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Mechanisms of Resistance

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

Jeffrey M. Rybak, C. Michael Dickens, Josie E. Parker, Kelly E. Caudle, Kayihura Manigaba, Sarah G. Whaley, Andrew T. Nishimoto, Arturo Luna-Tapia, Sujoy Roy, Qing Zhang, Katherine S. Barker, Glen E. Palmer, Thomas R. Sutter, Ramin Homayouni, Nathan P. Wiederhold, Steven L. Kelly, P. David Rogers
Jeffrey M. Rybak
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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C. Michael Dickens
bW. Harry Feinstone Center for Genomic Research, University of Memphis, Memphis, Tennessee, USA
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Josie E. Parker
cInstitute of Life Science, Swansea University Medical School, Swansea, Wales, United Kingdom
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Kelly E. Caudle
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Kayihura Manigaba
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Sarah G. Whaley
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Andrew T. Nishimoto
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Arturo Luna-Tapia
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Sujoy Roy
eFungus Testing Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Qing Zhang
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Katherine S. Barker
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Glen E. Palmer
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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Thomas R. Sutter
bW. Harry Feinstone Center for Genomic Research, University of Memphis, Memphis, Tennessee, USA
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Ramin Homayouni
dBioinformatics Program, University of Memphis, Memphis, Tennessee, USA
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Nathan P. Wiederhold
eFungus Testing Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Steven L. Kelly
cInstitute of Life Science, Swansea University Medical School, Swansea, Wales, United Kingdom
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P. David Rogers
aDepartment of Clinical Pharmacy, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USA
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DOI: 10.1128/AAC.00651-17
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ABSTRACT

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2. Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

INTRODUCTION

Candida species are among the most common causes of bloodstream infections in the United States and are associated with high rates of morbidity and mortality. While Candida albicans is the most commonly isolated causative agent of candidemia, the incidence of infections due to other non-albicans species of Candida has increased in recent decades (1–3). Of these non-albicans species, Candida parapsilosis is of particular concern as a human fungal pathogen. It exhibits the capacity to grow on a wide variety of surfaces, such as prosthetic materials and intravascular devices, and grows well within parenteral nutrition. It persists in hospital environments and may be transmitted nosocomially via hand carriage. Low-weight neonates and intensive care patients are among those at the highest risk of infections with C. parapsilosis (4). Relative to what is known about the mechanisms by which antifungal drug resistance develops in C. albicans, there is little information regarding the mechanisms by which antifungal drug resistance develops in C. parapsilosis. Uniquely among Candida species, C. parapsilosis demonstrates intrinsic reduced in vitro susceptibility to the echinocandins, presumably as a result of a naturally occurring polymorphism in FKS1 (5). While it is uncommon, clinical resistance to this class of antifungals has emerged in C. parapsilosis (6, 7). Azole resistance, on the other hand, is more common in C. parapsilosis, in which the rates of fluconazole resistance are approximately five times higher than those in C. albicans (8). Recent reports indicate that overexpression of the drug efflux pump Mdr1 as well as an increase in or mutation of the target of the azoles, Erg11, contributes to azole resistance in this species (9–12).

In this study, we investigated the mechanisms underlying antifungal drug resistance in a clinical isolate of C. parapsilosis by comparing it to a genetically matched, antifungal-susceptible isolate from the same patient. Through whole-transcriptome and whole-genome sequencing (WGS) analysis and allelic replacement, we demonstrate that a mutation in ERG3 leads to resistance to the azole antifungals via alternate sterol production. Additionally, we discover that this mutation in ERG3, as well as its disruption, also causes increased resistance to the echinocandins. This is the first report of an ERG3-related resistance mechanism in C. parapsilosis and is the first evidence in any Candida species that such mutations also influence susceptibility to the echinocandins.

