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Antimicrobial Agents and Chemotherapy, May 2009, p. 1772-1778, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.00020-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role for Fks1 in the Intrinsic Echinocandin Resistance of Fusarium solani as Evidenced by Hybrid Expression in Saccharomyces cerevisiae

Santosh K. Katiyar and Thomas D. Edlind^*

Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania

Received 6 January 2009/ Returned for modification 2 February 2009/ Accepted 14 February 2009

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The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.

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Serious fungal infections have increased in recent years as a consequence of increased immunosuppression associated with human immunodeficiency virus infection, organ and tissue transplants, and aggressive treatments for neoplastic and autoimmune disease. These infections typically are treated with ergosterol biosynthesis-inhibiting azole antifungals such as fluconazole. However, azoles have limitations: their activity is fungistatic, acquired resistance in normally susceptible yeast is not uncommon, and intrinsic low- to high-level resistance is demonstrated by many molds (3, 21). The membrane-disrupting antifungal amphotericin B is generally fungicidal and has broad-spectrum activity, and resistance to it is rare, but its use remains limited due to toxicity. The echinocandins (ECs) caspofungin (CSP), micafungin (MCF), and anidulafungin represent the most recently introduced group of antifungals. Importantly, ECs have fungicidal activity against most Candida species (including azole-resistant strains), fungistatic activity against Aspergillus species, and negligible toxicity (5, 20, 24). ECs act by inhibiting the synthesis of the cell wall polysaccharide β-1,3-glucan (7). This can result in cell lysis or more subtle cell wall changes that enhance susceptibility to innate immunity (25, 41).

Acquired EC resistance in susceptible fungi is associated with specific mutations in the integral membrane protein Fks1 (or its paralog Fks2) (7, 35). Fks1 is believed to represent the β-1,3-glucan synthase catalytic subunit, although this has not been formally proven since only crude membrane preparations retain catalytic activity. Most resistance-conferring mutations cluster within so-called hot spot 1, which corresponds, within the model yeast Saccharomyces cerevisiae, to Fks1 residues Phe639 to Pro647 (F639-P647) (1, 2, 6, 7, 13, 18, 19, 26, 33, 34, 40). Fortunately, acquired EC resistance is rare. On the other hand, the intrinsic EC resistance of ascomycetous molds such as Fusarium solani and Scedosporium prolificans, zygomycetous molds such as Rhizopus oryzae, and the basidiomycetous yeasts Cryptococcus neoformans and Trichosporon asahii represents a major limitation to EC clinical use. Many of these fungi have emerged in recent years as important opportunistic pathogens (3, 27, 32, 37), and a contributing factor may be their intrinsic resistance to antifungals, including ECs (10, 22, 30).

The basis for intrinsic EC resistance has been investigated in several of these fungi, but it remains unclear (14, 15, 28, 39). Notably, Ha et al. (14) reported that F. solani Fks1, when heterologously expressed in A. fumigatus, conferred a modest but potentially significant fourfold decrease in CSP susceptibility, the basis for which was not explored. However, expression in their system was abnormally low, and A. fumigatus itself exhibits some degree of intrinsic EC resistance (5), which together complicate the interpretation of this result. On the other hand, recent studies examining the basis for the intrinsically low EC susceptibility of Candida parapsilosis strongly implicated its Fks1 sequence; specifically, a hot spot 1 substitution equivalent to P647A (12).

FKS1 initially was identified as the S. cerevisiae gene whose null mutation confers susceptibility to the calcineurin inhibitor FK506 (7, 8). This susceptibility results from the requirement for the calcineurin-mediated expression of FKS2; relatedly, single fks1 and fks2 disruptants are viable, but double disruption is lethal (31). Here, we exploit this FK506 susceptibility to construct Fks1 hybrids, replacing hot spot 1 from S. cerevisiae with that from F. solani (and, for comparison, A. fumigatus and Candida glabrata) to further test the hypothesis that intrinsic EC resistance is mediated by Fks1 sequence.

