Impact of ERG3 mutations and expression of ergosterol genes controlled by UPC2 and NDT80 in Candida parapsilosis azole resistance  J. Branco, M. Ola, R.M. Silva, E. Fonseca, N.C. Gomes, C. Martins-Cruz, A.P. Silva, A. Silva-Dias, C. Pina-Vaz, C. Erraught, L. Brennan, A.G. Rodrigues, G. Butler, I.M. Miranda  Clinical Microbiology and Infection  Volume 23, Issue 8, Pages 575.e1-575.e8 (August 2017) DOI: 10.1016/j.cmi.2017.02.002 Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 The single nucleotide polymorphism (SNP) in ERG3 in Candida parapsilosis BC014RPSC disrupts synthesis of ergosterol. (a) Multiple sequence alignment of fungal Erg3 protein orthologues. R135 is conserved even in more distantly related species, suggesting a structural and/or functional role. (b) The R135I mutation replaces a polar positively charged amino acid (arginine) by a hydrophobic one (isoleucine). According to the predicted model for the Erg3 protein structure, this substitution would result in the loss of polar contacts with amino acids S159 and T197, disrupting the Erg3 three-dimensional structure by impairing contacts between α-helices. (c) The graphs show GC-MS chromatograms for two replicates of BC014S (red) and BC014RPSC (black). There is a clear ergosterol peak in the BC014S samples. However, in BC014RPSC, ergosterol is no longer generated, and Ergosta-7,22-dienol and Ergosta-7-enol accumulate instead. Clinical Microbiology and Infection 2017 23, 575.e1-575.e8DOI: (10.1016/j.cmi.2017.02.002) Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 Construction of Candida parapsilosis upc2Δ, ndt80Δ and upc2Δ/ndt80Δ mutants. (a) UPC2 and NDT80 were deleted in the azole-resistant BC014RPSC strain, using a SAT1-flipper cassette. Confirmation of deletion was performed by PCR using the following pairs of primers: CpUPC2gen_up and CpUPC2down_R, which amplified a 1.2-kb fragment in upc2Δ (lane 2); CpNDT80gen_up and CpNDT80down_R, which amplified a 1.2-kb fragment in ndt80Δ (lane 3). The same pair of primers was used to amplify UPC2 and NDT80 genes in the parental BC014RPSC strain, used as control (lanes 4 and 5, respectively). The confirmation of upc2Δ/ndt80Δ deletion used the same approach. Lane 1 represents the molecular size marker (NZYDNA Ladder III). (b) Growth kinetics of all strains used in this study assessed by 2-hourly measurements of OD600. The growth curve represents the average of three independent experiments, each one measured in triplicate. Clinical Microbiology and Infection 2017 23, 575.e1-575.e8DOI: (10.1016/j.cmi.2017.02.002) Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 3 Candida parapsilosis Upc2 and Ndt80 transcription factors are required for ERG genes expression. Quantitative RT-PCR analysis of ERG genes. Expression levels of ERG11, ERG25, ERG6, ERG2, ERG3 and ERG4 were determined in the parental strain BC014RPSC and in all deleted strains; TUB4 gene was used as normalizer. The expression values correspond to the average value plus standard deviation of five independent experiments. *p<0.05, **p<0.01, ***p<0.001. # indicates that all comparisons in the group were statistically significant. Clinical Microbiology and Infection 2017 23, 575.e1-575.e8DOI: (10.1016/j.cmi.2017.02.002) Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions