ABSTRACT
Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.
KEYWORDS: Candida parapsilosis, fluconazole, resistance, MRR1
INTRODUCTION
The triazole antifungal fluconazole remains an important antifungal agent in the treatment of candidemia and accounts for approximately 80% of all antifungal prescribing in the United States (1). Triazole antifungals are fungistatic against susceptible Candida species and act by binding to and competitively inhibiting sterol demethylase, a key enzyme in the fungal sterol biosynthesis pathway. This leads to reduced production of ergosterol and an accumulation of 14-α methyl-sterols that are thought to be deleterious to the fungal cell membrane.
Invasive candidiasis is among the most common nosocomial fungal infections and is associated with significant morbidity and mortality (2). Candida parapsilosis is the most common causative agent in non-albicans invasive Candida disease in pediatric and neonatal populations and is the most common Candida species isolated from intensive care units (ICUs) (3). In a CDC surveillance of candidemia between 2012 and 2016, all cause in-hospital mortality rose to 30%, and antifungal resistance was found in 7% of all Candida isolates, while C. parapsilosis was the only species with a notable increase in fluconazole resistance from 4.4% in 2012 to 14% in 2016 (4). The rates of fluconazole resistance in C. parapsilosis can vary depending on geographic region; however, on average, the rate remains around 3.4% worldwide (5).
Resistance to fluconazole has been well-studied in Candida albicans, where it has been shown to often be the result of multiple mechanisms working in concert. Two of these involve the gene (ERG11) encoding the triazole target, sterol-demethylase. Mutations in ERG11 can lead to amino acid substitutions in sterol-demethylase that impair the ability of fluconazole to inhibit its activity. Overexpression of ERG11, often due to activating mutations in the gene encoding the transcriptional regulator of sterol biosynthesis genes, Upc2, leads to increased production of this enzyme, requiring more triazole to inhibit its activity (6). Another mechanism of resistance involves the gene (ERG3) encoding sterol-desaturase. Mutations leading to loss of function of this enzyme preclude the accumulation of deleterious 14-α methyl sterols and allow the fungus to survive in the presence of fluconazole (7). While activating mutations in UPC2 have not been described in fluconazole-resistant C. parapsilosis clinical isolates, mutations in ERG3 and ERG11 have been reported, with the mutation in ERG11 leading to the Y132F amino acid substitution being quite common among resistant isolates (8–15).
Another well-characterized mechanism of fluconazole resistance in C. albicans involves drug efflux through the overexpression of the genes encoding the ATP-binding cassette (ABC) transporters Cdr1 and Cdr2 and the major facilitator superfamily (MFS) transporter Mdr1. Overexpression of CDR1 and CDR2 is due to activating mutations in the gene encoding the transcription factor Tac1 (transcriptional activator of CDR genes), whereas overexpression of MDR1 is due to activating mutations in the gene encoding the transcription factor Mrr1 (multidrug resistance regulator) (16, 17). While fluconazole-resistant clinical isolates of C. parapsilosis have been observed to overexpress homologs of C. albicans CDR1 and MDR1, these genes have not been shown to have any direct impact on fluconazole susceptibility (9, 18–20). Moreover, while mutations in the homologs of TAC1 and MRR1 have been observed in fluconazole-resistant C. parapsilosis clinical isolates (11, 15, 21, 22) and activating mutations in CpMRR1 have been evolved experimentally, such mutations have not yet been well characterized in clinical isolates (23).
Previously, we examined known mechanisms of fluconazole resistance for Candida spp. in a collection of 35 fluconazole-resistant C. parapsilosis clinical isolates (22). We found 11 resistant isolates with a mutation leading to the Y132F amino acid substitution in the fluconazole drug target, CpErg11, and three resistant isolates each overexpressing the gene encoding the putative drug transporter CpMdr1 (22). Among these three isolates, we identified three homozygous CpMRR1 mutations, including two novel mutations resulting in the I283R and A854V amino acid substitutions, and a previously identified mutation resulting in the R479K substitution. These isolates were also among those with the highest measured fluconazole resistance, with MICs of ≥64 μg/mL (22). To determine the direct impact of CpMDR1 expression on CpMRR1-mediated fluconazole resistance, CpMDR1 was deleted from the corresponding C. parapsilosis isolates. While in C. albicans the deletion of MDR1 from backgrounds known to have activating mutations in MRR1 is associated with a 2- to 4-fold decrease in fluconazole MIC, there was little to no change in fluconazole MIC upon CpMDR1 deletion in C. parapsilosis (22). In the present study, we show that activating mutations in CpMrr1 are a common contributor to fluconazole resistance in C. parapsilosis and elicit their effect not through overexpression of the gene encoding CpMdr1 but rather through overexpression of the gene encoding the MFS transporter, here named CpMdr1B, and unexpectedly in conjunction with overexpression of the gene encoding the ABC transporter, here named CpCdr1B.