RESULTS

Clinical isolates and susceptibility testing.The C. parapsilosis isolates used in this study were collected over the course of multiple hospitalizations from a patient with aortic valve endocarditis, as outlined elsewhere (13). These isolates were kindly provided by Jose Vazquez. The originally reported antifungal susceptibilities (determined using the methodologies previously recommended in National Committee for Clinical Laboratory Standards document M27-A2 [14]) both for the susceptible isolate collected at the initial admission (isolate 1) and for the subsequently collected resistant isolate (isolate 2) for several azoles, echinocandins, and amphotericin B are shown in Table 1. MIC values for fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, micafungin, and amphotericin B were independently determined for this study by the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio (Table 1). Isolate 1 was susceptible to all azoles tested when the result was read at both 24 and 48 h, and likewise, it was susceptible to amphotericin B and all echinocandins tested (when susceptibility was determined at 24 h). Interestingly, when susceptibility was determined at 24 h, isolate 2 appeared to be susceptible to all azoles tested, whereas when susceptibility was determined at 48 h, isolate 2 was resistant to all azoles tested. These results were consistent upon repeat testing. Isolate 2 also exhibited an increase in MIC for the echinocandins, reaching the intermediate range for micafungin and anidulafungin. No change in susceptibility to amphotericin B was observed. In separate experiments, growth curves for fluconazole MIC cultures were plotted at 24 and 48 h (Fig. 1). At both time points, isolate 2 phenocopied the erg3Δ/erg3Δ mutants.

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TABLE 1

Antifungal susceptibilities of C. albicans and C. parapsilosis isolates to azoles, echinocandins, and amphotericin B

FIG 1
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FIG 1

Fluconazole MIC growth curves. Isolates 1 and 2 and their related strains were grown for 24 h (A) and 48 h (B) in the presence of the indicated concentrations of fluconazole.

Transcriptional profiling reveals increased expression of ergosterol biosynthesis genes.Global changes in gene expression between isolates 1 and 2 were determined by RNA sequencing. A total of 378 genes (see Table S1 in the supplemental material) were observed to be reproducibly upregulated by a minimum of 1.5-fold in the resistant isolate compared to their level of expression in the susceptible isolate. Of note, only three of these genes (UPC2, ATC1, and CPAR2_400860) have been characterized in C. parapsilosis at this time. Gene Ontology term analysis performed using the Candida Genome Database (www.candidagenome.org ) revealed genes of the sterol metabolic process to be the most significantly enriched among all upregulated genes (P = 2.96e−5). Of these 14 genes, as shown in Table 2, the sterol regulatory transcription factor UPC2 and the C. parapsilosis orthologs of identified C. albicans Upc2 (CaUpc2) target genes (ERG1, ERG2, ERG5, ERG6, ERG11, ERG24, and ERG25) were included (15). Differential expression of key ergosterol biosynthesis genes, as well as the lack of a change in expression of the multidrug transporter genes CDR1, CDR2, and MDR1, was confirmed by quantitative reverse transcription-PCR (data not shown). An additional 32 genes were observed to be upregulated in the resistant isolate, but the extent of upregulation could not be reliably quantified due to the extremely low transcript counts for the susceptible isolate (Table S2). Genes which were reproducibly downregulated in the resistant isolate compared to their level of expression in the susceptible isolate are listed in Table S3.

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TABLE 2

Genes involved in sterol metabolic processes overrepresented among the upregulated genes in isolate 2 relative to isolate 1

Next-generation sequencing identified an SNP in ERG3 in the resistant isolate.To further characterize this matched isolate pair, whole-genome sequencing was used to identify single nucleotide polymorphisms (SNPs) occurring in isolate 2 compared to the sequence of isolate 1 (Fig. 2). A total of 462 SNPs involving 305 individual genes was detected. Many of the genes containing SNPs are involved in cell adhesion, biofilm formation, and cytoskeletal rearrangement. A homozygous nonsynonymous mutation was detected in ERG3, in which a glycine was replaced by an arginine at position 111. This polymorphism was confirmed by Sanger-based sequencing (data not shown).

FIG 2
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FIG 2

Schematic representation of RNA and whole-genome sequencing data for isolate 2 compared to isolate 1. Colored circle segments represent contig boundaries. The outer track represents RNA sequence data, such that the green lines indicate those genes which were overexpressed in isolate 2 by at least 1.5-fold relative to their levels of expression in isolate 1 and red lines indicate those genes which were underexpressed by 0.5-fold or less relative to their levels of expression in isolate 1. The inner track represents sequence variants which were found in isolate 2 but not in isolate 1 for the whole-genome sequence data. Overexpressed genes of the ergosterol biosynthetic process are labeled with S. cerevisiae ortholog gene names.

