reference. These two data sets were then com-pared vertically with one another to identify genes that areregulated in both cultures and genes that are uniquely regu-lated in either. From these comparisons, a total of 2,492 ORFswhose expression varied during the course of the analysis wereidentified. Within this group, three different data subsetsemerged: differentially upregulated genes in biofilm (41 ORFs;Table 1), differentially upregulated genes in planktonic cul-tures (25 ORFs; Table 2), and the remainder (2,426 ORFs)comprising a CGS (see Table S1 in the supplemental material)whose expression is similarly regulated in both types of cul-tures. The DNA microarray data set is derived from two inde-pendent experiments and showed a reproducibility of �95%.The genes expressed differentially in either biofilm or plank-tonic cultures were noted in both replicates. Only one repre-sentative set of data is presented here. Finally, we also com-

FIG. 2. Phloxine B staining of C. albicans biofilm formation. A setof five petri dishes corresponding to each datum point in biofilmformation was used for staining with 5 �M Phloxine B (34) and pho-tographed (Arcturus). Differences in staining are evident during the6 h of biofilm development. (A) T � 30; (B) T � 90; (C) T � 270.Magnification, �40.



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pared the gene expression profiles of those cells that adhere tothe substrate during the 30-min seeding period to the remain-ing 20% of the cells that did not adhere to the plates (nonad-herent cells) (see Table S2 in the supplemental material).

Differential gene expression in biofilms. A total of 41 ORFswere expressed more highly during the initial 6 h of biofilmdevelopment versus planktonic cultures; their relative levels ofgene expression are found in Table 1. Of these 41 ORFs, nine(SUL1, MET10, HAL21, HAL22, MET14, MET15, MET3,ECM17, and ORF19.1159) encode proteins directly involved insulfur metabolism. The changes in expression of this particularset of ORFs is striking and clearly indicate that not only par-ticular ORFs but also the entire sulfur assimilation pathway isupregulated in early biofilm cultures. Moreover, the kinetics ofthis induction is also striking, beginning within 30 min of sur-face contact (Table 1 and Table S2 in the supplemental mate-rial) and increasing through the 6 h of the experiment. Uniqueto the present study and not detected previously (18) are C.albicans HAL21 and HAL22 ORFs, which are also highly up-regulated in biofilm cells. HAL21 and HAL22 represent a geneduplication similar to that of HAL2/MET22 in Saccharomycescerevisiae (45). C. albicans HAL21 and HAL22 encode a phos-phatase that catalyzes the hydrolysis of either 3�-phosphoad-enosine-5�-phosphate (PAP) and/or 3�-phosphoadenosine 5�-phosphosulfate (PAPS) to AMP and are thus likely to functionin sulfur recycling. MET16, which encodes a 3�-phosphoadenyl-sulfate reductase that reduces 3�-phosphoadenylylsulfate toadenosine-3�,5�-bisphosphate and free sulfite, reported to beupregulated in mature (72 h) biofilms (18), is not upregulatedduring early biofilm formation. In the absence of concomitantupregulation of MET16, upregulation of HAL21/HAL22 sug-gests the activation of a futile sulfur cycle in biofilm cells. Inplants, accumulation of toxic intermediates such as APS andPAPS is controlled by HAL2, which transfers the sulfur atom

to an organic compound (47), typically glutathione (GSH) inmost organisms (42, 48). Functionally, GSH not only confersprotection against reactive oxygen species but also serves as astorage and transport form of cysteine.