RESULTS
CpMRR1 polymorphisms are common among fluconazole-resistant clinical isolates of C. parapsilosis.
In C. albicans, MRR1-activating mutations leading to increased expression of MDR1 and increased fluconazole resistance were found in around 20% of clinical isolates in one large collection of resistant isolates (6). To determine the frequency with which mutations in CpMRR1 occur within our collection, we first sequenced the CpMRR1 alleles for all 35 fluconazole-resistant C. parapsilosis clinical isolates (Table 1). We found mutations leading to amino acid substitutions in CpMrr1 to be present in 12 isolates (31%). These included the three isolates (Cp36, Cp30, and Cp29) previously found to be homozygous for mutations encoding either the I283R, R479K, or A854V substitutions (22). Seven isolates were heterozygous for the A854V substitution, with one of these also being heterozygous for a P255L substitution. One isolate was heterozygous for the G294E substitution, and one isolate presented heterozygous mutations for both a frameshift at K129 and a G982R substitution. These results suggest that mutations in CpMRR1 are relatively common among resistant clinical isolates of C. parapsilosis, especially those without CpERG11 mutation.
TABLE 1.
Amino acid substitutions in CpMrr1 identified in a collection of C. parapsilosis clinical isolates
Fluconazole resistance in clinical isolates of C. parapsilosis is mediated in part by activating mutations in CpMRR1.
While MRR1 mutations contribute to fluconazole resistance in C. albicans, they are not sufficient to impart high-level fluconazole resistance. To determine the direct contribution of mutations in CpMRR1 to fluconazole resistance in C. parapsilosis, we introduced the three mutations found to be homozygous within isolates in our collection into fluconazole-susceptible isolate Cp13 (fluconazole MIC, 0.25 μg/mL) using the plasmid-based CRISPR-Cas9 gene editing system pCP-tRNA (24). Introduction of the mutations leading to the I283R, R479K, and V854A substitutions in both alleles resulted in a 128- to 256-fold increase in fluconazole MIC and was sufficient to impart fluconazole resistance (MIC, 32 to 64 μg/mL) (Fig. 1). Conversely, correction of the mutations in both alleles to the wild-type CpMRR1 sequence in clinical isolates Cp36, Cp30, and Cp29 resulted in a 32- to 128-fold decrease in fluconazole MIC and was sufficient to impart fluconazole susceptibility (MIC, 1 to 2 μg/mL). These results indicate that, unlike C. albicans, CpMRR1 mutations are sufficient to impart high-level fluconazole resistance in clinical isolates of C. parapsilosis.
FIG 1.
Impact of CpMRR1 mutations on fluconazole susceptibility. Susceptibility testing was performed according to CLSI guidelines. Data shown are representative of three independent MIC measurements. MICs represented in micrograms per milliliter were measured at 24 h. Graphs are divided by specific isolates with complemented transformant strains, each with specific homozygous CpMrr1 amino acid substitution. “R” represents the clinical breakpoint for fluconazole resistance in C. parapsilosis; “S” represents the clinical breakpoint for fluconazole susceptibility for C. parapsilosis (34).
Increased expression of specific genes in clinical isolates of C. parapsilosis, including CpMDR1, CpMDR1B, and CpCDR1B is driven by activating mutations in CpMRR1.
In C. albicans, activation of Mrr1 leads to upregulation of a distinct repertoire of genes, including the gene encoding the transporter Mdr1 (17, 25). To determine which genes are differentially expressed in the presence of mutations in CpMRR1, we subjected isolates Cp36, Cp30, and Cp29 and their respective derivatives with the wild-type corrected CpMRR1 allele to transcriptional profiling by transcriptome sequencing (RNA-seq). We found 41 genes to be commonly upregulated and 23 to be downregulated among the three clinical isolates when mutations in CpMRR1 were present compared to the respective wild-type CpMRR1 derivatives (Fig. 2). Among upregulated genes were those homologous to genes upregulated in C. albicans strains carrying activating mutations in MRR1, including homologs of GRP2, LPG20, orf19.7306, orf19.7166, orf19.6586, OYE32, MDR1, and MRR1 (Table 2) (17).
