CaFT Profiling of Inhibitors of Ergosterol Biosynthesis

The CaFT strain pool includes heterozygotes for all but three genes involved in the ergosterol biosynthetic pathway (Figure 2A). This pathway represents a well-characterized target for antifungal agents (for a review see [15]) and provides an opportunity to determine the specificity of chemically induced HI in C. albicans by conventional methods (spot tests) and the CaFT.

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Figure 2. Characterization of Fluconazole-Induced HI by Spot Tests and CaFT Profiling

(A) Specificity of chemically induced HI by fluconazole as determined by spot tests. The heterozygous deletion strains corresponding to genes involved in the ergosterol biosynthesis pathway were tested against fluconazole at 0.75 μg/ml. Note that the two underlined strains, CYB5 (orf19.7049) and ERG4 (orf19.5379), were not present in the CaFT pool and that an ERG9 (orf19.3616) strain was not constructed. The HIS3 strain is used throughout this study as the wild-type control, since the HIS3 gene, as an auxotrophic marker, was used to construct all the heterozygous deletion strains (see Materials and Methods).

(B) CaFT profile of fluconazole at 0.162 μg/ml (with fitness, F, value of 0.80; i.e., IC₂₀). The x-axis represents the strains in the CaFT pool, and y-axis the normalized z-scores of the corresponding strain in this experiment. (Although the z-scores of both up- and down-barcodes are assessed independently for each strain, the higher of the two was selected for display.) Significant outliers are highlighted by filled symbols, as their z-scores deviate significantly from the population. The z-scores of two groups of strains, corresponding to other genes involved in ergosterol biosynthesis or potential efflux pumps, are highlighted by open symbols.

(C) CaFT profiles of fluconazole at different concentrations, with corresponding F values and ICs (in parentheses). In order to simplify a given CaFT profile, all the z-scores are displayed one-dimensionally according to the values but regardless of strain identities. Selected strains are marked so that their z-scores can be compared with the population and between experiments. Note that clotrimazole-induced HI of CDR1 and PDR16 was independently observed in the ScFT [13].

(D) Spot tests of heterozygous deletion strains identified in the CaFT (CDR1, PDR17, and ERG6) and the CDR2 and MDR1 strains (for their relevance, see the text) against multiple fluconazole concentrations.

(E) Spot tests of homozygous deletion strains against fluconazole. (The orf19 designations are as follows: ERG11 = orf19.922, NCP1 = orf19.2672, CDR1 = orf19.6000, PDR17 = orf19.5839, ERG6 = orf19.1631, CDR2 = orf19.5958, MDR1 = orf19.5604, FCR1 = orf19.6817.)

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Fluconazole is clinically used to treat C. albicans infections. It inhibits the sterol 14α-demethylase, which is encoded by ERG11. (Note that we use standard gene names as appear in the Candida Genome Database and MycoPathPD, with orf19 designations given in the appropriate figure legends, and that the prefix Sc is used to refer to S. cerevisiae genes.) The heterozygous deletion strains for genes involved in ergosterol biosynthesis were tested against fluconazole by spot tests at multiple concentrations. While none showed significant HI under the standard growth conditions, the ERG11 and NCP1 (encoding a NADP-cytochrome P450 reductase required for the Erg11p function) strains displayed specific hypersensitivity to fluconazole (Figure 2A). Similarly, both strains showed fluconazole-induced HI in the CaFT over a wide range of ICs, as their z-scores were significantly differentiated from the population (Figure 2B and 2C; a summary of all the CaFT results described is provided in Table S1.) Two additional hypersensitive strains detected are CDR1 and PDR17 (orthologous genes in S. cerevisiae are ScPDR5 and ScPDR16, respectively; note that the other ortholog of ScPDR16, orf19.1027, was not present in the pool). In addition, the ERG6 strain showed relative resistance as indicated by the significant negative z-scores at higher concentrations (Figure 2C). The responses of these strains to fluconazole were confirmed by the spot tests (Figure 2D). Although overexpression of efflux pump genes (CDR1, CDR2, and MDR1) and PDR16 (encoding a phosphatidylinositol transfer protein) was detected in fluconazole-resistant clinical isolates, only CDR1 and PDR16 have been correlated directly to resistance [16,17]. The strains for CDR2 and MDR1 showed no hypersensitivity to fluconazole in the CaFT (Table S1) or spot tests (Figure 2D), whereas homozygous deletion strains for CDR1 and PDR17 were markedly more susceptible to fluconazole (Figure 2E).

