1887

Abstract

is an important opportunistic fungal pathogen capable of causing both mucosal and disseminated disease. Infections are often treated with fluconazole, a front-line antifungal drug that targets the biosynthesis of ergosterol, a major component of the fungal cell membrane. Resistance to fluconazole can arise through a variety of mechanisms, including gain-of-function mutations, loss of heterozygosity events and aneuploidy. The clinical isolate P60002 was found to be highly resistant to azole-class drugs, yet lacked mutations or chromosomal rearrangements known to be associated with azole resistance. Transcription profiling suggested that increased expression of two putative drug efflux pumps, and , might confer azole resistance. However, ectopic expression of the P60002 alleles of these genes in a drug-susceptible strain did not increase fluconazole resistance. We next examined whether the presence of three copies of chromosome 4 (Chr4) or chromosome 6 (Chr6) contributed to azole resistance in P60002. We established that Chr4 trisomy contributes significantly to fluconazole resistance, whereas Chr6 trisomy has no discernible effect on resistance. In contrast, a Chr4 trisomy did not increase fluconazole resistance when present in the standard SC5314 strain background. These results establish a link between Chr4 trisomy and elevated fluconazole resistance, and demonstrate the impact of genetic background on drug resistance phenotypes in .

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2017-06-01
2020-08-07
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References

  1. Beck-Sague C, Banerjee S, Jarvis WR. Infectious diseases and mortality among US nursing home residents. Am J Public Health 1993;83:1739–1742 [CrossRef][PubMed]
    [Google Scholar]
  2. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007;20:133–163 [CrossRef][PubMed]
    [Google Scholar]
  3. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG et al. Hidden killers: human fungal infections. Sci Transl Med 2012;4:165rv13 [CrossRef][PubMed]
    [Google Scholar]
  4. Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends Microbiol 2001;9:327–335 [CrossRef][PubMed]
    [Google Scholar]
  5. Sanglard D, Odds FC. Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2002;2:73–85 [CrossRef][PubMed]
    [Google Scholar]
  6. Liu W, Tan J, Sun J, Xu Z, Li M et al. Invasive candidiasis in intensive care units in China: in vitro antifungal susceptibility in the China-SCAN study. J Antimicrob Chemother 2014;69:162–167 [CrossRef][PubMed]
    [Google Scholar]
  7. Manzoni P, Farina D, Leonessa M, Priolo C, Gomirato G. Use of prophylactic fluconazole in a neonatal intensive care unit: efficacy is similar to that described in adult high-risk surgical patients. Crit Care 2006;10:402 [CrossRef][PubMed]
    [Google Scholar]
  8. Geber A, Hitchcock CA, Swartz JE, Pullen FS, Marsden KE et al. Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrob Agents Chemother 1995;39:2708–2717 [CrossRef][PubMed]
    [Google Scholar]
  9. Abe F, Usui K, Hiraki T. Fluconazole modulates membrane rigidity, heterogeneity, and water penetration into the plasma membrane in Saccharomyces cerevisiae. Biochemistry 2009;48:8494–8504 [CrossRef][PubMed]
    [Google Scholar]
  10. Mishra NN, Prasad T, Sharma N, Gupta DK. Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients. Braz J Microbiol 2008;39:219–225 [CrossRef][PubMed]
    [Google Scholar]
  11. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004;39:309–317 [CrossRef][PubMed]
    [Google Scholar]
  12. Marchaim D, Lemanek L, Bheemreddy S, Kaye KS, Sobel JD. Fluconazole-resistant Candida albicans vulvovaginitis. Obstet Gynecol 2012;120:1407–1414 [CrossRef][PubMed]
    [Google Scholar]
  13. Vanden Bossche H, Marichal P, Willemsens G, Bellens D, Gorrens J et al. Saperconazole: a selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Mycoses 1990;33:335–352[PubMed]
    [Google Scholar]
  14. Kelly SL, Lamb DC, Loeffler J, Einsele H, Kelly DE. The G464S amino acid substitution in Candida albicans sterol 14α-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem Biophys Res Commun 1999;262:174–179 [CrossRef][PubMed]
    [Google Scholar]
  15. Selmecki A, Forche A, Berman J. Aneuploidy and isochromosome formation in drug-resistant Candida albicans. Science 2006;313:367–370 [CrossRef][PubMed]
    [Google Scholar]
  16. Ribeiro MA, Paula CR. Up-regulation of ERG11 gene among fluconazole-resistant Candida albicans generated in vitro: is there any clinical implication?. Diagn Microbiol Infect Dis 2007;57:71–75 [CrossRef][PubMed]
    [Google Scholar]
  17. Dunkel N, Liu TT, Barker KS, Homayouni R, Morschhäuser J et al. A gain-of-function mutation in the transcription factor Upc2p causes upregulation of ergosterol biosynthesis genes and increased fluconazole resistance in a clinical Candida albicans isolate. Eukaryot Cell 2008;7:1180–1190 [CrossRef][PubMed]
    [Google Scholar]
  18. MacPherson S, Akache B, Weber S, de Deken X, Raymond M et al. Candida albicans zinc cluster protein Upc2p confers resistance to antifungal drugs and is an activator of ergosterol biosynthetic genes. Antimicrob Agents Chemother 2005;49:1745–1752 [CrossRef][PubMed]
    [Google Scholar]
  19. Kelly SL, Lamb DC, Corran AJ, Baldwin BC, Kelly DE. Mode of action and resistance to azole antifungals associated with the formation of 14α-methylergosta-8,24(28)-dien-3β,6α-diol. Biochem Biophys Res Commun 1995;207:910–915 [CrossRef][PubMed]
    [Google Scholar]
  20. Dunkel N, Blass J, Rogers PD, Morschhäuser J. Mutations in the multi-drug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains. Mol Microbiol 2008;69:827–840 [CrossRef][PubMed]
    [Google Scholar]
  21. Coste A, Turner V, Ischer F, Morschhäuser J, Forche A et al. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 2006;172:2139–2156 [CrossRef][PubMed]
    [Google Scholar]
  22. Coste AT, Karababa M, Ischer F, Bille J, Sanglard D. TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 2004;3:1639–1652 [CrossRef][PubMed]
    [Google Scholar]
  23. Wang Y, Liu JY, Shi C, Li WJ, Zhao Y et al. Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans. Int J Antimicrob Agents 2015;46:552–559 [CrossRef][PubMed]
    [Google Scholar]
  24. Heilmann CJ, Schneider S, Barker KS, Rogers PD, Morschhäuser J. An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans. Antimicrob Agents Chemother 2010;54:353–359 [CrossRef][PubMed]
    [Google Scholar]
  25. Sasse C, Dunkel N, Schäfer T, Schneider S, Dierolf F et al. The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans. Mol Microbiol 2012;86:539–556 [CrossRef][PubMed]
    [Google Scholar]
  26. Jensen RH, Astvad KM, Silva LV, Sanglard D, Jørgensen R et al. Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations. J Antimicrob Chemother 2015;70:2551–2555 [CrossRef][PubMed]
    [Google Scholar]
  27. White TC. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 1997;41:1482–1487[PubMed]
    [Google Scholar]
  28. Selmecki A, Gerami-Nejad M, Paulson C, Forche A, Berman J. An isochromosome confers drug resistance in vivo by amplification of two genes, ERG11 and TAC1. Mol Microbiol 2008;68:624–641 [CrossRef][PubMed]
    [Google Scholar]
  29. Kwon-Chung KJ, Chang YC. Aneuploidy and drug resistance in pathogenic fungi. PLoS Pathog 2012;8:e1003022 [CrossRef][PubMed]
    [Google Scholar]
  30. Pfau SJ, Amon A. Chromosomal instability and aneuploidy in cancer: from yeast to man. EMBO Rep 2012;13:515–527 [CrossRef][PubMed]
    [Google Scholar]
  31. Chen G, Rubinstein B, Li R. Whole chromosome aneuploidy: big mutations drive adaptation by phenotypic leap. Bioessays 2012;34:893–900 [CrossRef][PubMed]
    [Google Scholar]
  32. Selmecki A, Forche A, Berman J. Genomic plasticity of the human fungal pathogen Candida albicans. Eukaryot Cell 2010;9:991–1008 [CrossRef][PubMed]
    [Google Scholar]
  33. Yona AH, Manor YS, Herbst RH, Romano GH, Mitchell A et al. Chromosomal duplication is a transient evolutionary solution to stress. Proc Natl Acad Sci USA 2012;109:21010–21015 [CrossRef][PubMed]
    [Google Scholar]
  34. Gallè F, Sanguinetti M, Colella G, Di Onofrio V, Torelli R et al. Oral candidosis: characterization of a sample of recurrent infections and study of resistance determinants. New Microbiol 2011;34:379–389[PubMed]
    [Google Scholar]
  35. Cohen NR, Lobritz MA, Collins JJ. Microbial persistence and the road to drug resistance. Cell Host Microbe 2013;13:632–642 [CrossRef][PubMed]
    [Google Scholar]
