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Antimicrob Agents Chemother. Nov 1997; 41(11): 2471–2479.
PMCID: PMC164147

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors.

Abstract

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abruzzo GK, Flattery AM, Gill CJ, Kong L, Smith JG, Krupa D, Pikounis VB, Kropp H, Bartizal K. Evaluation of water-soluble pneumocandin analogs L-733560, L-705589, and L-731373 with mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrob Agents Chemother. 1995 May;39(5):1077–1081. [PMC free article] [PubMed]
  • Balkovec JM, Black RM, Hammond ML, Heck JV, Zambias RA, Abruzzo G, Bartizal K, Kropp H, Trainor C, Schwartz RE, et al. Synthesis, stability, and biological evaluation of water-soluble prodrugs of a new echinocandin lipopeptide. Discovery of a potential clinical agent for the treatment of systemic candidiasis and Pneumocystis carinii pneumonia (PCP). J Med Chem. 1992 Jan;35(1):194–198. [PubMed]
  • Bartizal K, Abruzzo G, Trainor C, Krupa D, Nollstadt K, Schmatz D, Schwartz R, Hammond M, Balkovec J, Vanmiddlesworth F. In vitro antifungal activities and in vivo efficacies of 1,3-beta-D-glucan synthesis inhibitors L-671,329, L-646,991, tetrahydroechinocandin B, and L-687,781, a papulacandin. Antimicrob Agents Chemother. 1992 Aug;36(8):1648–1657. [PMC free article] [PubMed]
  • Bouffard FA, Zambias RA, Dropinski JF, Balkovec JM, Hammond ML, Abruzzo GK, Bartizal KF, Marrinan JA, Kurtz MB, McFadden DC, et al. Synthesis and antifungal activity of novel cationic pneumocandin B(o) derivatives. J Med Chem. 1994 Jan 21;37(2):222–225. [PubMed]
  • Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ, Robbins PW. Classification of fungal chitin synthases. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):519–523. [PMC free article] [PubMed]
  • Bulawa CE, Miller DW, Henry LK, Becker JM. Attenuated virulence of chitin-deficient mutants of Candida albicans. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10570–10574. [PMC free article] [PubMed]
  • Castro C, Ribas JC, Valdivieso MH, Varona R, del Rey F, Duran A. Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae. J Bacteriol. 1995 Oct;177(20):5732–5739. [PMC free article] [PubMed]
  • Debono M, Gordee RS. Antibiotics that inhibit fungal cell wall development. Annu Rev Microbiol. 1994;48:471–497. [PubMed]
  • Douglas CM, Foor F, Marrinan JA, Morin N, Nielsen JB, Dahl AM, Mazur P, Baginsky W, Li W, el-Sherbeini M, et al. The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12907–12911. [PMC free article] [PubMed]
  • Douglas CM, Marrinan JA, Li W, Kurtz MB. A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase. J Bacteriol. 1994 Sep;176(18):5686–5696. [PMC free article] [PubMed]
  • Drgonová J, Drgon T, Tanaka K, Kollár R, Chen GC, Ford RA, Chan CS, Takai Y, Cabib E. Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science. 1996 Apr 12;272(5259):277–279. [PubMed]
  • Elble R. A simple and efficient procedure for transformation of yeasts. Biotechniques. 1992 Jul;13(1):18–20. [PubMed]
  • el-Sherbeini M, Clemas JA. Nikkomycin Z supersensitivity of an echinocandin-resistant mutant of Saccharomyces cerevisiae. Antimicrob Agents Chemother. 1995 Jan;39(1):200–207. [PMC free article] [PubMed]
  • Fonzi WA, Irwin MY. Isogenic strain construction and gene mapping in Candida albicans. Genetics. 1993 Jul;134(3):717–728. [PMC free article] [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Goldman RC, Frost DJ, Capobianco JO, Kadam S, Rasmussen RR, Abad-Zapatero C. Antifungal drug targets: Candida secreted aspartyl protease and fungal wall beta-glucan synthesis. Infect Agents Dis. 1995 Dec;4(4):228–247. [PubMed]
  • Gow NA, Robbins PW, Lester JW, Brown AJ, Fonzi WA, Chapman T, Kinsman OS. A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6216–6220. [PMC free article] [PubMed]