The five strains identified with fluconazole in the CaFT represent different aspects of its MOA: the drug target (Erg11p) and its accessory protein (Ncp1p), the principal efflux pump (Cdr1p), and two additional factors, Pdr17p and Erg6p, that are likely involved in drug uptake [18,19]. Another triazole (voriconazole) and imidazoles (ketoconazole, clotrimazole, econazole, and sulconazole) yielded similar CaFT profiles (Figure S1). The profiles of additional inhibitors of ergosterol biosynthesis, including terbinafine, lovastatin, and dyclonine, are described in Text S1 (Figure S2 and Table S1), and their results largely corroborate those determined in the ScFT [13]. Inhibitors that do not act through specific protein targets were also examined, including amphotericin B, which binds preferentially to ergosterol in the plasma membrane of fungi, and two toxic ergosterol analogs (ECC69 and ECC1384). In each case, complex CaFT profiles affecting multiple aspects of metabolism and membrane-related functions were produced but with no clear target(s) resolved (Figure S3).

CaFT Profiling of Inhibitors of Other Enzymes and Protein Complexes

The CaFT profiles of enzyme inhibitors are usually concise. For example, ALG7 and AUR1 heterozygotes correspond to the target gene of tunicamycin and aureobasidin A, respectively, and each displayed highly significant and specific hypersensitivity to its cognate inhibitor (Figure S4). Similarly, the catalytic subunit (FKS1) and the regulatory subunit (RHO1) of glucan synthase were identified in the CaFT with their cognate inhibitors, caspofungin and ergokonin A (Figure S5). Brefeldin A is unusual in that it binds to the interface of two proteins, both of which are members of two protein families in C. albicans. Despite such apparent complexity, a single target pair, SEC7 and ARF2, was robustly identified in the CaFT (Figure S6).

Cerulenin specifically inhibits the condensation reaction associated with the α subunit of the fatty acid synthase (FAS), a heteromultimeric complex of α (Fas2p) and β (Fas1p) subunits. Although not examined in the ScFT, cerulenin elicited reproducibly specific hypersensitivity of the FAS1 strain but not of FAS2, even at the highest drug concentration tested (Figure 3A). These results were also demonstrated by spot tests (Figure 3B). In S. cerevisiae, the expression of ScFAS2 is regulated in an ScFas1p-dependent manner to control the stoichiometry of the FAS complex [20]. Our results suggest that a similar regulatory mechanism exists in C. albicans, with the level of Fas1p being the critical factor controlling the FAS complex (see Figure 3C and Discussion). Consistent with this model, only FAS1 exhibits HI under the standard growth conditions (Figure 3B and 3D).

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Figure 3. CaFT Profiling and Characterization of Cerulenin-Induced HI

(A) CaFT profiles of cerulenin, with highlighted strains as follows: FAS1 (orf19.979), FAS2 (orf19.5949), and MDR1 (orf19.5604). A homozygous deletion of MDR1 is hypersensitive to cerulenin [21]; however, the heterozygous deletion strain showed no specific hypersensitivity at the ICs tested in the CaFT.

(B) The spot tests of selected heterozygous deletion strains against cerulenin. Note that the hypersensitivity of the MDR1 strain was only seen at the highest concentration of cerulenin tested.

(C) A model for regulating stoichiometry of the FAS complex in S. cerevisiae. As shown by Wenz et al. [20], the expression of ScFAS2 is repressed by an unknown transcription repressor (rep. X), which is in turn derepressed by free β subunit (Fas1p). According to this model, ScFAS2 expression is dependent on free Fas1p to control the normal stoichiometry of the FAS complex. A similar regulatory mechanism in C. albicans may explain the observed cerulenin-induced HI of FAS1 but not FAS2 (see text for details).

(D) FAS1 is haploinsufficient under the standard growth conditions. In order to determine HI, cultures of the selected strains were first incubated for 6 h (reaching exponential growth) and then diluted to an OD₆₀₀ of 0.005. The fresh cultures were incubated for another 12 h, after which the OD was monitored for an additional 4 h.

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Failure to detect hypersensitivity of the FAS2 heterozygote to cerulenin reflects a more general difficulty in correctly identifying chemically induced HI within protein complexes, due to regulation of subunit stoichiometry, its assembly, or activation. To further investigate these potential issues, additional reference compounds that inhibit distinct protein complexes were examined.

Microtubules are comprised of α- and β-tubulin subunits encoded by TUB1 and TUB2, respectively. A potential binding site for benomyl, a microtubule depolymerizing agent, has been suggested in the core of S. cerevisiae β-tubulin [22], and the heterozygous deletion strains for both ScTUB1 and ScTUB2 are benomyl hypersensitive [23]. In the CaFT, however, only the TUB1 strain displayed significant hypersensitivity to benomyl, as well as to additional microtubule inhibitors, including nocodazole, mebendazole, and thiabendazole, while the TUB2 strain was marginally hypersensitive only to nocodazole (Figure 4A and unpublished data; note that TUB4, orf19.1238, was not represented in the CaFT). Spot tests confirmed the hypersensitivity of the TUB1 strain and the lack of benomyl-induced HI for TUB2 (Figure 4B). The lack of specific hypersensitivity of the TUB2 heterozygote to most, if not all, of the microtubule inhibitors tested raises the question of how the stoichiometry of α- and β-tubulin subunits is regulated in C. albicans. In yeast, overexpression of ScTUB2 results in lethality, whereas overexpression of ScTUB1 does not [24]. Moreover, mutations in ScTUB1 and ScTUB2 are known for their unlinked noncomplementation [25]. In contrast, only an ScTUB1 heterozygote is reported to be defective under the standard growth conditions [23,26]. In C. albicans, heterozygosity of both TUB1 and TUB2 confers growth defects (Figure 4C). Although stoichiometric regulation of tubulins in C. albicans is unknown, it may confound the chemically induced HI phenotypes associated with TUB1 and TUB2.

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Figure 4. CaFT Profiles of Microtubule Inhibitors

(A) CaFT profiles of nocodazole, benomyl, mebendazole and thiabendazole. Note that the ScFT experiments with these compounds have not been reported. Highlighted, in addition to TUB1 (orf19.7308) and TUB2 (orf19.6034), are BUB1 (orf19.2678), MAD2 (orf19.1040) and ESP1 (orf19.3356) strains that showed modest but reproducible hypersensitivity to nocodazole. The corresponding proteins are involved in mitotic checkpoint regulation and spindle pole body assembly [27]. Their relevance becomes apparent in the results described in Figure 7.

(B) Spot tests of heterozygous deletion strains highlighted in (A) against benomyl. Note the lack of hypersensitivity of TUB2 in spot tests is consistent with the CaFT results, implying that the failure to detect hypersensitivity of the TUB2 strain in the CaFT is not due to poor performance of the barcodes.

(C) TUB1 and TUB2 strains are haploinsufficient under the standard growth conditions (see Figure 3 legend for experimental details).

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Radicicol inhibits cell growth by competitively binding to the conserved chaperone, HSP90 [28], a key molecular chaperone that, together with its co-chaperones, facilitates proper folding of multiple client proteins [29]. Unlike S. cerevisiae, which contains two HSP90 proteins, ScHsc82p and ScHsp82p, C. albicans possesses only one. Despite sharing all the conserved amino acid residues in both yeast HSP90 proteins implicated in radicicol binding [28], the HSP90 heterozygous deletion strain lacked detectable hypersensitivity when tested in the CaFT (Figure 5A) or when examined either by spot tests (Figure 5B) or liquid IC determination (unpublished data). Instead, three (SGT1, CDC37, and CNS1) of the five potential Hsp90p-associated co-chaperones (CPR6 and orf19.7602 are also represented in the CaFT) displayed HI to radicicol (Figure 5A and 5B). Interestingly, the three yeast orthologs, ScSgt1p, ScCdc37p, and ScCns1p, all physically and genetically interact with ScHsc82p and/or ScHsp82p [30,31]. Although radicicol-induced HI of HSP90 was not detected, the CaFT profiles reflected other aspect of the target; that is, activation of Hsp90p by its co-chaperones (see Discussion).