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Strains, drugs, and reagents. Strains were obtained from the following sources: S. cerevisiae BY4742 (Research Genetics), A. fumigatus Af293 (no. A1100; Fungal Genetics Stock Center), F. solani (Nectria haematococca MPVI; no. 8124; Fungal Genetics Stock Center), S. prolicans 07-1208 (A. Fothergill, University of Texas Health Science Center at San Antonio), C. glabrata R-3562 (P. D. Rogers, University of Tennessee Health Science Center; henceforth referred to as strain CgS), and C. glabrata 20409.021 (M. Pfaller, University of Iowa; henceforth referred to as strain CgR). BY4742 deletant fks1453-649::URA3 (FKS1 codons 453 to 649 replaced with URA3) was described recently (12). Plasmid pRS416 was obtained from J. Nickels (Drexel University College of Medicine). Except where noted, the medium was YPD (1% yeast extract, 2% peptone, 2% dextrose). Synthetic defined medium without uracil/uridine (SD-ura) or with complete supplement mixture (SD+csm) was obtained as DOB from Qbiogene/MP Biomedicals (Solon, OH). FK506 was obtained from Tecoland (Edison, NJ), 5-fluoroorotic acid from RPI (Mount Prospect, IL), CSP (Cancidas) from Merck (Rahway, NJ), and MCF (Mycamine) from Astellas (Deerfield, IL). Drug stocks were prepared in dimethyl sulfoxide and stored at –20°C. DNA primers (listed in Table 1) were purchased from IDT (Coralville, IA).

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TABLE 1. DNA primers used in this study

DNA preparation, PCR, and sequencing. DNA was prepared by phenol extraction as described previously (9), or by a more rapid method involving the vortexing of cells with glass beads for 5 min, heating them in a boiling water bath for 3 min, and centrifugation at high speed to pellet debris. To generate deletants, mutants, and hybrids, PCR was performed with high-fidelity Phusion polymerase as recommended by the manufacturer (New England Biolabs, Ipswich, MA), with the indicated primers and template, 28 cycles, and annealing at 54°C. For screening and sequencing, PCR was performed with Taq polymerase (New England Biolabs) under the same conditions, generally with forward primer ScFKS1-425F or ScFKS1-586F and reverse primer ScFKS1-707R or URA3-117R. Aliquots were analyzed by gel electrophoresis. PCR products were treated with ExoSAP-IT (USB, Cleveland, OH) prior to sequencing (Genewiz, South Plainfield, NJ) with primer ScFKS1-425F or ScFKS1-586F.

Amplification and sequencing of S. prolificans FKS1. PCR primers conFKS1-311F and conFKS1-748R were designed based on Fks1 sequences (corresponding to S. cerevisiae residues 311 to 318 and 740 to 748, respectively) conserved among ascomycetous molds, particularly Fusarium species. S. prolificans genomic DNA was prepared from conidia germinated in YPD medium for 5 h. PCR was performed with Phusion polymerase, with annealing at 50°C for 3 cycles followed by annealing at 60°C for 29 cycles. The product was directly sequenced with the primers used for amplification. S. prolificans-specific primers then were designed (e.g., SpFKS1-714F) and used in conjunction with additional primers based on conserved sequences to complete the S. prolificans sequence corresponding to S. cerevisiae Fks1 codons 319 to 1457.

Construction of Fks1-F639Y mutant. The method used for the site-directed mutagenesis of the chromosomal FKS1 gene was described recently (12). Briefly, strain fks1453-635::URA3 was constructed by transforming BY4742 with a PCR product generated using pRS416 template and primers ScFKS1-453-URA3F and ScFKS1-635-URA3R, with selection on SD-ura plates. Following confirmation by PCR and sequencing, this strain was transformed with a PCR product generated using the BY4742 genomic DNA template and primers ScFKS1-375F and ScFKS1mut639R; the latter incorporated an AT mixture (W) into the second position of codon 639 to specify either F or Y. Transformants were selected on FK506 (1 µg/ml in YPD) plates (for the reconstitution of functional Fks1) and screened on SD-ura plates (for the loss of URA3). PCR products generated with primers ScFKS1-425F and ScFKS1-707R were sequenced to confirm FKS1 reconstitution and identify the F639 mutation.

Construction of Fks1 hybrids. The method used for hybrid Fks1 construction represents an extension of the site-directed mutagenesis method. To construct the fks1617-649::URA3 strain (step 1 of the two-step method), primers ScFKS1-617-URA3F and ScFKS1-649-URA3R were designed that incorporate 20 bases at their 3' ends to amplify URA3 and 40 bases at their 5' ends complementary to FKS1 sequences upstream of codon 617 or downstream of codon 649, respectively. The template was URA3-containing plasmid pRS416 (GenBank accession no. U03450). PCR products were purified (IsoPure; Denville, Metuchen, NJ), and 1 µg was used to transform BY4742 as described previously (9), with selection on SD-ura plates. Colonies were screened by replica plating on YPD with and without FK506 (1 µg/ml). PCR screening and DNA sequencing confirmed the desired deletion.