FIG 2.
Differentially expressed genes in clinical isolates with homozygous mutations leading to amino acid substitutions in CpMrr1 compared to the respective corrected strain as determined by RNA sequencing. Upregulated genes include genes with a ≥2-fold change for the clinical isolate with CpMrr1 mutation versus each clinical isolate with CpMrr1 corrected to wild type. Downregulated genes include genes with a ≤−2 fold change for the clinical isolate with CpMrr1 mutation versus each clinical isolate with CpMrr1 corrected to wild type. FDR P values ≤ 0.05.
TABLE 2.
Genes with differential expression in clinical isolates containing CpMrr1 mutation compared to wild type
| C. parapsilosis genea | C. albicans homologb | Fold changec |
Descriptiond | ||
|---|---|---|---|---|---|
| A854V vs WT | R479K vs WT | I283R vs WT | |||
| CPAR2_100480 | GRP2 | 518.37 | 4,861.44 | 201.2 | Similar to S. cerevisiae Gre2p (methylglyoxal reductase); expression increased in fluconazole- and voriconazole-resistant strains |
| GRP2 | GRP2 | 136.77 | 756.03 | 47.92 | Similar to S. cerevisiae Gre2p (methylglyoxal reductase); expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_105750 | 107.7 | 8.08 | 24.03 | Has domain(s) with predicted DNA binding activity | |
| CPAR2_103330 | orf19.2812 | 12.32 | 88.35 | 11.11 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_103600 | orf19.320 | 11.77 | 69.14 | 24.01 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_603010 | MDR1 | 10.51 | 218.69 | 7 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains; ortholog(s) localize to the endoplasmic reticulum |
| MDR1 | MDR1 | 9.41 | 43.78 | 14.33 | Member of the MDR family of major facilitator transporter superfamily; putative drug transporter; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_206630 | orf19.3544 | 8.66 | 75.34 | 15.97 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_103320 | 7.81 | 8 | 7.09 | Uncharacterized | |
| CPAR2_601840 | orf19.5517 | 7.61 | 57.44 | 8.14 | Putative alcohol dehydrogenase; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_401490 | GST2 | 6.53 | 30.58 | 5.36 | GST2/URE2 family protein; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_404080 | orf19.5860 | 4.9 | 9.56 | 6.12 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| SADH | ADH5 | 4.83 | 10.44 | 3.35 | Butyraldehyde dehydrogenase, carbonyl reductase involved in amino acid degradation pathways |
| CPAR2_701130 | PLB3 | 4.81 | 6.13 | 5.73 | Has domain(s) with predicted phospholipase activity and role in metabolic process, phospholipid catabolic process |
| CPAR2_404090 | orf19.345 | 4.64 | 5.13 | 6.38 | Putative succinate-semialdehyde dehydrogenase [NAD(P)+]; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_703250 | 4.6 | 24.67 | 5.76 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains | |
| LPG20 | LPG20 | 4.5 | 43.42 | 9.14 | Aldo-keto reductase family protein; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_805920 | orf19.1075.1 | 4.5 | 15.54 | 3.65 | Uncharacterized |
| CPAR2_304370 | CDR1 | 4.35 | 15.28 | 5.82 | Has domain(s) with predicted ATP binding, ATPase activity, ATPase-coupled transmembrane transporter activity, nucleoside-triphosphatase activity, nucleotide binding activity |
| OYE32 | OYE32 | 4.13 | 14.15 | 3.17 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains; ortholog(s) have role in cell redox homeostasis |
| CPAR2_702640 | orf19.6586 | 4.08 | 15.93 | 11.22 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_701460 | orf19.7306 | 4 | 9.32 | 7.67 | Aldo-keto reductase family protein; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_300590 | FLU1 | 3.33 | 3.91 | 2.96 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane localization |
| CPAR2_503210 | orf19.6943 | 3.23 | 5.29 | 2.91 | Uncharacterized |
