title: Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance.
abstract: OBJECTIVE
  Candida parapsilosis is a healthcare-related fungal pathogen particularly common among immunocompromised patients. Our understanding of antifungal resistance mechanisms in C. parapsilosis remains very limited. We previously described an azole-resistant strain of C. parapsilosis (BC014RPSC), obtained following exposure in vitro to posaconazole. Resistance was associated with overexpression of ergosterol biosynthetic genes (ERG genes), together with the transcription factors UPC2 (CPAR2-207280) and NDT80 (CPAR2-213640). The aim of this study was to identify the mechanisms underlying posaconazole resistance of the BC014RPSCstrain.
  
  
  METHODS
  To identify the causative mutation, we sequenced the genomes of the susceptible (BC014S) and resistant (BC014RPSC) isolates, using Illumina technology. Ergosterol content was assessed in both strains by mass spectrometry. UPC2 and NDT80 genes were deleted in BC014RPSCstrain. Mutants were characterized regarding their azole susceptibility profile and ERG gene expression.
  
  
  RESULTS
  One homozygous missense mutation (R135I) was found in ERG3 (CPAR2-105550) in the azole-resistant isolate. We show that Erg3 activity is completely impaired, resulting in a build up of sterol intermediates and a failure to generate ergosterol. Deleting UPC2 and NDT80 in BC014RPSCreduces the expression of ERG genes and restores susceptibility to azole drugs.
  
  
  CONCLUSIONS
  A missense mutation in the ERG3 gene results in azole resistance and up-regulation of ERG genes expression. We propose that this mutation prevents the formation of toxic intermediates when cells are treated with azoles. Resistance can be reversed by deleting Upc2 and Ndt80 transcription factors. UPC2 plays a stronger role in C. parapsilosis azole resistance than does NDT80.
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: J
authors.initials: J
authors.lastname: Branco
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland.
authors.forename: M
authors.initials: M
authors.lastname: Ola
authors.affiliation: Department of Medical Sciences, iBiMED & IEETA, University of Aveiro, Aveiro, Portugal.
authors.forename: R M
authors.initials: RM
authors.lastname: Silva
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: E
authors.initials: E
authors.lastname: Fonseca
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: N C
authors.initials: NC
authors.lastname: Gomes
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Martins-Cruz
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; CINTESIS-Centre for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: A P
authors.initials: AP
authors.lastname: Silva
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; CINTESIS-Centre for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Silva-Dias
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; CINTESIS-Centre for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Pina-Vaz
authors.affiliation: Institute of Food and Health, School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: C
authors.initials: C
authors.lastname: Erraught
authors.affiliation: Institute of Food and Health, School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: L
authors.initials: L
authors.lastname: Brennan
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; CINTESIS-Centre for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: A G
authors.initials: AG
authors.lastname: Rodrigues
authors.affiliation: UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland.
authors.forename: G
authors.initials: G
authors.lastname: Butler
authors.affiliation: Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal; CINTESIS-Centre for Health Technology and Services Research, Faculty of Medicine, University of Porto, Porto, Portugal. Electronic address: imiranda@med.up.pt.
authors.forename: I M
authors.initials: IM
authors.lastname: Miranda
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D000074429:Candida parapsilosis / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D025141:Drug Resistance, Fungal; D004875:Ergosterol / Q000032:analysis / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D020869:Gene Expression Profiling; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D020125:Mutation, Missense; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000072664:Provitamins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000073336:Whole Genome Sequencing
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D000072664:Provitamins; D014157:Transcription Factors; D010088:Oxidoreductases; D004875:Ergosterol
keywords: Antifungal drug resistance; Ergosterol biosynthetic pathway; Middle sporulation element; Opportunistic pathogen; Sterol-response element; Transcription factors