One of the most highly expressed genes in biofilm cells isMET3. The protein encoded by MET3 is the primary activatorof sulfur assimilation in the cell. Studies in the fungal pathogenCryptococcus neoformans indicate that MET3 also plays animportant, but not understood role in its virulence and growth.MET3 expression levels in C. neoformans are not regulated byexogenous methionine, although they are in S. cerevisiae (64).In C. albicans, it is reported that MET3 transcription is fullyrepressed by 1 mM methionine or cysteine (10) and that re-pression of MET3 expression is detected at 0.1 mM cysteine ormethionine. The F-12 media we use contain 0.2 and 0.03 mMcysteine and methionine, respectively, concentrations that areinhibitory for MET3 expression (10). As shown in Fig. 3, thelevels of MET3 transcripts in planktonic cells are low, a findingconsistent with repression of MET3 as described above. Incontrast, even in the presence of what should be inhibitoryconcentrations of methionine and cysteine, MET3 is induced23-fold by T � 30 in biofilm cells; thus, contact with theabiotic surface appears to override methionine/cysteine repres-sion of MET3. The activation and maintenance of these path-ways as a unique attribute in the transcriptome of cells growingas biofilms make these pathways an excellent drug target forthe prevention of biofilm formation; one such drug alreadydescribed elsewhere (3) might be azoxybacillin, a sulfite reduc-tase inhibitor and natural antifungal agent produced by Bacil-lus cereus. The potential use of this drug will be the target offurther investigation.

The identity of the ORFs induced in biofilm cells in thepresent study further suggests that sulfur assimilation in C.albicans differs substantively from that in S. cerevisiae and more

FIG. 3. Correlation of DNA microarray analysis and RT-PCR. Three genes, which represent a yeast-specific gene (YWP1) and two biofilmupregulated (MET3 and ORF19.6824) genes are detected by RT-PCR and compared to the signal values obtained from the GeneChip data.Amplified products are detected on a 1% agarose gel. Lanes: 0, T � 0; 1 to 5, T � 30 to T � 390 (planktonic); 6 to 10, T � 30 to T � 390 (biofilm).



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closely resembles sulfur assimilation pathways described inNeurospora crassa and Aspergillus nidulans (40). In these fila-mentous fungi, sulfur assimilation, especially through the for-mation of cysteine, follows two biosynthetic pathways: the me-thionine-cystathionine (CT) pathway and the O-acetyl-serine(OAS) pathway (59). In C. albicans ORF19.1159 encodes apresumptive homologue of the A. nidulans serine O-trans-acetylase [CYSA], the enzyme which converts serine to O-acetylserine as part of the OAS pathway (25); ORF19.7152encodes a protein with 85% identity to the O-acetylserinesulfhydrylase (CYSK) of Aspergillus, which converts O-acetyl-serine into cysteine incorporating a sulfur group. These ORFsencode enzymes that are key to the formation of cysteine via

the OAS pathway, a pathway not found in S. cerevisiae (46).Our assertion that this pathway is present in C. albicans isstrengthened by the observation that, unlike S. cerevisiae, C.albicans met15-null mutants can grow on methionine-depletedmedia (61). We note that the C. albicans CysA and CysKhomologues, ORF19.1159 and ORF19.7152, were not includ-ed/assayed in the previous study (18). Although the expressionof both of these ORFs is upregulated only in biofilm cultures,there was no differential upregulation of MET6 (homocysteinemethyltransferase) and SAM2 (S-adenosylmethionine syn-thase) suggesting again that the flow of sulfur under theseconditions favors the synthesis of cysteine (Fig. 4).

Complementing the induction of sulfur assimilation path-

TABLE 1. Genes and/or ORFs expressed differentially in biofilm cells

ORFaExpression (fold change)b at:

Gene; ORF (assembly 19)c

T � 30 T � 90 T � 150 T � 270 T � 390

Homology inS. cerevisiae

6.4694 1.0 2.8 3.3 4.5 1.0 SUL1; ORF19.102526.7984 22.9 22.0 16.0 26.9 12.6 MET3; ORF19.50256.3204 3.2 3.8 2.6 4.0 2.4 MET10; ORF19.40766.6837 2.4 2.2 2.3 2.9 1.9 ECM17; ORF19.40996.8195 4.1 5.4 4.0 5.0 2.2 MET14; ORF19.9466.4514 9.9 10.3 10.6 14.5 5.4 MET15; ORF19.56456.2502 3.3 5.2 5.0 4.3 1.7 HAL21; ORF19.996.2508 3.7 5.6 5.4 4.9 1.7 HAL22; ORF19.1056.3134 1.4 2.8 2.5 2.5 1.8 ORF19.1159; CYS A homolog6.6913 3.5 5.7 4.6 6.3 1.0 OPT3; ORF19.56736.1607 �1.9 1.0 1.0 2.4 2.8 GCS1; ORF19.50606.4839 3.2 6.6 5.7 8.1 9.3 NCE103; ORF19.17216.7898 22.6 17.0 18.8 7.5 10.3 PHO89; ORF19.45996.2299 6.4 8.6 7.9 5.7 8.9 ORF19.3936; similar to PHO816.5895 6.9 5.6 4.3 7.5 4.8 AAH1; ORF19.22516.7999 1.0 1.7 1.0 2.2 2.3 DIM1; ORF19.50106.254 1.0 1.0 1.0 2.2 2.7 KRR1; ORF19.82776.2709 1.0 1.4 1.0 2.3 3.1 RRP8; ORF19.36306.8114 3.8 5.7 9.4 12.6 24.3 ORF19.7214; similar to 1,3-glucosidase6.3733 1.2 1.7 1.3 3.8 3.9 ORF19.3088; similar to S. cerevisiae YDR1846.3068 1.6 2.8 2.4 3.1 �1.6 AGP3; ORF19.37956.2084 1.0 3.9 4.0 4.2 2.4 YMC2; ORF19.47336.6487 1.7 1.7 1.5 2.1 2.6 ABP140; ORF19.36766.1078 2.3 2.5 2.0 2.7 1.6 PUS7; ORF19.93226.972 2.1 1.6 1.7 2.5 2.2 POL5; ORF19.130426.9132 2.7 2.1 2.0 2.0 2.7 ORF19.5905; similar to S. cerevisiae YBL028C6.4849 6.0 5.9 4.2 2.1 3.4 ORF19.5777; similar to S. cerevisiae YBR096W6.3387 1.0 1.0 1.0 2.7 3.2 ORF19.5049; similar to S. cerevisiae YLR0036.337 1.0 1.5 1.4 2.6 2.3 ORF19.2527; similar to S. cerevisiae YNL035c6.2076 1.3 1.3 1.2 2.1 2.0 ORF19.1388; similar to S. cerevisiae YER002

No homology inS. cerevisiae

6.1742 5.0 26.2 32.4 24.4 79.9 AMO2; ORF19.31526.7581 1.7 2.8 3.4 6.5 3.5 ASM3; ORF19.60376.241 2.2 1.0 3.5 98.4 86.2 POL93; ORF19.60786.258 1.0 2.4 2.4 1.6 2.1 SSP96; ORF19.51456.2306 75.6 104.7 93.1 85.6 49.2 ORF19.15396.8807 9.1 7.9 8.4 12.1 55.3 ORF19.68246.4376 2.3 4.4 3.9 4.1 14.5ORF ORF19.17976.283 6.3 7.8 7.4 5.5 1.0 ORF19.44246.102 1.0 2.7 8.8 2.3 3.8 LDG3; ORF19.6486.6.6784 1.0 2.5 2.1 2.9 2.5 ORF19.66756.7184 1.3 2.5 2.6 3.2 2.9 ORF19.6920

a Assembly 6.b Positive or negative values represent the fold changes of up- or downregulated genes, respectively.c Gene names may also be found at http://agabian.ucsf.edu.



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ways is the upregulation of AGP3, an ORF that encodes aputative serine transporter protein and thus would provide acarbon acceptor for sulfur assimilation (7) through the OASpathway. Similarly, OPT3, a probable oligopeptide transporter,based on its similarity to ISP4 of Arabidopsis, displays kineticsof expression similar to that of SUL1 during early biofilmformation (Table 1), suggesting that OPT3 may play a role inthe transport of sulfur-containing compounds (e.g., GSH orGSH-linked xenobiotics), as OPT1 does in S. cerevisiae (8).

