Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis

Strains and media.All C. parapsilosis isolates used in this study are listed in Table 4. Isolates were kept as frozen stocks in 40% glycerol at −80°C and subcultured on YPD (1% yeast extract, 2% peptone, 1% dextrose) agar plates at 30°C. YPD liquid medium was used for routine growth of the strains, while YPM (1% yeast extract, 2% peptone, 1% maltose) liquid medium was used for induction of the MAL2 promoter in constructed strains. Nourseothricin (200 μg/ml) was added to YPD agar plates for selection of isolates containing the SAT1-flipper cassette (32). One Shot Escherichia coli TOP10 chemically competent cells (Invitrogen) were used for plasmid construction. These strains were grown in Luria-Bertani (LB) broth or on LB agar plates supplemented with 100 μg/ml ampicillin (Sigma) or 50 μg/ml kanamycin (Fisher BioReagents), when needed.

Drug susceptibility testing.Drug susceptibility testing was performed by broth microdilution according to reference standard methods described in CLSI document M27-A3 (33). Testing was performed at least in duplicate for each isolate and each antifungal agent. The starting inoculum was between 0.5 × 10³ and 2.5 × 10³ cells/ml, and all testing was performed in RPMI 1640 with 0.2% glucose buffered with 0.165 M MOPS (morpholinepropanesulfonic acid) and adjusted to pH 7.0. The plates were incubated at 35°C, and MICs were read visually for the echinocandins and azoles and were considered the lowest concentration that resulted in 50% inhibition of growth compared to that for the drug-free growth control at both 24 and 48 h. For amphotericin B, the MIC was read as the lowest concentration that resulted in complete inhibition of growth at each of these time points.

The susceptibility tests with fluconazole were also repeated, and the results were verified by broth microdilution using a microplate spectrophotometer with optical density readings as the endpoint. Each strain was tested at least in triplicate. Cultures were diluted to 2.5 × 10³ cells/ml in sterile RPMI 1640 (Sigma, St. Louis, MO) with 2% glucose buffered with 0.165 M MOPS and adjusted to pH 7.0. Plates were incubated at 35°C for 24 and 48 h with shaking. The optical density at 600 nm was read with a BioTek Synergy 2 microplate reader (Fisher Scientific, Waltham, MA); the background reading due to the medium was subtracted from all readings. The relative growth was calculated as the percentage of cell growth in drug-containing medium relative to the cell growth in the absence of drug; the results were plotted as percent inhibition versus the fluconazole concentration.

Construction of plasmids.All primers used are listed in Table 5. An ERG3 deletion construct for C. parapsilosis was generated by amplifying an ApaI-XhoI-containing fragment consisting of flanking regions upstream from positions −280 to +51 relative to the start codon of C. parapsilosisERG3 using primers ERG3-A and ERG3-B, as well as a NotI-SacII-containing fragment consisting of downstream flanking regions from positions +1047 to +1868 using primers ERG3-C and ERG3-D. These upstream and downstream fragments of ERG3 were cloned upstream and downstream, respectively, of the SAT1-flipper cassette in plasmid pSFS2 to result in plasmid p77ERG3. Additionally, the coding region of ERG3 was amplified from either isolate 1 or isolate 2 with primers ERG3-A and ERG3-E. Each of these ApaI-XhoI-containing fragments replaced the upstream sequence in cassette p77ERG3 to introduce the entire gene, creating plasmids p77ERG3comp and p76ERG3.

Similarly, an ERG3 deletion construct for C. albicans was generated by amplifying an ApaI-XhoI-containing fragment consisting of flanking regions upstream from positions −350 to +39 relative to the start codon of C. albicansERG3 using primers CaERG3A-F and CaERG3B-R, as well as a NotI-SacII-containing fragment consisting of flanking regions from positions +997 to +1677 downstream of the start codon using primers CaERG3C-F and CaERG3D-R. These upstream and downstream fragments were cloned upstream and downstream, respectively, of the SAT1-flipper cassette in plasmid pSFS2, resulting in pCaERG3M1.

Candida parapsilosis transformation. The C. parapsilosis strains were transformed by electroporation as described previously but with some modifications (32). Cells were grown for 6 h in 2 ml YPD liquid medium, and then 4 μl of this cell suspension was passed to 50 ml of fresh YPD liquid medium and grown overnight at 30°C in a shaking incubator. When the culture's optical density at 600 nm reached 2.0, cells were collected by centrifugation, resuspended in 1 ml 10× TE (Tris-EDTA) buffer, 1 ml lithium acetate, and 8 ml of deionized water, and then reincubated at 30°C for 1 h. Freshly prepared 1 M dithiothreitol was added to the cell suspension, and the cells were incubated for an additional 30 min. The cells were then washed twice with ice-cold water and then once with ice-cold 1 mM sorbitol. Finally, the cells were resuspended in 100 μl of fresh ice-cold 1 mM sorbitol. The gel-purified ApaI-SacI fragment from the appropriate plasmid was mixed with 40 μl of competent cells, and the mixture was transferred into a chilled 2-mm electroporation cuvette. The reaction was carried out at 1.5 kV, using a CelljecT Pro electroporator (Thermo). Immediately thereafter, 1 ml of YPD containing 1 M sorbitol was added and the mixture was transferred to a 1.5-ml centrifuge tube. Cells were allowed to recover at 30°C for 6 h. Finally, 100 μl was removed and plated to YPD agar plates containing 200 μg/ml nourseothricin and 1 M sorbitol. Transformants were selected after at least 48 h of growth at 30°C.