To generate the fks1-617-649 hybrids (step 2 of the two-step method), forward (ScAfFKS1-617F and ScFsFKS1-617F) and reverse (ScAfFKS1-649R and ScFsFKS1-649R) primers were designed that incorporate ca. 20 bases at their 3' ends to amplify fungal (A. fumigatus or F. solani) FKS1 sequences equivalent to codons 617 through 649 (determined by BLAST alignments) and ca. 40 bases at their 5' ends complementary to S. cerevisiae FKS1 sequences upstream or downstream of codons 617 and 649, respectively. These primers were used for PCR with the respective A. fumigatus or F. solani genomic DNA template. Purified product (1 µg) was transformed into the fks1617-649::URA3 strain described above, with selection on FK506 (1 µg/ml in YPD) plates for the reconstitution of functional FKS1. Colonies were screened on SD-ura medium for the loss of URA3. Alternatively, colonies from the FK506 plate were pooled and ca. 50,000 cells plated on SD+csm with 5-fluoroorotic acid (0.075%) to select for the loss of URA3. PCR and sequencing were used to confirm the desired fks1-Af617-649 (fks1 reconstituted with A. fumigatus residues 617 to 649) and fks1-Fs617-649 (fks1 reconstituted with F. solani residues 617 to 649) hybrid constructs. The construction of hybrid fks1-Fs617-649-F639 was as described above, except the reverse primer (ScFsFKS1-649R-F639) encoded F639 in place of the wild-type Y.

The construction of the fks1-453-649 hybrids used the previously described step 1 strain fks1453-649::URA3 (12). Step 2 was as described above, except the forward primers were designed with 3' ends to amplify C. glabrata FKS2 (ScCgFKS12-453F) or F. solani FKS1 (ScFsFKS1-453F) sequences downstream of their codon 453 equivalents and with 5' ends complementary to S. cerevisiae FKS1 sequences upstream of codon 453.

Susceptibility testing. Broth microdilution assays were used to determine MICs essentially as described previously (17). Briefly, fresh cultures derived from single colonies were diluted to 3 x 10³ cells per ml in YPD, and 100 µl was aliquoted to wells of a 96-well plate, except for row A wells, which received 200 µl. Drug (0.3 to 1 µl, representing an 200-fold dilution of the stock) was added to well A to the desired concentration and was serially twofold diluted into wells B through G; well H served as a drug-free control. Plates were incubated at 30°C (35°C for C. glabrata) for 24 h, and the absorbance at 630 nm was read with a microplate reader. The MIC was defined as the lowest concentration inhibiting growth 80% relative to that of the drug-free control. Assays were repeated at least twice, with comparable results.

Nucleotide sequence accession numbers. The S. prolificans sequences determined in the course of this work were deposited in GenBank under accession nos. EU337013 and ABY53595.

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Amplification and sequencing of S. prolificans FKS1. FKS1 sequences have been reported for F. solani (14) and related Fusarium species (29; www.broad.mit.edu) but not for any Scedosporium species. Phylogenetic analysis suggests a close relationship between these two genera (11). Therefore, PCR primers for amplifying S. prolificans FKS1 were designed based on Fusarium FKS1 sequences that are conserved among ascomycetous molds, as deduced from BLAST analyses, and with the consideration of S. prolificans codon usage. These primers were used with S. prolificans genomic DNA to amplify products that were confirmed by DNA sequencing to encode an Fks1 homolog. Additional S. prolificans-specific primers then were designed to complete the sequence corresponding to residues 311 to 1468 (S. cerevisiae Fks1 numbering is used here and throughout). BLAST analysis confirmed that S. prolificans Fks1 (GenBank accession no. EU337013) and F. solani Fks1 (accession no. ABC59463) are closely related, with 84% identity on the amino acid level; there is 71% identity between S. prolificans and S. cerevisiae Fks1 sequences. FKS1 is single copy in Fusarium species and other ascomycetous molds, and our PCR results suggest this is true for S. prolificans as well.