| CPAR2_402120 | orf19.1430 | 3.22 | 3.24 | 2.88 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_807720 | POX1-3 | 3.21 | 2.82 | 3.27 | Ortholog(s) have role in fatty acid beta-oxidation, long-chain fatty acid catabolic process and peroxisome localization |
| PDR16 | PDR16 | 3 | 3.61 | 2.82 | Phosphatidylinositol transfer protein; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_301750 | orf19.4779 | 2.9 | 3.23 | 2.03 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane localization |
| CPAR2_103670 | orf19.2446 | 2.88 | 8.42 | 5.36 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_109500 | orf19.6066 | 2.77 | 3.75 | 2.78 | Ortholog(s) have 4-hydroxybenzaldehyde dehydrogenase activity, carboxylate reductase activity |
| STP4 | STP4 | 2.72 | 2.91 | 2.8 | Putative transcription factor with zinc finger DNA-binding motif; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_200870 | orf19.6600 | 2.67 | 2.9 | 2.05 | Ortholog(s) have phosphatidic acid transfer activity and role in cardiolipin metabolic process, phospholipid translocation, phospholipid transport, positive regulation of phosphatidylcholine biosynthetic process |
| CPAR2_702300 | orf19.7166 | 2.65 | 5.79 | 2.86 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains; ortholog(s) localize to the Golgi apparatus and endoplasmic reticulum |
| CPAR2_700540 | orf19.7235 | 2.57 | 3.36 | 3.72 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| MRR1 | MRR1 | 2.47 | 9.22 | 3.36 | Regulator of MDR1 transcription; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_400630 | orf19.4609 | 2.45 | 3.32 | 2.88 | Protein of unknown function; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_701900 | orf19.1985 | 2.27 | 3.61 | 2.83 | Has domain(s) with predicted protein serine/threonine kinase activity, transferase activity, transferring phosphorus-containing groups activity and role in protein phosphorylation |
| CPAR2_602390 | orf19.3442 | 2.25 | 2.68 | 2.63 | Putative oxidoreductase; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_211640 | orf19.5785 | 2.17 | 3.08 | 4.66 | Uncharacterized |
| CPAR2_201400 | orf19.6348 | 2.1 | 4.02 | 2.84 | Putative ubiquitin thiolesterase; predicted role in ubiquitin-dependent protein catabolism; expression increased in fluconazole- and voriconazole-resistant strains |
| LAP4 | LAP4 | 2.04 | 4.43 | 3.12 | Similar to aminopeptidase I; expression increased in fluconazole- and voriconazole-resistant strains |
| CPAR2_203940 | −2.02 | −2.92 | −4.19 | Uncharacterized | |
| CPAR2_800070 | PGA28 | −2.13 | −4.8 | −4.36 | Ortholog of C. albicans SC5314: C7_03110W_A/PGA28 |
| CPAR2_106690 | DIP5 | −2.13 | −4.9 | −2.23 | Ortholog(s) have l-aspartate transmembrane transporter activity, l-glutamate transmembrane transporter activity, dicarboxylic acid transmembrane transporter activity |
| CPAR2_500170 | ZRT1 | −2.2 | −2.34 | −2.81 | Has domain(s) with predicted metal ion transmembrane transporter activity, role in metal ion transport, transmembrane transport and membrane localization |
| CPAR2_407280 | orf19.1370 | −2.34 | −5.5 | −3.43 | Ortholog of C. albicans SC5314: C2_09800C_A |
| CPAR2_102140 | ADH4 | −2.47 | −4.56 | −3.33 | Has domain(s) with predicted oxidoreductase activity and role in metabolic process |
| CPAR2_702930 | orf19.6475 | −2.57 | −4.77 | −2.99 | Ortholog of Candida metapsilosis: CMET_1690 |
| CPAR2_403560 | orf19.1318 | −2.57 | −2.89 | −3.05 | Ortholog of C. albicans SC5314: C4_03580W_A |
| CPAR2_601420 | orf19.3475 | −2.59 | −3.21 | −3.51 | Ortholog of C. albicans SC5314: C6_02330W_A |
| CPAR2_106680 | PUT4 | −2.65 | −3.87 | −2.91 | Has domain(s) with predicted amino acid transmembrane transporter activity, role in amino acid transmembrane transport, transmembrane transport and membrane localization |
| CPAR2_701480 | orf19.7300 | −2.71 | −11.84 | −7.32 | Ortholog of C. albicans SC5314: CR_09040W_A |
| CPAR2_403880 | −2.74 | −3.95 | −3.7 | Uncharacterized | |
| CPAR2_502450 | MRV4 | −3.29 | −5.43 | −6.65 | Ortholog of C. albicans SC5314: C5_04210C_A/MRV4 |
| CPAR2_103080 | GLX3 | −4.2 | −5.87 | −5.28 | Ortholog(s) have glyoxalase III activity, protein folding chaperone activity |
| HGT10 | HGT10 | −4.99 | −9.23 | −4.09 | Ortholog(s) have solute/proton symporter activity, role in glycerol transport, transmembrane transport and plasma membrane localization |
| TNA1 | TNA1 | −5.8 | −3.37 | −2.99 | Ortholog(s) have carboxylic acid transmembrane transporter activity and role in carboxylic acid transport, quinolinic acid transmembrane transport |
| CPAR2_502460 | MRV2 | −6.41 | −49.42 | −19.66 | Ortholog of S. cerevisiae: YDL218W, C. albicans SC5314: C5_04190W_A/MRV2 |
| CPAR2_701510 | SLP3 | −6.68 | −9.98 | −11.84 | Has domain(s) with predicted membrane localization |
| NAG4 | NAG3 | −8.41 | −4.32 | −6.87 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane localization |
| CPAR2_802720 | orf19.3232 | −11.99 | −5.19 | −3.3 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane localization |
| CPAR2_108370 | HGT1 | −12.9 | −5.89 | −13.54 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane, membrane localization |
| CPAR2_102120 | orf19.2633.1 | −20.74 | −5.08 | −11.07 | Uncharacterized |
| CPAR2_108340 | HGT2 | −33.69 | −8.52 | −7.04 | Has domain(s) with predicted transmembrane transporter activity, role in transmembrane transport and integral component of membrane, membrane localization |
CPAR2 identifier from genome annotation of C. parapsilosis, CDC317, Candida Genome Database. Gene name provided where available.
Candida albicans homologs are identified where possible from Candida Genome Database. Gene names provided may be true orthologs and best hits.
Fold change (≥2 or ≤−2) for clinical isolates with CpMRR1 mutation versus each clinical isolate with CpMRR1 corrected to wild type. Performed in 3 biological replicates with FDR P values or ≤ 0.05.
Gene descriptions taken from Candida Genome Database.
Interestingly, two homologs of C. albicans MDR1 (orf19.5604) were among those genes upregulated in the presence of a CpMRR1 mutation, CpMDR1 (CPAR2_301760), which shares 66% predicted peptide sequence identity with C. albicans Mdr1, as well as a second homolog sharing 54.0% predicted peptide sequence identity that we have designated CpMDR1B (CPAR2_603010). Also upregulated was a gene encoding a homolog of the C. albicans ABC transporter gene CDR1, which we have designated CpCDR1B (CPAR2_304370). This gene shares 77% predicted peptide sequence identity with C. albicans Cdr1 and is distinct from the previously named C. parapsilosis CpCDR1 (CPAR2_405290), which shares 79% predicted peptide sequence identity. For relative comparison of the expression levels of these genes, in clinical isolate Cp29, which carries a mutation leading to the A854V amino acid substitution, average reads per kilobase per million (RPKM) values for CpCDR1, CpCDR1B, CpMDR1, and CpMDR1B were 43.4, 341.9, 468.4, and 966.3, respectively.
In C. albicans, Cdr1 (Candida drug resistance) is an ATP-binding cassette transporter, and homologs are known to be a major determinant of fluconazole resistance in other Candida species (26, 27). However, in C. albicans, expression of CDR1 is not known to be regulated by Mrr1 but rather by the zinc-cluster transcription factor Tac1 (16). Additionally, some of the genes associated with upregulation in C. albicans Mrr1 activation were downregulated in the isolates that contained CpMRR1 mutations, including the homologs of HGT2, HGT1, GLX3, and ADH4. These results highlight similarities and differences in the genes regulated by CpMRR1 in C. parapsilosis and MRR1 in C. albicans and raise the possibility that both MFS and ABC transporters may participate in CpMRR1-mediated fluconazole resistance in C. parapsilosis.
Overexpression of CpMDR1B and CpCDR1B results in increased resistance to fluconazole in C. parapsilosis.
To determine if increased expression of the genes encoding the CpMdr1, CpMdr1B, and CpCdr1B transporters are capable of influencing fluconazole resistance, these genes were overexpressed by placing them under the control of the strong CpTEF1 promoter, resulting in a 58-, 15-, and 20-fold increase in relative expression of CpMDR1, CpMDR1B, and CpCDR1B, respectively (Fig. 3A). These levels of overexpression not only replicated the expression observed in the highest expressing resistant clinical isolate, Cp30, but also resulted in a 16-fold increase in fluconazole resistance for the CpMDR1B and CpCDR1B overexpression strains. Conversely, no change in fluconazole MIC was observed for the strain overexpressing CpMDR1 (Fig. 3B). These results indicate that overexpression of CpMDR1B and CpCDR1B to levels comparable to those observed in resistant clinical isolates is capable of influencing fluconazole susceptibility.
FIG 3.
Constitutive overexpression (OE) of CpCDR1B, CpMDR1, and CpMDR1B in a susceptible clinical isolate. (A) Relative fold change of efflux pump expression as measured by reverse transcriptase quantitative PCR (RT-qPCR). Expression is measured compared to the average for the susceptible isolate Cp13. Error bars represent standard deviation for three independent experiments. (B) Fluconazole MICs for overexpressing strains. Susceptibility testing was performed according to CLSI guidelines. Data shown are representative of three independent MIC measurements. Values are represented in micrograms per milliliter measured at 24 h. “R” represents the clinical breakpoint for fluconazole resistance in C. parapsilosis; “S” represents the clinical breakpoint for fluconazole susceptibility for C. parapsilosis.
Disruption of CpMDR1B and CpCDR1B, but not CpMDR1, increases susceptibility of fluconazole in resistant clinical isolates carrying activating mutations in CpMRR1.
In order to determine the direct contributions of CpMDR1, CpMDR1B, and CpCDR1B to CpMrr1-mediated fluconazole resistance, we disrupted these genes individually and in combination in a resistant clinical isolate. We used the isolate Cp29 as the background strain, as it overexpresses all three of these genes and is homozygous for the most commonly observed CpMRR1 substitution, A854V. To accomplish this, the pCP-tRNA method was used to insert a STOP-codon containing a segment of DNA at the 5′ end for each desired open reading frame. Disruption of CpMDR1 resulted in a single dilution decrease in fluconazole MIC, whereas disruption of either CpMDR1B or CpCDR1B resulted in a modest reduction in fluconazole MIC from 64 μg/mL to 16 μg/mL. Combined disruption of both CpMDR1B and CpCDR1B was sufficient to impart fluconazole susceptibility resulting in a reduction in MIC from 64 μg/mL to 4 μg/mL (Table 3). No additional effect was observed for deletion of CpMDR1 either in combination with CpMDR1B or CpCDR1B or when all three genes were disrupted. Notably, similar effects were also observed for voriconazole MICs. These data indicate that activating mutations in CpMRR1 increases fluconazole resistance, not through overexpression of CpMDR1 but primarily through the increased expression of CpMDR1B and CpCDR1B.
TABLE 3.
Triazole MICsa for efflux pump disruption strains made in CpMrr1 activating mutation containing background
| Strainb | MIC (μg/mL) |
||||
|---|---|---|---|---|---|
| Fluconazole | Voriconazole | Isavuconazole | Itraconazole | Posaconazole | |
| Cp29 | 64 | 0.5 | 0.125 | 0.125 | 0.125 |
| Cp29 mdr1 | 32 | 0.5 | 0.125 | 0.125 | 0.06 |
| Cp29 mdr1b | 16 | 0.25 | 0.125 | 0.125 | 0.06 |
| Cp29 cdr1b | 16 | 0.25 | 0.06 | 0.125 | 0.06 |
| Cp29 mdr1,mdr1b | 16 | 0.25 | 0.125 | 0.125 | 0.06 |
| Cp29 mdr1,cdr1b | 16 | 0.25 | 0.06 | 0.125 | 0.06 |
| Cp29 mdr1b,cdr1b | 4 | 0.06 | 0.06 | 0.125 | 0.06 |
| Cp29 dr1,mdr1b,cdr1b | 4 | 0.06 | 0.06 | 0.125 | 0.06 |
DISCUSSION