Also upregulated during early stages of biofilm developmentare NCE103, a carbonic anhydrase-like gene (22), and GCS1,a gene encoding -glutamylcysteine synthase (4). Both of theseenzymes are indirectly related to the sulfur pathway and oxi-dative metabolism. Mutant strains of NCE103 in S. cerevisiaeare unable to grow under normal aerobic conditions, butcomplementation with the coding region of a carbonic anhy-drase gene from Medicago sativa rescue this deficiency (13). Inrats, carbonic anhydrase III also functions as a phosphataseupon glutathiolation of one of its cysteine residues (9). Gcs1,the rate-limiting enzyme in GSH biosynthesis, is associatedwith oxidative metabolism via the de novo production of GSH.In Schizosaccharomyces pombe, Gcs1 is under complex regula-

tion by GSH feedback inhibition, the availability of L-cysteine,and the carbon source (32) and is associated with oxidativemetabolism via the de novo production of GSH. Transcriptsfrom both NCE103 and GCS1 are highly upregulated in bio-films; however, the kinetics of GCS1 induction differs from thatof the sulfur assimilation pathways and NCE103. AlthoughNCE103 is induced immediately upon attachment of cells tothe abiotic surface, the signal values of the NCE103 transcriptincrease over time, and induction of GCS1 transcription is notobserved until 4 h into biofilm growth. At this stage the cellshave started to form a second layer and more intricate cell-cellinteractions (Fig. 1); more than 50% of the available glucosehas been consumed in both biofilm and planktonic cultures andthe phloxine B staining pattern of the cells changes (Fig. 2).Taken together, these observations suggest that alterations inmembrane structure and permeability and a requirement forGSH-related metabolic activities occur during the transitionalperiod of the early to intermediate stage of biofilm formation.The potential role of GSH in the transient acquisition of drugresistance in biofilms merits some comment. In both mamma-lian tumor cells (51) and in the eukaryotic pathogen, Plasmo-dium falciparum (44), changes in GSH levels result in altered

TABLE 2. Genes or ORFs expressed differentially in planktonic cells

ORFaExpression (fold change)b at:

Gene; ORF (assembly 19)c

T � 30 T � 90 T � 150 T � 270 T � 390

Homology inS. cerevisiae

6.4985 3.1 2.4 2.0 2.1 2.5 IPL1; ORF19.34746.5762 1.8 1.0 4.0 11.4 32.9 BIO32; ORF19.35676.7295 2.4 1.0 1.0 4.5 5.4 HSP78; ORF19.8826.1952 2.1 2.5 1.9 2.8 4.9 SUA72; ORF19.35196.8933 140.1 24.3 16.1 1.0 16.2 INO1; ORF19.75856.2039 8.9 2.9 2.6 6.9 26.2 ITR2; ORF19.35266.7648 5.7 10.3 8.8 12.5 16.2 EXO70; ORF19.65126.677 4.2 3.7 6.4 21.7 14.1 CGR1; ORF19.102376.7846 3.4 2.0 2.1 3.2 6.4 ORF19.6408; similar to S. cerevisiae

YNL064C6.7979 2.0 1.9 1.8 3.8 3.7 ORF19.5030; similar to S. cerevisiae

YDR068W6.6497 1.9 1.0 2.3 3.4 2.9 ORF19.428; similar to S. cerevisiae

YJL057C6.7758 2.3 1.0 1.9 5.0 5.2 ORF19.3021; similar to S. cerevisiae

YMR114C6.3406 2.3 �2.8 1.0 7.9 10.6 ORF19.5125; similar to S. cerevisiae

YLR149c6.9062 1.0 �6.5 �11.3 3.8 6.5 ORF19.5975

No homology inS. cerevisiae

6.1227 2.0 1.0 1.3 2.1 6.5 FMO2; ORF19.84776.4504 1.3 �2.3 �2.2 4.4 7.8 CRW3; ORF19.56356.4753 �2.1 �2.0 �2.0 2.3 4.3 SOD33; ORF19.21086.3288 �2.3 �13.7 �35.3 �6.1 �2.9 YWP1; ORF19.36186.2131 2.4 3.8 3.3 2.8 4.4 ORF19.10496.2106 1.2 1.0 1.0 2.1 2.2 ORF19.14826.8 5.9 7.1 7.9 11.6 8.3 ORF19.436.81 2.6 1.0 2.2 3.7 8.8 ORF19.37426.4587 2.2 1.0 2.6 5.8 6.5 ORF19.28066.1755 1.0 8.2 8.5 8.5 6.6 ORF19.121726.444 1.0 �1.7 �1.6 2.3 3.7 ORF19.633