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Figure 5. CaFT Profiles and Spot Tests of Radicicol

(A) CaFT profiles of radicicol with genes encoding co-chaperones of Hsp90p highlighted, including SGT1 (orf19.4089), CDC37 (orf19.5531), CNS1 (orf19.6052), orf19.7602 (an ortholog of ScAHA1), and CPR6 (orf19.7654) ([30] and references therein). Radicicol also elicited HI of ERG27 (orf19.3240), CDR1 (orf19.6000, an efflux pump), and PRS1 (orf19.1575, involved in cell integrity/stress signaling as reported [32]).

(B) Spot tests of radicicol against heterozygous deletion strains corresponding to HSP90 and its co-chaperones.

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Additional inhibitors of protein complexes tested with informative mechanistic CaFT profiles included cytochalasin D, roridin A, and verrucarin A. Cytochalasin D inhibits both association and dissociation of actin filaments in vitro. The CaFT results reveal a particular aspect of actin polymerization affected in vivo by cytochalasin D, namely, the branching of actin cables to produce cortical actin, as multiple members of the ARP2/3 complex showed markedly HI (Figure S7). The structurally related mycotoxins roridin A and verrucarin A both noticeably affected multiple subunits of the initiation factor eIF3 complex (Figure S8).

CaFT Profiling and MOA Studies of 5-Fluorouracil, 5-Fluorocytosine, and Tubercidin

The preceding examples demonstrate the specificity of chemically induced HI and the biological relevance of information contained within CaFT profiles; that is, small molecules that selectively inhibit proteins or protein complexes typically elicit specific CaFT profiles comprising the target proteins and/or other factors that functionally interact with the targets. The base analogs, 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU), on the other hand, do not exert inhibitory effects directly on specific proteins. We tested whether both analogs and tubercidin (7-deazaadenosine) elicit specific HI indicative of their MOAs. 5-FC and 5-FU are pro-drugs whose MOA has been well characterized in S. cerevisiae and to a lesser extent in C. albicans (see below). 5-FC, once inside the cell, is converted to 5-FU by cytosine deaminase, and 5-FU to 5-FUMP by uracil phosphoribosyltransferase (UPRT). Both enzymes are part of the pyrimidine salvage pathway (Figure 6A). 5-FU, in the ScFT, has been shown to induce HI of several genes involved in rRNA processing and ribosomal biogenesis [11,13]. In the CaFT, 5-FU, 5-FC, and tubercidin elicited responses of two distinct groups of heterozygous deletion strains, reflecting collectively a common MOA. While the majority of hypersensitive strains correspond to genes whose S. cerevisiae orthologs are involved in biogenesis of the 60S ribosomal subunit, resistant strains predominantly correspond to those encoding protein subunits of the U3 snoRNP complex, which is required for 18S rRNA processing (Figure S9). As ribosomal RNAs account for over 80% of the total cellular RNA, the preferential effects on rRNA biogenesis by these inhibitors may simply reflect the relative abundance of rRNAs. Another analog of uracil, 6-azauracil (6-AU, known to inhibit the IMP dehydrogenase involved in purine biosynthesis [33]), on the other hand, did not produce a ribosomal biogenesis–related profile (Figure S9A). The distinct CaFT profiles between 6-AU and 5-FC, 5-FU, or tubercidin suggest that a more specific mechanism may be involved (see Discussion).

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Figure 6. Genetic Characterization of 5-FC and 5-FU in C. albicans

(A) Pyrimidine salvage pathway, and transport and metabolism of 5-FC (see text for details).

(B) A hypothesis that fluorinated uracil blocks the formation of pseudouridine in rRNA. Note that pseudouridines are a prerequisite for ribosomal RNA processing [34].

(C and D) Functional characterization of potential permeases or transporters involved in the uptake of 5-FC (C) and 5-FU (D). A homozygous deletion strain for FCY2 (distinguished by the asterisk) and conditional shut-off (tetracycline repressible promoter, GRACE) strains [6] for genes indicated were tested in the absence (i.e., the non-repressing conditions, top panels) or the presence (i.e., the repressing conditions, bottom panels) of 100 μg/ml tetracycline against both compounds at the concentrations indicated. Note that 1) in (C) the FCY2 homozygous deletion strain was resistant to 5-FC, and 2) in (D) only the FCY21 GRACE strain exhibited slightly increased susceptibility to 5-FU under the non-repressing condition, suggesting that overexpression of FCY21 (from the tetracycline promoter, as observed in other cases, unpublished observations) may marginally facilitate the uptake of 5-FU.

