FKS1 Mutations Responsible for Selective Resistance of Saccharomyces cerevisiae to the Novel 1,3-{beta}-Glucan Synthase Inhibitor Arborcandin C

Arborcandin C is a novel antibiotic with potent antifungal activitythat inhibits 1,3-ß-glucan synthase in fungi. We examinedspontaneous Saccharomyces cerevisiae mutants which are selectivelyresistant to arborcandin C and revealed that a single aminoacid replacement in Fks1p of Asn⁴⁷⁰ with Lys or of Leu⁶⁴² withSer confers selective resistance on Fks1p mutants.

INTRODUCTION

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Introduction

The 1,3-ß-glucan polymer is a major component of thecell wall in yeast and filamentous fungi (11) and is synthesizedby 1,3-ß-glucan synthase (GS). In Saccharomyces cerevisiae,GS consists of at least two subunits: a putative catalytic subunitencoded by related genes FKS1 and FKS2 (7, 10) and a regulatorysubunit, a GTP-bound protein encoded by RHO1 (16). Several typesof GS inhibitors, such as echinocandins (5, 13), papulacandins(20), and FR901469 (8), have been identified. GS inhibitorsare a new class of antifungal antibiotics with clinical significancebecause of their strong fungicidal activity and low toxicity(1, 3).

Arborcandins are recently isolated cyclic lipopeptides whichinhibit GS from Candida albicans and Aspergillus fumigatus (14)and which also have growth-inhibitory activity (14). Althoughwe reported that arborcandin C noncompetitively inhibited GS(14), the molecular target of the inhibition still remains tobe elucidated. In this study, we isolated selective arborcandinC-resistant S. cerevisiae mutants with antibiotic-resistant-GSactivity and revealed that two single-amino-acid mutations inFks1p conferred selective resistance to arborcandin C.

Standard procedures were used for DNA manipulations (17). Geneticmanipulations for yeast were carried out as described previously(9). Yeast strains and plasmids used in this study are listedin Table 1. All yeast strains were derivatives of S. cerevisiaeYPH250 (18). Yeast cells were grown either in YPDAU medium (2%peptone, 1% yeast extract, 20 µg of adenine sulfate/ml,20 µg of uracil/ml, 2% glucose) or SD medium (9) supplementedappropriately. FKS1 was cloned by screening the genomic DNAlibrary of the wild-type strain (YPH250) for complementing fks1 ${Delta}$ ::LEU2on SD medium containing 1 µg of FK506/ml. An arborcandinC resistance gene was cloned by screening the genomic DNA librariesof the arborcandin C-resistant mutants ACR79-5 and ACR1A3 forgenes that conferred growth in SD medium containing 5 µgof arborcandin C/ml on YPH250. The DNA sequence was determinedby automated DNA sequencer model ABI3700 with a dye terminatorcycle sequencing kit (Applied Biosystems).

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TABLE 1. Yeast strains and plasmids used in this study

Growth inhibition was determined by the broth microdilutionmethod in a 96-well microplate using YPDAU medium. Briefly,10⁴ yeast cells were inoculated into 150 µl of mediumin each well and incubated at 30°C for 16 to 24 h in thepresence or absence of GS inhibitors. The optical density at595 nm of the exponentially growing culture was measured bymicroplate reader (ARVOsx; Perkin-Elmer).

Preparation and measurement of activity of GS from wild-typeand mutant S. cerevisiae strains were performed according tothe procedure described by Inoue et al. (10) and Cabib and Kang(4), respectively. The solubilized membrane fraction (10) wasused as GS.

Isolation of selective arborcandin C-resistant mutants.

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Isolation of selective...

Arborcandin C is structurally different from echinocandins andpapulacandins (14). To examine whether arborcandin C has a moleculartarget different from those of known GS inhibitors, we tooka genetic approach. Spontaneously resistant S. cerevisiae mutantswere isolated at a concentration of 1 µg of arborcandinC/ml on a YPDAU medium plate. We selected two mutants, ACR79-5and ACR1A3, which are resistant only to arborcandin C, not toany other type of GS inhibitors, for further study. As the echinocandin-typeand papulacandin-type inhibitors, we used pneumocandin A₀ (2)and F-10748 C₁ (15), respectively. As shown in Table 2, bothmutants were 100-fold more resistant to arborcandin C than theparental wild-type strain and there was a modest change in thesusceptibility to pneumocandin A₀ and F-10748 C₁. We did notobserve significant differences between the wild-type strainand the mutants in their phenotypes, such as their cell morphologyand calcofluor white sensitivity phenotypes, which reflect structuralchanges in the cell wall (data not shown).

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TABLE 2. Susceptibilities of selective arborcandin C-resistant mutants to GS inhibitors

To determine whether the selective resistance of the mutantsto arborcandin C was due to a change of sensitivity of GS, thesensitivity of GS from the mutants was tested. As shown in Table3, GS from either of the resistant mutants was highly resistantto arborcandin C. The 50% inhibitory concentration of arborcandinC against the mutant's GS was more than 100-fold higher thanthat of arborcandin C against the wild-type enzyme. On the otherhand, the sensitivity of both mutant enzymes to the other typesof GS inhibitors tested was not significantly changed. Theseresults suggested that the selective resistance of the mutantswas due to the selective resistance of GS.

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TABLE 3. Sensitivity of GS from selective arborcandin C-resistant mutants to GS inhibitors

Cloning of mutated genes responsible for selective arborcandin C resistance.

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Cloning of mutated genes...