  • Hirano H, Tanaka K, Ozaki K, Imamura H, Kohno H, Hihara T, Kameyama T, Hotta K, Arisawa M, Watanabe T, et al. ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4396–4403. [PMC free article] [PubMed]
  • Holm C, Meeks-Wagner DW, Fangman WL, Botstein D. A rapid, efficient method for isolating DNA from yeast. Gene. 1986;42(2):169–173. [PubMed]
  • Inoue SB, Takewaki N, Takasuka T, Mio T, Adachi M, Fujii Y, Miyamoto C, Arisawa M, Furuichi Y, Watanabe T. Characterization and gene cloning of 1,3-beta-D-glucan synthase from Saccharomyces cerevisiae. Eur J Biochem. 1995 Aug 1;231(3):845–854. [PubMed]
  • Kamada Y, Qadota H, Python CP, Anraku Y, Ohya Y, Levin DE. Activation of yeast protein kinase C by Rho1 GTPase. J Biol Chem. 1996 Apr 19;271(16):9193–9196. [PubMed]
  • Kelly R, Register E, Hsu MJ, Kurtz M, Nielsen J. Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein. J Bacteriol. 1996 Aug;178(15):4381–4391. [PMC free article] [PubMed]
  • Kurtz MB, Abruzzo G, Flattery A, Bartizal K, Marrinan JA, Li W, Milligan J, Nollstadt K, Douglas CM. Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies. Infect Immun. 1996 Aug;64(8):3244–3251. [PMC free article] [PubMed]
  • Kurtz MB, Bernard EM, Edwards FF, Marrinan JA, Dropinski J, Douglas CM, Armstrong D. Aerosol and parenteral pneumocandins are effective in a rat model of pulmonary aspergillosis. Antimicrob Agents Chemother. 1995 Aug;39(8):1784–1789. [PMC free article] [PubMed]
  • Kurtz MB, Cortelyou MW, Kirsch DR. Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene. Mol Cell Biol. 1986 Jan;6(1):142–149. [PMC free article] [PubMed]
  • Kurtz MB, Douglas C, Marrinan J, Nollstadt K, Onishi J, Dreikorn S, Milligan J, Mandala S, Thompson J, Balkovec JM, et al. Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis. Antimicrob Agents Chemother. 1994 Dec;38(12):2750–2757. [PMC free article] [PubMed]
  • Kurtz MB, Douglas CM. Lipopeptide inhibitors of fungal glucan synthase. J Med Vet Mycol. 1997 Mar-Apr;35(2):79–86. [PubMed]
  • Kurtz MB, Heath IB, Marrinan J, Dreikorn S, Onishi J, Douglas C. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother. 1994 Jul;38(7):1480–1489. [PMC free article] [PubMed]
  • Mazur P, Baginsky W. In vitro activity of 1,3-beta-D-glucan synthase requires the GTP-binding protein Rho1. J Biol Chem. 1996 Jun 14;271(24):14604–14609. [PubMed]
  • Mazur P, Morin N, Baginsky W, el-Sherbeini M, Clemas JA, Nielsen JB, Foor F. Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell Biol. 1995 Oct;15(10):5671–5681. [PMC free article] [PubMed]
  • Mio T, Yabe T, Sudoh M, Satoh Y, Nakajima T, Arisawa M, Yamada-Okabe H. Role of three chitin synthase genes in the growth of Candida albicans. J Bacteriol. 1996 Apr;178(8):2416–2419. [PMC free article] [PubMed]
  • Mio T, Yamada-Okabe T, Yabe T, Nakajima T, Arisawa M, Yamada-Okabe H. Isolation of the Candida albicans homologs of Saccharomyces cerevisiae KRE6 and SKN1: expression and physiological function. J Bacteriol. 1997 Apr;179(7):2363–2372. [PMC free article] [PubMed]
  • Popolo L, Gilardelli D, Bonfante P, Vai M. Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae. J Bacteriol. 1997 Jan;179(2):463–469. [PMC free article] [PubMed]
  • Qadota H, Python CP, Inoue SB, Arisawa M, Anraku Y, Zheng Y, Watanabe T, Levin DE, Ohya Y. Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science. 1996 Apr 12;272(5259):279–281. [PubMed]
  • Sudoh M, Nagahashi S, Doi M, Ohta A, Takagi M, Arisawa M. Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition. Mol Gen Genet. 1993 Nov;241(3-4):351–358. [PubMed]
  • Sudoh M, Watanabe M, Mio T, Nagahashi S, Yamada-Okabe H, Takagi M, Arisawa M. Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase. Microbiology. 1995 Oct;141(Pt 10):2673–2679. [PubMed]
  • Swoboda RK, Bertram G, Budge S, Gooday GW, Gow NA, Brown AJ. Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans. Infect Immun. 1995 Nov;63(11):4506–4514. [PMC free article] [PubMed]

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