(E) Suppression of 5-FC (top) and 5-FU (bottom) antifungal activity by genetic depletion of Fur1p. The FUR1 and HIS3 (the control) GRACE strains were grown in the presence (100 μg/ml, +TET) or absence (−TET) of tetracycline with 5-FC or 5-FU at the concentrations indicated. Individual growth was normalized with growth of the HIS3 strain without antifungal drug in the presence or absence of tetracycline. Note that when the expression of FUR1 was repressed, the strain was markedly resistant to both compounds, whereas it was modestly hypersensitive under the non-repressing conditions, likely due to overexpression of FUR1 from the tetracycline promoter. (The orf19 designations are as follows: FCY2 = orf19.1357, FCY21 = orf19.333, NNT1 = orf19.4118, DIP5 = orf19.2445, and FUR1 = orf19.2640.)

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The CaFT profiles of 5-FU and 5-FC also provide mechanistic insights into their uptake and metabolism. Counterintuitively, 5-FC is over 200-fold more potent than 5-FU (Figure 6C and 6D). The underlying mechanism for this difference is revealed in their CaFT profiles. FCY2 encodes a permease for cytosine and purines. The corresponding heterozygous deletion strain displayed specific and significant resistance to 5-FC, but not to 5-FU or tubercidin (Figure S9A). A homozygous deletion of FCY2 was highly and specifically resistant to 5-FC (Figures 6C, 6D, and S9B), suggesting a direct and specific role of Fcy2p in transporting 5-FC. In contrast, 5-FU may enter the cell by passive diffusion (Figure 6D), hence the relatively low potency. Similarly, the nucleoside transporter Nnt1p (absent in S. cerevisiae) was identified as responsible for the uptake of tubercidin (Figure S9B). Another notable resistant strain identified in the CaFT profiles of both 5-FC and 5-FU corresponds to FUR1 (Figure S9A). FUR1 encodes UPRT that converts both pro-drugs to 5-FUMP (Figure 6A). A loss of function of FUR1 is expected to confer resistance to both 5-FC and 5-FU, as demonstrated by the high level resistance of a FUR1 conditional shut-off strain under the repressing conditions (Figure 6E). These results predict specific resistance mechanisms that are known for 5-FC. Since the pyrimidine salvage pathway (Figure 6A) is not essential when the de novo pathway is intact, any loss-of-function mutations in genes involved in the former may have no effect on viability, and yet they may confer specific resistance, as demonstrated by mutations in the uptake and/or metabolism of this compound in S. cerevisiae (e.g., [35]) and, more importantly, in clinical isolates of C. albicans (e.g., [36,37]). This may also account for the primary resistance in C. albicans; that is, the inherent resistance to 5-FC without prior exposure, which is observed in ∼3% of clinical isolates [38].

Novel Antifungal Compounds That Affect Microtubule Dynamics

Mechanisms inferred from screening known antifungal agents in the fitness tests of S. cerevisiae and C. albicans imply that MOA information could similarly be obtained for new compounds. To test this possibility, we examined a group of chemically related active compounds with unknown MOA (Figure 7A). CaFT profiles identified the TUB1 strain as strikingly hypersensitive to all compounds examined (Figure 7B). Distinct secondary hypersensitivity, including heterozygotes compromised for mitotic checkpoint and cortical actin function, enabled compounds to be grouped into three sub-classes (Figure 7A and 7B). Selected compounds from each class were examined in spot tests, which largely confirmed the CaFT results (Figure 7C). Compared with known microtubule inhibitors (Figure 4), the CaFT profiling suggested that these compounds inhibit growth, likely by affecting the microtubule function.