  36. Taff HT, Mitchell KF, Edward JA, Andes DR. Mechanisms of Candida biofilm drug resistance. Future Microbiol 2013;8:1325–1337 [CrossRef][PubMed]
    [Google Scholar]
  37. Luna-Tapia A, Tournu H, Peters TL, Palmer GE. Endosomal trafficking defects can induce calcium-dependent azole tolerance in Candida albicans. Antimicrob Agents Chemother 2016;60:7170–7177 [CrossRef][PubMed]
    [Google Scholar]
  38. Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J. Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 2003;48:959–976 [CrossRef][PubMed]
    [Google Scholar]
  39. Hill JA, Ammar R, Torti D, Nislow C, Cowen LE. Genetic and genomic architecture of the evolution of resistance to antifungal drug combinations. PLoS Genet 2013;9:e1003390 [CrossRef][PubMed]
    [Google Scholar]
  40. Lamb DC, Corran A, Baldwin BC, Kwon-Chung J, Kelly SL. Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients. FEBS Lett 1995;368:326–330 [CrossRef][PubMed]
    [Google Scholar]
  41. Rodloff C, Koch D, Schaumann R. Epidemiology and antifungal resistance in invasive candidiasis. Eur J Med Res 2011;16:187–195 [CrossRef][PubMed]
    [Google Scholar]
  42. Neves-Junior A, Cartágenes-Pinto AC, Rocha DA, de Sá LF, Junqueira ML et al. Prevalence and fluconazole susceptibility profile of Candida spp. clinical isolates in a Brazilian tertiary hospital in Minas Gerais, Brazil. An Acad Bras Cienc 2015;87:1349–1359 [CrossRef][PubMed]
    [Google Scholar]
  43. Pfaller MA, Jones RN, Messer SA, Edmond MB, Wenzel RP. National surveillance of nosocomial blood stream infection due to Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program. Diagn Microbiol Infect Dis 1998;31:327–332 [CrossRef][PubMed]
    [Google Scholar]
  44. Perea S, López-Ribot JL, Kirkpatrick WR, McAtee RK, Santillán RA et al. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 2001;45:2676–2684 [CrossRef][PubMed]
    [Google Scholar]
  45. Ford CB, Funt JM, Abbey D, Issi L, Guiducci C et al. The evolution of drug resistance in clinical isolates of Candida albicans. Elife 2015;4:e00662 [CrossRef][PubMed]
    [Google Scholar]
  46. Li X, Yang F, Li D, Zhou M, Wang X et al. Trisomy of chromosome R confers resistance to triazoles in Candida albicans. Med Mycol 2015;53:302–309 [CrossRef][PubMed]
    [Google Scholar]
  47. Perepnikhatka V, Fischer FJ, Niimi M, Baker RA, Cannon RD et al. Specific chromosome alterations in fluconazole-resistant mutants of Candida albicans. J Bacteriol 1999;181:4041–4049[PubMed]
    [Google Scholar]
  48. Harrison BD, Hashemi J, Bibi M, Pulver R, Bavli D et al. A tetraploid intermediate precedes aneuploid formation in yeasts exposed to fluconazole. PLoS Biol 2014;12:e1001815 [CrossRef][PubMed]
    [Google Scholar]
  49. Rustchenko-Bulgac EP. Variations of Candida albicans electrophoretic karyotypes. J Bacteriol 1991;173:6586–6596 [CrossRef][PubMed]
    [Google Scholar]
  50. Iwaguchi S, Homma M, Tanaka K. Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans. J Gen Microbiol 1990;136:2433–2442 [CrossRef][PubMed]
    [Google Scholar]
  51. Pfaller MA, Rhine-Chalberg J, Redding SW, Smith J, Farinacci G et al. Variations in fluconazole susceptibility and electrophoretic karyotype among oral isolates of Candida albicans from patients with AIDS and oral candidiasis. J Clin Microbiol 1994;32:59–64[PubMed]
    [Google Scholar]
  52. Hickman MA, Zeng G, Forche A, Hirakawa MP, Abbey D et al. The 'obligate diploid' Candida albicans forms mating-competent haploids. Nature 2013;494:55–59 [CrossRef][PubMed]
    [Google Scholar]
  53. Selmecki A, Bergmann S, Berman J. Comparative genome hybridization reveals widespread aneuploidy in Candida albicans laboratory strains. Mol Microbiol 2005;55:1553–1565 [CrossRef][PubMed]
    [Google Scholar]
  54. Hirakawa MP, Martinez DA, Sakthikumar S, Anderson MZ, Berlin A et al. Genetic and phenotypic intra-species variation in Candida albicans. Genome Res 2015;25:413–425 [CrossRef][PubMed]
    [Google Scholar]
  55. Burrack LS, Applen SE, Berman J. The requirement for the Dam1 complex is dependent upon the number of kinetochore proteins and microtubules. Curr Biol 2011;21:889–896 [CrossRef][PubMed]
    [Google Scholar]
  56. Reuss O, Vik A, Kolter R, Morschhäuser J. The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 2004;341:119–127 [CrossRef][PubMed]