Sequence alignment to identify a potential basis for F. solani and S. prolificans EC resistance. Eight Fks1 residues in two separate regions have been implicated in acquired EC resistance through the characterization of mutant S. cerevisiae (33, 34), Candida albicans (1, 19, 26, 34), Candida glabrata (2, 19, 40), Candida krusei (6, 18, 34), and Candida tropicalis (6, 13 and S. Katiyar and T. Edlind, unpublished data). An alignment of these residues from wild-type and mutant Fks1 is shown in Fig. 1, along with corresponding Fks1 residues from F. solani and S. prolificans. It is apparent from this analysis that the Y639 equivalent residue within hot spot 1 could account for the intrinsic EC resistance of F. solani and S. prolificans, since an F639Y equivalent mutation in C. albicans Fks1 is associated with acquired resistance (19). Additional BLAST searches indicate that Y639 is not found in any fully EC-susceptible organism for which Fks1 sequences are available. However, as previously noted (19), a Y639 equivalent residue occurs in the single Fks1 of additional molds whose genomes have been sequenced (Fusarium graminearum, Fusarium verticillioides, and Neurospora crassa) and in one of the three Fks genes of Candida guilliermondii. Each of these demonstrates EC resistance or reduced susceptibility (4, 19, 36).

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FIG. 1. Fks1 and Fks2 hotspots 1 and 2, which are implicated in EC resistance, aligned with the equivalent regions of F. solani and S. prolificans Fks1. Dots represent identity to S. cerevisiae Fks1. Mutated residues associated with EC resistance are underlined, with the mutations indicated underneath (see the text for references). All mutations were spontaneously acquired, except for the A. fumigatus mutation, which was introduced by transformation (38). Arrowheads () indicate the Y639-equivalent residues of F. solani and S. prolificans Fks1, which are proposed here to be the basis for their intrinsic EC resistance. The C. tropicalis and S. prolificans Fks1 sequences are incomplete, and hence are not numbered.

Site-directed mutagenesis of S. cerevisiae Fks1 F639Y. Although an F639Y equivalent mutation was identified in a C. albicans isolate (19), no wild-type parent was available to unambiguously test its role in EC resistance. Therefore, we employed our recently described PCR-based site-directed mutagenesis method (12) to construct an S. cerevisiae chromosomal fks1-F639Y mutant and compared its EC susceptibility to that of its otherwise identical FKS1 parent. CSP and MCF MICs determined by broth microdilution increased 16- and 8-fold, respectively (Table 2). An F639F (i.e., wild-type Fks1) strain generated in parallel by the same method had wild-type EC susceptibilities, as expected.

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TABLE 2. EC susceptibilities of Fks1 mutants, hybrids, and parent strains

Strategy for generating hybrid Fks1. The genetic manipulation of F. solani to directly test the role of its Fks1 in EC resistance is not yet feasible. Heterologous expression in S. cerevisiae represents an attractive alternative. Plasmids often are used for this purpose; however, artifactual expression could result from a variable plasmid copy number or the use of nonnative promoters, which could result in artifactual EC susceptibility. Furthermore, the large size of the FKS1 gene and the presence of multiple introns in most species makes manipulation difficult. Since sequence analysis (Fig. 1) identified Fks1 hot spot 1 as a strong candidate for the basis of F. solani EC resistance, we elected to express this region as a hybrid within S. cerevisiae Fks1. To eliminate expression artifacts, a two-step method was envisioned for constructing these hybrids within the native chromosomal FKS1 gene. In step 1 (Fig. 2), a partial fks1 deletant in haploid S. cerevisiae strain BY4742 is constructed that replaces codons 617 to 649 with the URA3 marker. In contrast to parent strain BY4742, the fks1617-649::URA3 deletant should grow on SD-ura but not on medium containing FK506, a calcineurin inhibitor that blocks the expression of the backup gene FKS2 (31). In step 2 (Fig. 2), this deletant is transformed with a PCR product with terminal S. cerevisiae FKS1 sequences to direct homologous recombination and an internal F. solani FKS1 sequence that precisely corresponds to the codon 617 to 649 deletion. Plating on FK506-containing medium selects for functional, reconstituted Fks1, and screening on SD-ura medium confirms URA3 loss. PCR and DNA sequencing are used for the final confirmation of the desired fks1-Fs617-649 hybrid construct.

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FIG. 2. (A) Strategy for generating hybrid S. cerevisiae Fks1 incorporating the hot spot 1 region from F. solani Fks1. Step 1 is the construction in S. cerevisiae BY4742 of the internal deletion fks1617-649::URA3. Step 2 is the use of the step 1-generated deletant as the host for the construction of hybrid fks1-Fs617-649, which replaces S. cerevisiae Fks1 codons 617 to 649 with the corresponding region from F. solani. The drawing is not to scale. (B) Alignment of S. cerevisiae Fks1 residues 617 to 649 with the equivalent regions from A. fumigatus and F. solani.

A. fumigatus hybrid replacing Fks1 residues 617 to 649. As an initial test, we constructed and tested Fks1 hybrids replacing S. cerevisiae residues 617 to 649 with the equivalent region from A. fumigatus (Fig. 2B). Although MICs for ECs against A. fumigatus typically are high, morphological disruption (minimum effective concentration) due to apical growth inhibition occurs at much lower concentrations (16), as does the inhibition of A. fumigatus cell-free β-1,3-glucan synthase (23). EC susceptibilities for the fks1-Af617-649 hybrid were compared to those for the wild-type BY4742 parent and the fks1617-649::URA3 deletant that was used to generate the hybrid (Table 2). The wild-type CSP MIC decreased 16-fold for the deletant, which is consistent with previous studies of a different fks1 strain (8). This decrease, which was not observed with MCF, may reflect an intrinsically higher CSP susceptibility of Fks2 compared to that of Fks1 (31). The reconstitution of fks1 with A. fumigatus residues 617 to 649 yielded a functional Fks1, as indicated by the full reversal of FK506 susceptibility (not shown). With respect to EC susceptibility, the CSP hypersusceptibility of fks1 was partially reversed in the fks1-Af617-649 hybrid, but it remained clearly EC susceptible, with EC MICs that were modestly (two to fourfold) lower than those for BY4742. This is consistent with hot spot analysis (Fig. 1); i.e., mutations associated with EC resistance are not present in A. fumigatus Fks1.

F. solani hybrids replacing Fks1 residues 617 to 649. The strategy for generating fks1-Fs617-649 hybrids was as described above, except that two different reverse primers were used to incorporate either wild-type Y639 (predicted to confer EC resistance) or the mutation F639 (predicted to confer EC susceptibility). Indeed, with wild-type hybrid fks1-Fs617-649, the MICs for CSP and MCF were eight- and fourfold higher than those obtained for parent BY4742 (Table 2). As predicted, however, this increase was dependent on the presence of Y639, since replacement with F639 in hybrid fks1-Fs617-649-F639 conferred EC hypersusceptibility. Specifically, the CSP and MCF MICs for the Y639 hybrid were 32- and 8-fold greater, respectively, than those obtained for the F639 hybrid. Both hybrids were fully functional, as indicated by their FK506 resistance (not shown).

C. glabrata and F. solani hybrids replacing Fks1 residues 453 to 649. Typically, C. glabrata is highly EC susceptible. Clinical isolate CgR, however, demonstrates an eightfold reduced CSP susceptibility (Table 2). Fks2 encoded by this strain includes a hot spot 1 mutation equivalent to F639V (19), along with a previously undetected L588P mutation (data not shown), and a DNA fragment including these mutations transformed a CSP-susceptible strain to resistance (19). To further test our approach, we generated hybrids replacing Fks1 residues 453 to 649 with the equivalent region of C. glabrata Fks2 derived from CgR and, for comparison, the closely related EC-susceptible strain CgS (with F639 and L588 equivalent residues). The CSP MIC for the fks1-CgS453-649 hybrid (fks1 reconstituted with CgS residues 453 to 649) was comparable to that for BY4742 (Table 2). This is consistent with C. glabrata Fks2 hot spot analysis (Fig. 1) and the extensive conservation (82% identity) of residues 453 to 649 between S. cerevisiae Fks1 and C. glabrata Fks2. In contrast, the CSP MIC increased eightfold in the fks1-CgR453-649 hybrid (fks1 reconstituted with CgR residues 453 to 649) (Table 2). This increase is equivalent to the difference observed between CgS and CgR themselves, further validating the hybrid approach.

On the other hand, a comparable hybrid replacing Fks1 residues 453 to 649 with the equivalent region of F. solani Fks1 (fks1-Fs453-649) generated unexpected results, with EC MICs two- to fourfold lower than those for the S. cerevisiae BY4742 parent (Table 2). This contrasts with the results for the hybrid containing residues 617 to 649 presented above (four- to eightfold higher MICs) and may reflect reduced Fks1 function (suggested by partial FK506 susceptibility; data not shown) due to the limited conservation between residues 453 to 649 of S. cerevisiae and F. solani Fks1 (48% identity). Nevertheless, a role for Y639 in F. solani EC resistance again was supported by the eightfold increased susceptibility of the F639-containing fks1-Fs453-649-F639 hybrid (Table 2).