To further characterize the MOA of these compounds, multiple microtubule-based secondary assays were performed. Microtubules form the core structural component of the nuclear mitotic spindle and are necessary for chromosomal movement. In the yeast, the movement of nuclei to the bud neck before mitosis and the subsequent separation of nuclei depend on cytoplasmic microtubules [39,40]. In the wild-type cells, these events occur in a highly coordinated cell cycle–dependent manner such that the large-budded cells are almost never observed without the nucleus at, or extended through, the bud neck. As nuclear migration defects associated with microtubule perturbation can readily be visualized by DAPI staining of DNA, we examined the terminal phenotypes associated with a TUB1 conditional shut-off strain and compared them with those observed in the wild-type C. albicans cells chemically inhibited with benomyl, nocodazole, and representative compounds from each sub-class (ECC85, ECC248, and ECC275). One hour after TUB1 repression, cell division and nuclear migration were largely arrested in the majority of the cells examined, as large-budded cells were predominantly observed, with nuclear staining being restricted to the mother cell (Figure 8A). To visualize microtubules, a C. albicans strain carrying a Tub1p-GFP fusion (under the TUB1 promoter, otherwise indistinguishable from the wild-type parental strain) was used to compare the effects of known microtubule inhibitors versus these ECC compounds. In the mock-treated (1% DMSO) cells, two distinct sub-cellular microtubule structures were observed depending on the cell cycle: the mitotic spindles (visualized in the large-budded cells) and the spindle-pole bodies (visualized prominently in the small-budded cells) (Figure 8B). As expected, benomyl and nocodazole ablated the mitotic spindles, resulting in patches or aggregates of tubulin. Defects in nuclear division and G2 cell cycle arrest, as well as a more pronounced filamentous growth, were also observed (Figure 8C and 8D). These results are in agreement with the previous observations [41,42] and establish a strong phenotypic correlation between genetic depletion and chemical inhibition of the tubulin function. The defects caused by ECC85 (Figure 8E), ECC248 (Figure 8F), and ECC275 (unpublished data) were similar to those, described above, seen for cells treated with nocodazole, benomyl, or genetically depleted for TUB1. However, cells treated with the control compounds fluconazole (Figure 8G) and amphotericin B (unpublished data) lacked microtubule and/or cell cycle defects. To address whether the representative compounds of each ECC sub-class directly inhibit microtubule polymerization, their effects on in vitro bovine microtubule polymerization were examined. ECC85, ECC248, and ECC275 each displayed a dose-dependent inhibition of microtubule polymerization. Moreover, the IC50 (of V_max) of ECC85 and ECC248 was approximately three times lower that of nocodazole (Figure S10). Collectively, these data provide in vitro and in vivo evidence that these compounds perturb the function of microtubules in C. albicans.

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Figure 7. CaFT Profiling and Characterization of Novel Antifungal Compounds

(A and B) Chemical structures (A) and the CaFT profiles (B) of novel and structurally related compounds that are predicted to affect microtubule dynamics.

(C) Spot tests of representative compounds (ECC220, ECC284, and ECC275) confirm hypersensitivity detected in the CaFT. For orf19 designation, see Figure 4.

https://doi.org/10.1371/journal.ppat.0030092.g007

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Figure 8. MOA Characterization of Compounds That Affect Microtubule Dynamics

(A) The phenotypic defects associated with a TUB1 conditional shut-off strain [6] examined under the repressing conditions (with time after the switch indicated on each photo). Under the non-repressing conditions, this strain was indistinguishable from the mock-treated wild-type cells (unpublished data). Nuclear migration was visualized by DAPI straining with morphology viewed under Nomarski optics. In (B–G), a strain carrying a Tub1p-GFP fusion was used.

(B) The microtubule/spindle structures (as visualized by GFP-Tub1 fusion), nuclear migration/division, and morphology of the mock-treated cells.

(C–G) The microtubule/spindle structures, nuclear migration, and morphology of cells treated with benomyl (C), nocodazole (D), ECC85 (E), ECC248 (F), and fluconazole (G). The concentrations (μg/ml, in parentheses) and incubation times are indicated on each photo.

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Genome annotation.

The C. albicans genome sequence at 10.9X coverage was determined by the Stanford Genome Technology Center (http://www-sequence.stanford.edu/). A precise genome annotation for C. albicans [3] was not publicly available during the course of this project. Instead, a list of 7,680 open reading frames (ORFs) encoding proteins ≥100 amino acids provided in an earlier release was used to initiate an internal annotation effort. To select ORFs for construction of heterozygous deletion strains, only those satisfying either of the following conditions were initially chosen: 1) ORFs (n = 4,068) with clear homologs (BLAST e-value < 1 × 10⁻²⁰) at amino acid level in other fungal species, or 2) ORFs (n = 1,635) with no clear fungal homolog but ≥ 600 nucleotides in length. Recent S. cerevisiae annotation efforts demonstrate that such rules provide 99% and 98% confidence of a bona fide gene locus rather than a spurious ORF [5,55]. The high degree of conservation in gene structure between S. cerevisiae and C. albicans, including average length, intron structure, intron-occurring frequency, GC-contents, and promoter elements [3], strongly reinforces the applicability of such gene-coding “rules” to C. albicans genome annotations.