    [Google Scholar]
  57. Gerstein AC, Rosenberg A, Hecht I, Berman J. diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays. Microbiology 2016;162: [CrossRef][PubMed]
    [Google Scholar]
  58. Clinical and Laboratory Standards Institute Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. CLSI Document M27-A Wayne, PA: 1997
    [Google Scholar]
  59. Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS One 2012;7:e37135 [CrossRef][PubMed]
    [Google Scholar]
  60. Andrews S. 2010; FastQC: A Quality Control Tool for High Throughput Sequence Data. www.bioinformatics.babraham.ac.uk/projects/fastqc
  61. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359 [CrossRef][PubMed]
    [Google Scholar]
  62. Tan Z, Hays M, Cromie GA, Jeffery EW, Scott AC et al. Aneuploidy underlies a multicellular phenotypic switch. Proc Natl Acad Sci USA 2013;110:12367–12372 [CrossRef][PubMed]
    [Google Scholar]
  63. Sewell DL, Pfaller MA, Barry AL. Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole. J Clin Microbiol 1994;32:2099–2102[PubMed]
    [Google Scholar]
  64. Koga-Ito CY, Lyon JP, Resende MA. Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates. Rev Inst Med Trop Sao Paulo 2008;50:7–10 [CrossRef][PubMed]
    [Google Scholar]
  65. Zomorodian K, Bandegani A, Mirhendi H, Pakshir K, Alinejhad N et al. In vitro susceptibility and trailing growth effect of clinical isolates of Candida species to azole drugs. Jundishapur J Microbiol 2016;9:e28666 [CrossRef][PubMed]
    [Google Scholar]
  66. Magee BB, Magee PT. WO-2, a stable aneuploid derivative of Candida albicans strain WO-1, can switch from white to opaque and form hyphae. Microbiology 1997;143:289–295 [CrossRef][PubMed]
    [Google Scholar]
  67. Selmecki AM, Dulmage K, Cowen LE, Anderson JB, Berman J. Acquisition of aneuploidy provides increased fitness during the evolution of antifungal drug resistance. PLoS Genet 2009;5:e1000705 [CrossRef][PubMed]
    [Google Scholar]
  68. Forche A, Alby K, Schaefer D, Johnson AD, Berman J et al. The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains. PLoS Biol 2008;6:e110 [CrossRef][PubMed]
    [Google Scholar]
  69. Clark FS, Parkinson T, Hitchcock CA, Gow NA. Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance. Antimicrob Agents Chemother 1996;40:419–425[PubMed]
    [Google Scholar]
  70. Neyfakh AA, Bidnenko VE, Chen LB. Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci USA 1991;88:4781–4785 [CrossRef][PubMed]
    [Google Scholar]
  71. Gerstein AC, Berman J. Shift and adapt: the costs and benefits of karyotype variations. Curr Opin Microbiol 2015;26:130–136 [CrossRef][PubMed]
    [Google Scholar]
  72. Bennett RJ, Forche A, Berman J. Rapid mechanisms for generating genome diversity: whole ploidy shifts, aneuploidy, and loss of heterozygosity. Cold Spring Harb Perspect Med 2014;4:a019604 [CrossRef][PubMed]
    [Google Scholar]
  73. Sheltzer JM, Amon A. The aneuploidy paradox: costs and benefits of an incorrect karyotype. Trends Genet 2011;27:446–453 [CrossRef][PubMed]
    [Google Scholar]
  74. Siegel JJ, Amon A. New insights into the troubles of aneuploidy. Annu Rev Cell Dev Biol 2012;28:189–214 [CrossRef][PubMed]
    [Google Scholar]
  75. Pavelka N, Rancati G, Zhu J, Bradford WD, Saraf A et al. Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast. Nature 2010;468:321–325 [CrossRef][PubMed]
    [Google Scholar]
  76. Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA et al. Identification of aneuploidy-tolerating mutations. Cell 2010;143:71–83 [CrossRef][PubMed]
    [Google Scholar]
  77. Shah AH, Singh A, Dhamgaye S, Chauhan N, Vandeputte P et al. Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans. Biochem J 2014;460:223–235 [CrossRef][PubMed]
    [Google Scholar]
  78. Dowell RD, Ryan O, Jansen A, Cheung D, Agarwala S et al. Genotype to phenotype: a complex problem. Science 2010;328:469 [CrossRef][PubMed]
    [Google Scholar]
  79. Vu V, Verster AJ, Schertzberg M, Chuluunbaatar T, Spensley M et al. Natural variation in gene expression modulates the severity of mutant phenotypes. Cell 2015;162:391–402 [CrossRef][PubMed]
    [Google Scholar]
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