a Assembly 6.b Positive or negative values represent the fold changes of up- or downregulated genes, respectively.c Gene names may also be found at http://agabian.ucsf.edu.



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sensitivity of these cell types to a variety of drugs. Although thisphenomenon remains to be explored in C. albicans, thegenomic expression data support the hypothesis that, duringbiofilm development, alterations in GSH levels may play a rolein the drug resistance phenotypes of cells growing as biofilms.

Of the remaining 28 ORFs upregulated in biofilm, 17 havehomologues in S. cerevisiae, 5 of which are homologous toORFs of unknown function; of these 17, 10 encode nuclearproteins (29). Not directly involved in sulfur assimilation butrepresenting a significant second subset of genes expressed inbiofilms are ORFs associated with phosphate metabolism:PHO89, PHO81, and ORF19.4424, a PHO2-like gene. In S.cerevisiae PHO89 encodes a membrane Pi transporter thatmediates cation-coupled Pi transport acting in concert with apH-dependent (alkaline) plasma membrane Na�-ATPase(ENA1/Pmr2p) (17). A C. albicans homologue of ENA1, en-coded by ORF19.5170, is preferentially upregulated in biofilmcells, along with the alkaline pH-regulated gene PHR1. Otherhomologues include ORFs associated with pseudohyphalgrowth (ORF19.5777); mitochondrial transport (YMC2);rRNA processing/Ribi regulon (KRR1, RRP8, and DIM1);tRNA modification (PUS7); an actin filament-binding protein(ABP140); an adenine deaminase (AAH1); and ORF19.7214(similar to S. cerevisiae YBR056), a glucan 1,3-�-glucosidase.Of the 28 ORFs differentially expressed in early biofilm for-mation (Table 1), 11 do not have a counterpart in S. cerevisiae;several of these, however, are similar to genes found in otherorganisms: ORF19.1797, which encodes a protein that containsa GutQ domain characteristic of many phosphosugar isomer-ases and phosphosugar-binding proteins required for bacteriallipopolysaccharide assembly (60); ORF19.4424, which encodes

a protein with a domain similar to one found in the SurEsurvival protein of bacteria and is most closely related to anacid phosphatase of Yarrowia lipolytica; AMO2, which encodesa protein with similarity to a putative peroxisomal copperamino oxidase from A. niger (16); and SSP96, a protein con-taining a flavin-binding monooxygenase-like domain.ORF19.6675 displays a low degree of similarity to lipoic acidsynthase, and ORF19.6824, a 467-amino-acid protein with apotential helix-loop-helix DNA-binding domain, is upregu-lated only in biofilm cells. ORF19.6824 appears to be specificfor C. albicans, since Southern blot analyses with a collection ofDNA from 11 different Candida species and S. cerevisiae failedto show positive hybridization, even at low stringency (data notshown). LDG3 encodes a leucine-aspartic acid-glycine richprotein of unknown function, which belongs to one of thelargest gene families in C. albicans (A. Kuo, unpublished data);LGD3 is the only member of this gene family upregulated inbiofilms. POL93, which is very similar to TCA8, encodes an RTthat is upregulated nearly 100-fold at T � 270 and is classifiedas a gypsy-like element with an internal primer site similar toa region of the Candida tRNAi

Met (21).Differential gene expression in planktonic cultures. Al-

though the cells growing as biofilms under the condition usedare almost uniformly hyphal, as also observed in biofilm de-rived from RPMI1640 medium (10), equivalent planktonic cul-tures consist of a mixed population of hyphal, pseudohyphal,and yeast forms in F-12 media. This qualitative observationwas quantitated by comparing the relative gene expressionlevels of HWP1 and YWP1, hyphal- and yeast-form specificgenes, respectively (55). This dichotomy in population struc-ture confounds interpretation of elevated levels of gene ex-