C. albicans heterozygote strain construction and the CaFT strain pool composition.

Heterozygous deletion strains were constructed essentially as in S. cerevisiae, with some modifications [6,56,57]. Briefly, individual strains were constructed by replacing the entire ORF of a particular gene with a cassette of HIS3 gene flanked by distinct upstream and downstream barcodes, termed up-tag and down-tag, respectively. Each tag is in turn flanked by two primer sequences that are common to all the up- or down-tags (Figure 1). The nucleotide sequences for up- and down-tags, as well as the flanking common primers, were as used in the S. cerevisiae deletion project [57]. This configuration provided two very powerful features to these double-barcoded heterozygote strains: 1) it enables PCR amplification of large numbers of distinct up- or down-tags using two sets of common PCR primers, and 2) it allows the identification of each heterozygote strain (and the corresponding gene) via the identities (i.e., the sequences) of a unique pair of up- and down-tags. More specifically, PCR amplification of a deletion cassette containing a HIS3 auxotrophic marker was performed using 99-nucleotide (nt) oligos containing 1) 5′ sequence of 43 nt directing homologous recombination at the 5′ or 3′ of the target gene, 2) internal strain-identifying unique 20-nt barcodes and flanking common primer sequences for the ultimate PCR amplification of the barcodes in the fitness test assay, and 3) 18 nt of 3′ sequence, which anneals to the 5′ or 3′ of the CaHIS3 gene and facilitates PCR amplification of the disruption cassette for transformation. Transformants were genotyped for correct integration into the target locus by PCR to confirm the expected 5′ and 3′ junctions. Genes chosen for disruption were selected based on prior knowledge that their closest homolog is 1) known to be essential in S. cerevisiae, 2) a member of a gene family known to display a growth phenotype when deleted in S. cerevisiae, or 3) conserved in A. fumigatus or other fungal organisms. In total, this procedure was applied to 2,868 distinct genes, which represented approximately 45% of the C. albicans genome. They included all the experimentally demonstrated essential genes we reported previously [6]. These 2,868 heterozygote strains were cultured individually and manually mixed in approximately equal proportions to generate a pool of C. albicans strains, aliquots of which were frozen and used in all the experiments. Although the full collection of strains comprising the CaFT is not available at this time, individual strains pertinent to the profiles presented in this study are accessible to investigators, following the standard Merck Material Transfer Agreement and clearance procedures.

Construction of homozygous deletion strains.

Heterozygous deletion strains were used to construct homozygous deletions with the PCR-based method described [6], except that the coding region of the second allele was replaced by the SAT-1 gene, which confers resistance to nourseothricin.

Antifungal compounds.

Compounds were purchased from Sigma-Aldrich (http://www.sigmaaldrich.com/), with the following exceptions: caspofungin from Merck (http://www.merck.com/), fluconazole from Pfizer (http://www.pfizer.com/), and aureobasidin A from Takara/Fisher (http://www.fishersci.com/). ECC220 is available from AKos Consulting and Solutions GmbH (Basel, Switzerland; http://www.akosgmbh.eu/), ECC22 from Interchim (Montlucon, France; http://www.interchim.com/), ECC248 from ASINEX (Moscow, Russia; http://www.asinex.com/), and ECC275 from Ambinter (Paris, France; http://www.ambinter.com/).

The CaFT DNA microarrays.

We custom-built a set of two DNA microarrays using Amersham CodeLink Activated Slides (http://www.gelifesciences.com/). These microarrays contain DNA oligos identical to the up- or down-tags, with each oligonucleotide duplicated side by side. Each array contains 16 sub-arrays (of 16 × 24 spots), for a total of 3,072 duplicated features, corresponding to all the strains in the CaFT pool, and various controls.

The CaFT experiments and data analysis.