RNA isolation and sequencing.RNA was isolated using the hot phenol method described previously (34). RNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Products), and RNA integrity was verified using a Bioanalyzer 2100 instrument (Agilent Technologies). Barcoded libraries were prepared using a Lexogen mRNA Sense kit for Ion Torrent according to the manufacturer's standard protocol. Libraries were sequenced on the Ion Torrent Proton sequencer. Individual sample fragments were concatenated to form the fastq file for the whole sample. The files were then run through FastQC software to check the data quality. Any reads with a Phred score of <20 were trimmed. Reads were then aligned to the C. parapsilosis CDC317 reference transcriptome using the RNA-Star long method. After alignment, transcriptome alignment counts were gathered. The read counts for each sample were normalized using the transcripts per kilobase million (TPM) method.

Southern hybridization.Genomic DNA (gDNA) for use with Southern blotting was prepared as described previously (35), digested with appropriate restriction endonucleases, separated on a 1% agarose gel, stained with ethidium bromide, and transferred by vacuum blotting to a nylon membrane. After UV cross-linking, gDNA was detected using an Amersham ECL Direct nucleic acid labeling and detection system according to the manufacturer's instructions.

Whole-genome sequencing.Genomic DNA for library preparation was isolated using a Qiagen Genomic-tip 100/G kit (Qiagen) following the manufacturer's instructions for genomic DNA preparation. Paired-end DNA sequence libraries for isolate 1 and for isolate 2 were prepared and sequenced using the Ion Proton Torrent sequencer, and the sequences were aligned to the C. parapsilosis CDC317 reference genome sequence. The GATK Best Practices work flow was used, incorporating the following tools: bwa, v0.5.9 (aln and sample); Picard tools (http://picard.sourceforge.net ), v1.107 (SortSam and MarkDuplicates); samtools, v0.1.19; and GATK, v2.8. (RealignerTargetCreateor, IndelRealigner, BaseRecalibrator, PrintReads, ReduceReads, HaplotypeCaller). A population variant file for C. parapsilosis was downloaded from the Broad Institute website (https://www.broadinstitute.org/fungal-genome-initiative ) and used as the –knownSites file in the GATK BaseRecalibrator step. The snpEff, v3.5, genetic variant annotation tool was used to annotate SNP and indel variants using the c_parapsilosis_CDC317 snpEff database. The GATK CombineVariants and SelectVariants tools were used to select SNPs found in isolate 2 but not in isolate 1 and SNPs that were heterozygous in isolate 1 and homozygous in isolate 2. The JBrowse Genome Browser, v1.11.6, was used to visualize sequence alignments at genomic positions in order to validate variant calls (36). Following installation of the C. parapsilosis CDC317 reference genome using the current chromosomal features file available at the Candida Genome Database (www.candidagenome.org ), the whole-genome sequencing (WGS) data for the parental isolate 1 were uploaded and aligned to the reference chromosome map using Ymap (lovelace.cs.umn.edu/Ymap ). WGS data for isolate 2 were then installed utilizing isolate 1 as the parental strain to allow loss-of-heterozygosity (LOH) analysis (16).

Sequence analysis of ERG3.The coding sequence of ERG3 in C. parapsilosis was amplified by PCR from C. parapsilosis genomic DNA using the primers listed in Table 5, cloned into pCR-BLUNTII-TOPO using a Zero Blunt TOPO PCR cloning kit, and transferred into Escherichia coli TOP10 cells with selection on LB agar plates containing 50 μg/ml kanamycin. Plasmid DNA was purified (QIAquick PCR purification kit; Qiagen) and sequenced on an ABI model 3130XL genetic analyzer using sequencing primers, resulting in a full-length sequence from both strands of ERG3. The sequencing was performed using a total of six sets of clones derived from three independent PCRs.

Sterol analysis.Nonsaponifiable lipids were extracted using alcoholic KOH. Samples were dried in a vacuum centrifuge (Heto) and were derivatized by adding 100 μl 90% N,O-bis(trimethylsilyl)trifluoroacetamide–10% tetramethylsilane (TMS) (Sigma) and 200 μl anhydrous pyridine (Sigma) and heating for 2 h at 80°C. TMS-derivatized sterols were analyzed and identified using GC-MS (with a Thermo 1300 GC coupled to a Thermo ISQ mass spectrometer; Thermo Scientific) with reference to the retention times and fragmentation spectra for known standards. GC-MS data files were analyzed using Xcalibur software (Thermo Scientific) to determine the sterol profiles for all isolates and for integrated peak areas (21).

Accession number(s).The RNA sequencing data have been deposited in the Gene Expression Omnibus repository under accession number GSE98986 . The NCBI BioProject accession number for the WGS data is PRJNA361149 . The coding sequence of ERG3 in isolate 2 described in this study has been deposited in GenBank under accession number KT277771 .