FIG. 4. Potential second cysteine synthesis pathway in C. albicans. The pathways for the synthesis of homocysteine, methionine, cysteine, andS-adenosylmethionine are shown. In S. cerevisiae (genes in green) the sulfate assimilation pathway flows toward the production of methionine andS-adenosylmethionine. The existence of an alternative route such as the OAS pathway described for A. nidulans is found in C. albicans biofilm cells(ORFs in red).



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pression for the 25 genes differentially expressed in planktonicculture; nevertheless, these bear mention. Of the 25 ORFs, 19display an increase in fold change during the entire 6 h ofanalysis; 10 of the 19 have a homologue in S. cerevisiae, includ-ing genes involved in metabolism (BIO32 and ITR2), cell cycle(EXO70), yeast-mycelial transition (CGR1), heat shock(HSP78), protein trafficking (ORF19.6408), unknown function(ORF19.3021 and ORF19.5125), and two transcription factors(SUA72 and ORF19.5975). The SUA7 gene in S. cerevisiae hastwo homologues in C. albicans: SUA71 and SUA72. AlthoughSUA72 is upregulated in planktonic cultures, SUA71 is down-regulated (see Table S1 in the supplemental material), sug-gesting distinct roles for these two almost identical (73% iden-tity at the amino acid level) putative transcription factors.ORF19.5975 is a potential zinc finger protein and is similar toS. cerevisiae Adr1, a putative transcription factor, with respectto the position of zinc ligand and conserved DNA contactresidues (52).

The remaining nine ORFs displaying increased mRNA lev-els during the first 6 h of planktonic growth and which have nohomologues in S. cerevisiae include: FMO2, a putative bacterialFAD-containing monooxygenase; CRW3, a potential cell sur-face antigen with a CFEM (for common in several fungalextracellular membrane proteins) domain present in proteinsinvolved in fungal pathogenesis (35); SOD33, a protein likelysimilar to a secreted Cu/Zn superoxide dismutase from N.crassa; and six additional ORFs with unknown function (Table2). It is noteworthy that the expression of five ORFs in theplanktonic category cluster with the yeast-form specific geneYWP1: ORF19.1482, ORF19.5975, CRW3, SOD33, andORF19.633. Of the remaining five planktonic ORFs (of thetwenty-five), four show constant fold changes through the en-tire 6 h, and one (INO1 inositol-1-phosphate synthase) isdownregulated (Table 2).

CGS. The major change in both planktonic and biofilm cul-tures upon initiation of this protocol is the induction of hyphalmorphogenesis. During the 6-h growth period of this experi-ment, a total of 2,426 ORFs were differentially regulated inboth planktonic and biofilm cultures: 1,660 ORFs were up-regulated, and 766 were downregulated. A total of 13% of theCGS ORF set (2,426) are ORFs of unclassified function, andalmost 20% of the total CGS ORFs have no significant homol-ogy to any S. cerevisiae genome sequences (Table 3). Threedifferent patterns of expression in the CGS were noted: groupA, ORFs with initial low fold change values that increased overtime (T � 390/T � 30, x � 2) (n � 568); group B, ORFs withconstant fold change values during the entire 6 h of the exper-iment (T � 390/T � 30, x � 1) (n � 1,120); group C, ORFswith initial increased fold change values that later decreased (T� 390/T � 30, x � 0.5) (n � 738) (Table S1 in the supplemen-tal material). Functional assignments of ORFs within the threegroups were made using MIPS categories based on homologywith S. cerevisiae genes (http://mips.gsf.de/proj/yeast/catalogues/funcat/index.html) (Table 3). Overall, the largest proportionof the CGS belongs to group B. Within the classes of geneexpression profiles, for groups B and C the largest functionalcategory is that of metabolism followed by protein fate, cellulartransport, cell cycle, and DNA processing. Group C ORFs inthe categories of cell cycle, DNA processing, and protein fatedecreased at later times, as might be expected as the overallcell density increased in the cultures. There is a relative in-crease in the number of ORFs in group A in the metabolismcategory due to the expression of ORFs related to amino acid,nitrogen, and sulfur metabolism; this group also includes genesinvolved in cell rescue, defense, and virulence, including SAP5,SAP6, RBT5, ECM33, ACE2, ALS3, HWP1, TPK2; PLB3,PLB4, PLB5, and ORF19.1586, similar to a bacterial phos-phatidylinositol-specific phospholipase C. A more detailed com-