For each compound tested, a prior IC curve was determined using the CaFT strain pool (with an initial OD₆₀₀ of 0.025) in liquid RPMI medium (in 96-well format), grown at 30 °C for 15 h. Based on the IC curve, 5-ml cultures of the CaFT pool (with the initial OD₆₀₀ of 0.025) were treated with the selected compound at multiple concentrations, together with mocks. After 15 h of growth at 30 °C, the fitness (F) values of compound-treated cultures were determined, F = OD_{(compound-treated)} / OD_(mock); that is, the inverse of IC. Cultures of desired F values were selected and diluted to OD₆₀₀ 0.05 with the medium containing the compound at the original concentrations. After another 23 h of growth, all cultures were collected and cell pellets frozen. Following extraction (MasterPure Yeast DNA Purification Kit; Epicentre, http://www.epibio.com/), genomic DNA preparations from compound-treated and mock cultures were PCR amplified with Cy3- or Cy5-labeled common primers. Labeled tags from compound-treated and mock cultures were mixed and hybridized against the corresponding DNA microarray (Figure 1). (The up- and down-tags were amplified and hybridized separately.) The intensities of signal were obtained for each barcode of compound-treated and mock cultures from a single DNA microarray. They were first converted to a log₂ scale before further analysis. For each tag, the log-fold-dropout (LFD) was computed as the average differences between the mock and the compound-treated. Following the compilation of results for 57 diverse compounds (referred to as the reference set), a statistical profile was computed for each tag by modeling the distribution of LFD as a mixture of a normal component (random variation of the tag when the strain growth is not preferentially affected by the compound) and a uniform distribution (representing unusual LFDs for a given tag). This model was optimized using the EM algorithm [58], and the parameters of the normal component were stored as a model of the intrinsic variability of a given tag through the complete experiment. For any tag in any given experiment, the computed LFDs are converted to a z-score by using the parameters of the corresponding tag profile. To facilitate the comparison of experiments with different LFDs distribution, we applied a multiplicative correction factor to the LFDs that corresponds to 1/σ_z, where σ_z is the standard deviation of the z-scores for that experiment. The z-scores were then recomputed from these normalized LFDs and are referred to as normalized z-scores. To avoid result overinterpretation, the z-scores are not converted to p-values and are considered the final quantitative result of the experiment. This approach has the advantage of accounting for the nonspecific sensitivity of some tags/strains to chemical insults and for unexpectedly variable tags/strains, and thus greatly reduces the frequency of false positives. Contrary to other approaches (e.g., [59]), our analyses do not rely on a correlation between signal variability and signal strength, but rather are modeled on a tag-by-tag basis, taking advantage of multiple experiments as pseudo-replicates, and rely on the EM algorithm to identify outliers. We hold that using a set of compound-treated cultures to establish a statistical model is a superior approach to using a set of replicated mock cultures since the latter would not model nonspecific responses to variations such as growth conditions and chemical insult (e.g., broadly hypersensitive stains).

C. albicans spot tests.

Individual heterozygous strains were first grown in liquid medium (without compound) to OD₆₀₀ ∼2. Cultures were diluted to OD₆₀₀ 0.2 and transferred to 96-well microtiter plate, followed by 1:5 serial dilutions. Aliquots (3 μl) of diluted cultures were spotted onto solid media (YPD) containing 1% DMSO (mock) and inhibitory compounds at the concentrations indicated with 1% DMSO. Plates were incubated at 30 °C, and photographed after 2 d (unless otherwise noted in the figure).

In vitro tubulin polymerization assay.

Cytoskeleton CytoDYNAMIXTM Screen 3 (http://www.cytoskeleton.com/) was employed in these assays, in which purified tubulin (>99% pure tubulin isolated from bovine brain, from Cytoskeleton catalog # TL238) was used to follow the in vitro microtubule polymerization process. To initiate the assay, different concentrations of ECC85, ECC248, ECC275, or nocodazole were added to the standard assay solution, which contains 3.5 mg/ml purified bovine tubulin in 80 mM PIPES (pH 6.9), 1 mM MgCl₂, 1 mM EGTA, 1 mM GTP, 5% glycerol, and 1% DMSO. The assay mixtures were incubated at 37 °C for 60 min, and the polymerization process was monitored by turbidity measurements at OD₃₄₀ at 1-min intervals.