Analysis of copy number variation and loss of heterozygosity.The Yeast Mapping Analysis Pipeline (Ymap; lovelace.cs.umn.edu/Ymap ) was used to identify chromosomal copy number variation (CNV) and loss of heterozygosity (LOH) between isolate 1 and isolate 2 (16). CNV and SNP analyses of isolate 1 revealed no segmental or chromosomal CNV and scattered regions of increased heterozygosity across each chromosome (Fig. 3A). Using isolate 1 as a parental reference isolate, the WGS data for isolate 2 were then assessed. Unlike its parent isolate, isolate 2 exhibited segmental tetraploidy in a single chromosome (Contig005809). In this region, containing 113 genes (Table S4), complete duplication across approximately 233,000 bases was observed (Fig. 3B). Additionally, LOH analysis revealed that while relatively little allelic variation was present in isolate 1, much of this variation was lost in isolate 2 (Fig. S1).

FIG 3
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FIG 3

Horizontal tracks represent the C. parapsilosis contig as labeled. The black line running horizontally through each contig represents the predicted local copy number, with the center set being equal to 2 and divergences up or down indicating an increased or decreased copy number at that locus, respectively. A region of segmental tetraploidy is denoted with an asterisk. (A) Gray vertical lines represent local allelic variation, with the darker gray indicating higher concentrations of heterozygosity. (B) Red vertical lines represent a loss of heterozygosity relative to that for the parent isolate (isolate 1), with darker red indicating a greater degree of change in heterozygosity.

Loss of Erg3 activity confers increased resistance to echinocandins in C. parapsilosis and, to a lesser extent, C. albicans.We first deleted both alleles of ERG3 in both the susceptible (isolate 1) and the resistant (isolate 2) isolates. As expected, the loss of ERG3 resulted in resistance to all azoles tested (Table 1). Surprisingly, deletion of ERG3 also resulted in MICs that fell within the intermediate to resistant range for the echinocandins (Table 1). In order to determine if this also occurs in the related species C. albicans, we tested the echinocandin susceptibilities of two independent ERG3 deletion mutants constructed in two different backgrounds. In both cases, deletion of ERG3 resulted in negligible increases in echinocandin MICs (Table 1).

The G111R substitution in Erg3 confers reduced susceptibility to azoles and echinocandins.In order to directly assess the role of the G111R amino acid substitution in Erg3 in the reduced susceptibility to azoles and echinocandins, we introduced the wild-type (WT) ERG3 alleles from the susceptible isolate (isolate 1) into its resistant counterpart (isolate 2), creating a homozygous mutant, and performed testing for susceptibility to azoles and echinocandins. Introduction of the wild-type ERG3 alleles into isolate 2 restored susceptibility to all azoles tested (Table 1). Surprisingly, susceptibility to the echinocandins was also restored to the levels observed in isolate 1.

The G111R substitution in Erg3 leads to alterations in sterol biosynthesis.As the mutation in ERG3 is not a nonsense mutation, we questioned if, alternatively, the activity of Erg3 is reduced by this mutation, leading instead to a less functional protein. Perturbations in this protein would be evident with increases in accumulated ergosta-7,22-dienol, episterol, and ergosta-7-enol. Sterol profiles were obtained using gas chromatography (GC)-mass spectrometry (MS) and are shown in Table 3. Isolate 1 exhibited a reasonably normal sterol profile in which ergosterol comprised the highest percentage. On the other hand, isolate 2 exhibited profiles in which ergosta-7-enol and ergosta-7,22-dienol represented the largest portion of the total cell sterol fraction. This is consistent with a reduction in sterol desaturase activity. Upon replacement of the mutant ERG3 alleles in isolate 2 with the wild-type allele from isolate 1, normal sterol profiles were restored. Deletion of ERG3 in both isolate 1 (susceptible isolate) and isolate 2 (resistant isolate) resulted in profiles consistent with the complete loss of Erg3 activity.