TABLE 3. Functional categories of CGSa

DescriptionNo. of genes in functional category (%)b

Group A Group B Group C

Metabolism 117 (15) 194 (11.5) 146 (13.5)Energy 28 (3.5) 28 (1.7) 29 (2.7)Cell cycle and DNA processing 31 (4) 166 (9.8) 116 (10.8)Transcription 65 (8) 92 (5.4) 54 (5)Protein synthesis 5 (0.5) 32 (1.9) 15 (1.4)Protein fate 34 (4) 133 (7.8) 136 (12.6)Protein with binding function or cofactor requirement 5 (0.5) 10 (0.6) 2 (0.1)Protein activity regulation 0.0 5 (0.3) 5 (0.5)Cell transport, transport facilitation, and transportation

routes117 (15) 163 (9.6) 125 (11.6)

Cellular communication/signal transduction mechanism 11 (1) 20 (1.2) 9 (0.8)Cell rescue, defense, and virulence 42 (5) 45 (2.7) 20 (1.9)Interaction with the cellular environment 15 (2) 73 (4.3) 32 (3)Transposable elements, viral and plasmid proteins 1 (0.1) 2 (0.1) 2 (0.2)Cell fate 19 (2) 62 (3.7) 24 (2.2)Biogenesis of cellular components 27 (3) 101 (6) 48 (4.5)Cell type differentiation 15 (2) 79 (4.6) 33 (3)Subcellular localization 2 (0.2) 4 (0.2) 0Unclassified proteins 102 (13) 212 (12.5) 120 (11)No good homologies in S. cerevisiae 161 (20) 272 (16) 165 (15)

a C. albicans ORFs or genes were assigned according to the MIPS classification of the S. cerevisiae homologues.b Group A, ORFs with initial low fold change values that increased over time; group B, ORFs with constant fold change values; group C, ORFs with initial increased

fold change values that later decreased.



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parison between groups C and A reveals that ORFs related toglycolysis and gluconeogenesis predominate in group C,whereas those associated with the oxidation of fatty acids andionic homeostasis are upregulated in group A.

ORFs with no homology to S. cerevisiae but which encodeinteresting amino acid motifs are also present in group A: forexample, ORF19.5760 encodes a 392-amino-acid protein witha Gly-Ser domain at the carboxy terminal similar to that foundin lustrin A, human and mouse cornified envelope loricrin, andthe extracellular matrix protein keratin (54); ORF19.7104 en-codes a mucin-like protein similar to microfilarial sheath pro-tein Shp3 from the nematode Litomosoides (27).

Genes differentially expressed within 30 min of adherence.Adherence with a hard surface induces the expression of genesinvolved in the priming of plant fungal pathogens to signalsthat promote host invasion (33). Perhaps related to this phe-nomenon, C. albicans displays a thigmotropic response (63)hypothesized to assist the organism in foraging for suitablesources of nutrients (49) and, when pathogenic, in defining theroute of tissue penetration (23). It has also been reported thatwhen C. albicans encounters an abiotic surface the transcrip-tion of genes encoding efflux pumps MDR1 and CDR1 arerapidly induced, concomitant with the display of enhancedresistance to fluconazole (41). Given this context, we sought todetermine whether contact sensing, a common feature in thebiology of saprophytic fungi (49), results in a discrete change ingene expression in C. albicans. To answer this question, weanalyzed alterations in the C. albicans transcriptome as a con-sequence of adherence to the abiotic surface by directly com-paring gene expression values in T � 30 adherent cells versusT � 30 nonadherent cells (Table S2 in the supplemental ma-terial). Surprisingly, within the 30-min period allowed for at-tachment and adherence, the transcription of 554 ORFs, ornearly 9% of the genome, was altered by a factor of �2: 300ORFs were upregulated in adherent cells. None of these con-tact-associated changes in gene expression correspond to thosenoted in the plant pathogen Colletotrichum gloeosporioises.Changes in the expression levels of the transporters CDR1,CDR2, and MDR1 were not observed; however, the experimen-tal protocols used by Mateus et al. (41) and by us differ signif-icantly. The most highly induced gene of known function inCandida biofilm cells is MET3 (23-fold at T � 30). Roughlyone-third of the 300 genes upregulated upon contact to theabiotic surface have no known function (see Table S2 in thesupplemental material); of the remainder, 57% are homol-ogous to a subset of genes of the Ribi (for ribosome biogene-sis) regulon of S. cerevisiae. This regulon consists of 236 genesprimarily encoding proteins involved in ribosome biogenesis,subunits of RNA polymerases I and III, enzymes involved inribonucleotide metabolism, tRNA synthases, translation fac-tors, and proteins of unknown function predominantly local-ized in the nucleolus (31) and is coordinately and transientlydownregulated in response to environmental or genetic per-turbation (19, 28, 43). In C. albicans adhesion to polystyreneresults in the upregulation at T � 30 of a majority of the genehomologues in the S. cerevisiae regulon with the exception ofgenes encoding tRNA synthases and modifying enzymes, genesinvolved in nucleotide metabolism and translation initiationfactors (see Table S2 in the supplemental material). Becausethe expression of members of this regulon is inherently unsta-

ble (24), the levels of the mRNAs measured in our study mayalternatively reflect a decrease in their rate of degradation. Ingeneral, the regulon is repressed by a wide variety of subopti-mal growth conditions, by environmental factors that threatencell viability, and by defects in the secretory pathway (19, 38,43). Conversely, transcription of the regulon increases whenstarved yeast cells are provided with glucose (62) and is gen-erally regulated by pathways such as those which coordinatecell size, progression through the cell cycle, and membraneintegrity, with nutrient availability (31, 38). Overall, expressionof this regulon in S. cerevisiae seems to be associated withfavorable growth conditions; its immediate induction in C.albicans upon adherence to the polystyrene surface may eitherprovide C. albicans with the ability to rapidly respond to achange in environment and/or indicate that growth in the formof a biofilm is preferred.

Other genes of known function upregulated during the first30 min of incubation include sets that encode proteins involvedin sulfur metabolism (CYS3, MET3, MET10, MET14, MET15,ECM17, MXR1, SPE2, and GCS1), phosphate metabolism(PHO8, PHO81, PHO84, PHO86, and PHO89), and iron as-similation (ARN1, CFL9, FTH1, and FRP2).

Final remarks. Although the induction of sulfur assimilationpathways and the homologues of the Ribi regulon representthe most dramatic differences between planktonic and biofilmcells, involving the coordinate upregulation of functionally re-lated subsets of ORFs, there are additional changes that arealso likely to be important in understanding the process ofbiofilm development. Activation of enzymes involved in oxida-tive mechanisms such as NCE1O3 and GCS1, together withthe expression of newly identified ORFs with no homology inthe S. cerevisiae genome, creates new avenues to the search ofnew pathways during biofilm formation. Surprisingly, none ofthe genes implicated in azole resistance are upregulated duringthe first 6 h, but a potential role for GSH in drug resistance isa consideration. The challenge now lies in understanding thefunctional relevance of these major shifts in the readout of thegenomic repertoire when cells grow as communities.

ACKNOWLEDGMENTS

This study was supported by NIH/NIDCR grants R21 DE15290 andPO1 DE07946 from the National Institutes of Health.

We thank John Lehnen for proofreading the manuscript.

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