To identify the mutated genes in ACR79-5 and ACR1A3, we screenedthe genomic DNA library of each mutant using the centromere-typevector (pCE112) for plasmids conferring arborcandin C resistanceon the wild-type strain. Four and three arborcandin C-resistantcolonies were isolated by the transformation with the librariesof ACR79-5 and ACR1A3, respectively. All plasmid DNAs recoveredfrom the resistant transformants conferred arborcandin C resistance(data not shown). Partial sequence analysis of the plasmidsrevealed that the minimum region conferring arborcandin C resistancefrom both the mutants contained an FKS1 locus as a completeopen reading frame (data not shown). Therefore, it was stronglysuggested that the gene mutated in both the resistant mutantswas FKS1.

Mutation of Asn⁴⁷⁰ to Lys or Leu⁶⁴² to Ser in Fks1p confers selective resistance to arborcandin C.

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Mutation of asn470 to...

By DNA sequence analysis of the FKS1 open reading frame, themutations in ACR79-5 and ACR1A3 were determined to be the replacementof Asn⁴⁷⁰ with Lys and of Leu⁶⁴² with Ser in Fks1p, respectively(Fig. 1). We subcloned each of the mutated regions to wild-typeFKS1 and examined the effect of each mutation in the fks1 ${Delta}$ P_GAL10-FKS2cells. In the fks1 ${Delta}$ P_GAL10-FKS2 strain, FKS2 gene expressionwas controlled by the GAL10 promoter and suppressed in the presenceof glucose. Therefore, the effect of the substitution in Fks1pcan be analyzed under the depletion of endogenous Fks1p andFks2p in the fks1 ${Delta}$ P_GAL10-FKS2 cells in glucose medium. The introductionof mutated FKS1 (FKS1^N470K or FKS1^L642S) to the fks1 ${Delta}$ P_GAL10-FKS2strain conferred selective resistance to arborcandin C, as shownby arborcandin C-resistant mutants ACR79-5 and ACR1A3 (Table2). Moreover, the GSs from cells with FKS1^N470K or FKS1^L642Sconferred selective resistance to arborcandin C (Table 3). Theseresults show that Asn⁴⁷⁰ and Leu⁶⁴² of Fks1p are critical aminoacids in the GS-inhibitory activity of arborcandin C and stronglysuggest that the target molecule of arborcandin C is Fks1p,the putative catalytic subunit of GS.

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FIG. 1. Nucleotide sequence analysis of FKS1 open reading frames of ACR79-5 and ACR1A3. (A) Partial nucleotide sequences of FKS1 from wild-type and arborcandin C-resistant mutants. Arrows, points of nucleotide mutations found in the resistant mutants. (B) Predicted amino acid sequences of Fks1p from the resistant mutants. The substituted residues in the resistant mutants are underlined.

The results we obtained also suggested that Asn⁴⁷⁰ and Leu⁶⁴²in Fks1p are topologically proximate. The model of membranetopology of Fks1p was predicted by hydropathy plot analysis(6). In the model, Asn⁴⁷⁰ and Leu⁶⁴² exist in the transmembranedomain and cytoplasmic loop domain, respectively, and Asn⁴⁷⁰is topologically distant from Leu⁶⁴² (Fig. 2A). On the otherhand, in the model predicted by the PHDhtm server (http://cubic.bioc.columbia.edu/predictprotein),Asn⁴⁷⁰ and Leu⁶⁴² exist at the first and third extracellularregions, respectively, and it is possible that these amino acidresidues are close to each other (Fig. 2B). Our results supportthe model of Fks1p by the PHDhtm server and contribute to theunderstanding of the exact structure of Fks1p.

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FIG. 2. Model of the predicted membrane topology of the N-terminal region of Fks1p. Solid line, polypeptide chain; boxes, putative transmembrane helices. (A) Model predicted by hydropathy plot analysis. (B) Model predicted by the PHDhtm server.

Amino acid residues corresponding to Asn⁴⁷⁰ and Leu⁶⁴² and theprimary structure of the regions around those amino acid residuesare well conserved among arborcandin-sensitive fungi (Fig. 3).However, these amino acid residues are also conserved in Cryptococcusneoformans, which is resistant to arborcandins (14). AlthoughC. neoformans has GS and although the unique FKS1 gene of thisorganism is essential for its growth (19), C. neoformans isresistant to all the known GS inhibitors (8, 12, 20). A commonmechanism of resistance to these GS inhibitors, including arborcandins,may exist in C. neoformans.

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FIG. 3. Amino acid sequence alignment of Fks proteins from various fungi. The amino acid sequences of S. cerevisiae Fks1p, residues 459 to 483 and 632 to 656, are aligned to the relevant sequences of Fks proteins from C. albicans, A. fumigatus, and C. neoformans (GenBank accession numbers D88815, U79728, and AF102882, respectively). Gray boxes and filled boxes, conserved residues among Fks proteins and mutated residues in arborcandin C-resistant mutants, respectively.

Isolation of the selective arborcandin C-resistant mutants andthe identification of amino acids responsible for the resistanceprovide new insights into the interaction between Fks1p andGS inhibitors. Based on the results of this study, future studieswill monitor the direct interaction between arborcandin C andthe N-terminal putative extracellular region. Structural analysisof the N-terminal region of Fks1p would also help to improvethe antifungal activity of arborcandins.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

Whole nucleotide sequences of FKS1 open reading frames of thewild type, ACR79-5, and ACR1A3 are available in the GenBankdatabase under the accession numbers AY395693, AY395694, andAY395695, respectively.

FOOTNOTES

* Corresponding author. Mailing address: Lead Discovery Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. Phone: 81-3-3492-3131. Fax: 81-3-5436-8578. E-mail: takaoo{at}shina.sankyo.co.jp.

REFERENCES

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