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TABLE 3

Sterol composition of each isolate and each ERG3 mutant

DISCUSSION

Most of what is known about the genetic basis of antifungal resistance has been determined from C. albicans. Mechanisms of azole resistance in this species include overexpression of the gene encoding the target enzyme, Erg11, and nonsynonymous mutations in both ERG11 and ERG3. Additionally, upregulation of drug efflux pumps represents an additional mechanism of azole resistance; ABC transporters Cdr1 and Cdr2 confer resistance to multiple azoles, while the major facilitator superfamily transporter Mdr1 confers resistance to fluconazole, ketoconazole, and voriconazole (17). Echinocandins, on the other hand, function by inhibiting β-d-glucan synthase activity, encoded by FKS1 in C. albicans and FKS1 and FKS2 in Saccharomyces cerevisiae (18). Although mutations within FKS genes have been linked to echinocandin resistance in several species of Candida (19), there are also additional mechanisms by which this resistance may occur. These include the activation of signaling pathways which regulate the stress response and cell wall integrity, as well as synthesis of ancillary cell wall components, such as chitin and mannan (20).

In the present study, RNA sequencing revealed the genes that are differentially expressed between these two genetically related clinical isolates. We did not observe overexpression of any gene encoding a characterized multidrug transporter, suggesting that azole resistance was unlikely due to reduced intracellular accumulation of drug. Furthermore, the observed 1.6- to 2.6-fold increased expression of ERG11 in the resistant isolate compared to its level of expression in the susceptible isolate alone would not be expected to produce high-level pan-azole resistance, particularly in the absence of an accompanying ERG11 mutation. Lastly, we did not observe any change in gene expression that would indicate a root cause for the observed reduction in echinocandin susceptibility, such as an increase in glucan synthase or chitin synthase expression.

Notably among the upregulated genes are several involved in the ergosterol biosynthesis pathway. This biosynthetic pathway results in the production of an essential fungal membrane component, ergosterol, and inhibition of the azole target sterol demethylase leads to the production of toxic sterol intermediates (21). The azoles exploit this and function specifically by inhibiting the enzyme 14α-lanosterol demethylase, encoded by ERG11, leaving the cell unable to produce ergosterol (22). The genes which were observed to be upregulated within the resistant isolate, ERG1, ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, ERG27, and DAP1, encode various proteins involved in sterol processing within this pathway and are located both upstream and downstream of the azole target, Erg11. Additionally, the gene encoding the sterol regulatory transcription factor Upc2 was upregulated. These observations are consistent with a loss of sterol desaturase activity, leading to decreased membrane ergosterol and activation of Upc2. In this way, upregulation of these critical genes represents a response to facilitate increased conversion of lanosterol to ergosterol in the event that production declines or is disrupted. This is further supported by the results of whole-genome sequencing of the isolate pair. Among the relatively small number of the SNPs detected in the resistant isolate which did not occur in the susceptible isolate, a nonsynonymous mutation was identified in ERG3. This gene is located downstream in the ergosterol biosynthesis pathway relative to the positions of the genes found to be upregulated by RNA sequencing. The amino acid substitution, at position 111 of the predicted protein, changes a glycine to an arginine.

A mutation within ERG3 has been documented in clinical isolates as well as passage-derived isolates of C. albicans and linked to both amphotericin B resistance and high-level azole resistance (23, 24). When azoles inhibit the function of 14α-demethylase and, therefore, the production of ergosterol, ERG3 encodes an enzyme which converts the nontoxic 14α-methylated sterol intermediates into the toxic sterol 14α-methyl-ergosta-8,24(28)-dien-3,6-diol. A reduction in or loss of this enzyme results instead in the accumulation of nontoxic ergosta-7,22-dienol. This leads to high levels of resistance to the azole drug class.

The loss of ergosterol in the yeast cell membrane, the target of amphotericin B, has been reported to result in resistance to this antifungal (25–27). However, using standard CLSI methods, we did not observe a reproducible increase in the amphotericin B MIC at 24 or 48 h for the C. parapsilosis or C. albicans mutants deleted for ERG3 or for the C. parapsilosis mutant expressing the G111R mutation. The majority of ERG3 mutations reported in the literature result in premature stop codons; however, there have been cases in which the strains instead exhibit nonsynonymous mutations, as observed in the isolate pair evaluated in the present study (23, 28–30).