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Acquired EC resistance in normally susceptible fungal species fortunately is a rare occurrence, but nevertheless it has provided considerable information regarding the putative target of this important new antifungal group. The large majority of resistance-conferring mutations involve hot spot 1 of integral membrane protein Fks1 (S. cerevisiae residues 639 to 647), and the remainder involve hot spot 2 (residues 1354 to 1357). The mechanism by which these mutations confer resistance remains to be determined.

Nevertheless, these mutation data are useful in that they can lead to testable hypotheses regarding the basis for intrinsic EC resistance, which represents the more significant clinical problem. For example, P647 is a highly conserved Fks1 residue but is replaced with an A in C. parapsilosis. The hypothetical role of this substitution in the intrinsically low EC susceptibility of this yeast was tested successfully in S. cerevisiae (12). Previously, an Fks1 hot spot 1 mutation equivalent to F639Y was implicated in the acquired EC resistance of a C. albicans clinical isolate (19). The Fks1 of multiple Fusarium species and, as shown here, S. prolificans naturally include the equivalent of Y639, and we hypothesized that this contributes to their intrinsic EC resistance.

The ideal test of this hypothesis would involve generating a Y639F mutation in one of these molds, but technically this would be very challenging. Alternatively, the full-length Fks1 from a resistant fungus could be heterologously expressed in a susceptible fungus; however, variation in expression and, perhaps more importantly, function would lead to unreliable EC susceptibility data. Function is used here in the general sense to refer not only to β-1,3-glucan synthase activity but also Fks1 processing, transport, stability, etc.

To address these issues, we developed a PCR-based approach in which the hot spot 1-containing region of F. solani Fks1 was incorporated into S. cerevisiae Fks1 with either wild-type Y639 or F639. This hybrid approach ensures normal levels of FKS1 expression and facilitates the testing of specific Fks1 regions and residues hypothesized to play a role in EC susceptibility. The approach was validated by studies with EC-susceptible A. fumigatus and paired EC-susceptible and -resistant C. glabrata isolates. An important caveat is that the functionality of these Fks1 hybrids, assessed by FK506 susceptibility (which blocks the expression of the backup gene FKS2), was dependent on the length of the hybrid region and its source. Thus, hybrids replacing S. cerevisiae Fks1 residues 453 to 649 with corresponding regions from C. glabrata and F. solani varied from fully to partially functional, respectively, and the latter provided unreliable EC susceptibility data. On the other hand, hybrids replacing residues 617 to 649 with corresponding regions from F. solani or A. fumigatus appeared to be fully functional.

The four- to eightfold reduced EC susceptibility of this F. solani hybrid of residues 617 to 649 compared to that of wild-type S. cerevisiae supports our hypothesis that the Fks1 hot spot 1 region of this opportunistic mold contributes to its intrinsic resistance. More specifically and significantly, our data implicate residue Y639, since F639 replacement within both the 617 to 649 and 453 to 649 hybrids conferred 8- to 32-fold reduced EC susceptibility. Although further studies are required, it is likely that Y639 contributes to the intrinsic EC resistance of other Fusarium species and, based on the sequence presented here, of S. prolificans.

It should be emphasized that these results do not rule out a potential contribution of other regions of F. solani Fks1 to its EC resistance. Furthermore, they do not rule out potential contributions of non-Fks1 factors, such as the poor uptake or degradation of ECs. Indeed, one or more of these are likely to be at work, since the increase associated with Y639 is considerably less than the ca. 80-fold differential in EC susceptibility between F. solani and fungi such as C. albicans (14).

In addition to their role in deciphering the basis for intrinsic EC resistance, S. cerevisiae Fks1 mutants and hybrids such as those described here could prove useful in drug discovery. Specifically, they could be used to screen for EC derivatives that retain activity against the F639Y mutant and fks1-Fs617-649 hybrid. The likelihood of identifying such derivatives is reasonably high, based on our observation that residue 639 substitutions in all cases affected susceptibility to CSP more than to MCF. This suggests that this residue directly interacts with one of the side chains that differ between these two ECs. Consequently, specific modifications to this side chain could reduce the negative effects of Y639 on EC susceptibility.

 
We thank A. Fothergill, J. Nickels, M. Pfaller, P. D. Rogers, and the Fungal Genetics Stock Center for strains and the Broad Fungal Genome Initiative for Fusarium sequences.

 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129. Phone: (215) 991-8377. Fax: (215) 848-2271. E-mail: tedlind{at}drexelmed.edu

Published ahead of print on 2 March 2009.

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Antimicrobial Agents and Chemotherapy, May 2009, p. 1772-1778, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.00020-09
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