The finding that the G111R amino acid substitution also explained the reduced susceptibility to the echinocandins observed in this isolate was unexpected, as, until now, alterations in sterol desaturase activity have not been associated with this phenotype. It is important to note that deletion of ERG3 had a similar effect on echinocandin susceptibility in C. parapsilosis, but this was not the case when ERG3 was deleted in C. albicans. It is possible that a mutation in ERG3 that impairs sterol desaturase activity, combined with the naturally occurring polymorphism in FKS1 in C. parapsilosis, uniquely impacts echinocandin susceptibility in this Candida species.

Our finding of an ERG3 mutation as a cause of azole resistance in this clinical isolate is supported by the recent work of Branco et al. (31). In that study, a C. parapsilosis strain that was evolved to become resistant to posaconazole in the laboratory was found to have a mutation in ERG3 (R135I) similar to the mutation found in the present study that resulted in resistance to fluconazole, voriconazole, and posaconazole. Echinocandin susceptibilities, however, were not reported.

The matched isolate pair described here provided an opportunity to evaluate factors contributing to a unique case of antifungal resistance. We reasoned that the mechanism(s) underlying this resistance would be detectable by differences in gene expression and/or by whole-genome sequence analysis. Here we have demonstrated that a mutation in ERG3, which encodes a key enzyme for the production of the membrane sterol ergosterol, increases resistance not only to the azoles but also to the echinocandins in a clinical isolate. This is the first report of a mutation in ERG3 influencing susceptibility to the echinocandins and of a single mechanism that affects susceptibility to these two important classes of antifungals.

MATERIALS AND METHODS

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.

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TABLE 4

C. parapsilosis strains and isolates used in this study

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.

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TABLE 5

Primers used in this study

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 .

ACKNOWLEDGMENTS

We thank Elizabeth L. Berkow for her contributions to the construction of the C. parapsilosis ERG3 mutants used in this study, Joachim Morschhäuser for his invaluable expertise and for generously providing the pSFS2 plasmid used in the gene disruption and complementation experiments, and Shirlean Goodwin for her assistance with the improvement of genomic DNA isolation and sequencing methods.

The research contained in this report was supported by National Institutes of Health (NIH) grant R01 AI058145 (to P.D.R.). Partial support for DNA sequencing and analysis was provided by the Memphis Research Consortium (to T.R.S.).

FOOTNOTES

    • Received 31 March 2017.
    • Returned for modification 19 April 2017.
    • Accepted 7 June 2017.
    • Accepted manuscript posted online 19 June 2017.
  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00651-17 .

  • Copyright © 2017 American Society for Microbiology.

All Rights Reserved .

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Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis
Jeffrey M. Rybak, C. Michael Dickens, Josie E. Parker, Kelly E. Caudle, Kayihura Manigaba, Sarah G. Whaley, Andrew T. Nishimoto, Arturo Luna-Tapia, Sujoy Roy, Qing Zhang, Katherine S. Barker, Glen E. Palmer, Thomas R. Sutter, Ramin Homayouni, Nathan P. Wiederhold, Steven L. Kelly, P. David Rogers
Antimicrobial Agents and Chemotherapy Aug 2017, 61 (9) e00651-17; DOI: 10.1128/AAC.00651-17

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Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis
Jeffrey M. Rybak, C. Michael Dickens, Josie E. Parker, Kelly E. Caudle, Kayihura Manigaba, Sarah G. Whaley, Andrew T. Nishimoto, Arturo Luna-Tapia, Sujoy Roy, Qing Zhang, Katherine S. Barker, Glen E. Palmer, Thomas R. Sutter, Ramin Homayouni, Nathan P. Wiederhold, Steven L. Kelly, P. David Rogers
Antimicrobial Agents and Chemotherapy Aug 2017, 61 (9) e00651-17; DOI: 10.1128/AAC.00651-17
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KEYWORDS

antifungal agents
azoles
Candida parapsilosis
echinocandins
Oxidoreductases
Candida
antifungal resistance
antimicrobial activity
antimicrobial agents
ergosterol
molecular genetics
mycology

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