Polymorphisms leading to amino acid substitutions in C. parapsilosis CpMrr1 have been previously observed among fluconazole-resistant (MIC, ≥8 μg/mL) and -susceptible dose-dependent (MIC, 4 μg/mL) clinical isolates (Fig. 4) (11, 13, 15, 19, 21–23). Additionally, the presence of certain amino acid substitutions, specifically, I283R, R479K, A854V, G583R, and K873N, have been associated with increased expression of CpMDR1 and increased resistance to fluconazole and voriconazole (22, 23, 28). CpMRR1 mutations have previously been evolved in the laboratory and experimentally shown to confer resistance to fluconazole (23, 28). Among fluconazole-resistant clinical isolates, CpMRR1 mutations were first reported in nine isolates collected from a population-based surveillance study of candidemia by the CDC (11). Only six of these exhibited ≥10-fold increases in CpMDR1 expression relative to the average expression of susceptible controls (11). Our initial examination of the 35 clinical isolates with fluconazole MIC values of ≥8 μg/mL in our collection revealed that none carry a mutation in CpERG3, whereas 11 carry a mutation in CpERG11 leading to the Y132F substitution. Three isolates were observed to have CpMDR1 expression ≥10-fold above the average of susceptible controls, and all three were homozygous for mutations in CpMRR1 (22). However, deletion of CpMDR1 in these isolates had no effect on fluconazole resistance in one isolate and reduced resistance by only one dilution in the remaining two isolates (128 to 64 μg/mL and 64 to 32 μg/mL).
FIG 4.
Amino acid substitutions identified in Candida parapsilosis CpMrr1. Substitutions identified in C. parapsilosis clinical isolates with fluconazole MICs of ≥8 μg/mL (11, 13, 15, 19, 21, 22). Red labeled domains were identified through the Candida Genome Database and a search of the InterPro database. I283R, R479K, and A854V were identified as homozygous mutations within our collection.
Our findings here delineate the direct contribution of CpMRR1 activating mutations to fluconazole resistance in clinical isolates and suggest that such mutations represent a relatively common mechanism of resistance in C. parapsilosis. Indeed, we observed CpMRR1 mutations in 12 of our 35 resistant isolates, with heterozygous mutations being generally associated with lower fluconazole MICs (16 to 32 μg/mL), whereas isolates with homozygous mutations exhibited MICs of 64 μg/mL. Only one isolate (isolate Cp39) carried a mutation in both CpMRR1 (leading to the G294E substitution) and CpERG11 (leading to the Y132F substitution). The data presented in this study clearly demonstrate that the CpMRR1 mutations leading to the I283R, R479K, and A854V substitutions confer resistance to fluconazole. Furthermore, these findings support the notion that the levels of resistance observed in 11 isolates in this collection can be fully explained by the mutations present in CpMRR1.
While homologs of C. albicans CDR1 and MDR1 have been observed to be overexpressed in resistant C. parapsilosis clinical isolates, their contribution to fluconazole resistance has been unclear. Transcriptional profiling of the clinical isolates with CpMRR1 mutations leading to I283R, R479K, or A854V substitutions compared to that of their respective strains with CpMRR1 edited to the wild-type sequence revealed changes in gene expression with similarities to those observed when Mrr1 is activated in C. albicans, as well as similarities to the transcriptional profiles of isolates previously experimentally evolved to have resistance to voriconazole. These include overexpression of CpMDR1, CpMDR1B (CPAR2_603010), and CpCDR1B (CPAR2_304370). Importantly, a CDR1 homolog has been shown to be regulated by an Mrr1 homolog in the related species Clavispora lusitaniae (also referred to as Candida lusitaniae) and has been shown to contribute to Mrr1-mediated fluconazole resistance. Moreover, there are similarities between the genes observed to be upregulated in that species when Mrr1 is activated and those that we observe here with the activation of CpMrr1 (29, 30). In contrast, in C. albicans, Mrr1 regulates fluconazole resistance primarily through overexpression of MDR1 with no additional MFS or ABC transporter having been identified as a target of CaMrr1, influencing fluconazole susceptibility (17).
Our observation that overexpression of CpMDR1 had no impact on fluconazole susceptibility and that deletion of CpMDR1 in clinical isolate Cp29 resulted in only a single dilution reduction in fluconazole MIC was consistent with our previous findings that deletion of CpMDR1 had little to no effect on fluconazole susceptibility in isolates in which it was highly overexpressed (22). Indeed, CpMDR1B appears to be more functionally similar to C. albicans MDR1, as overexpression at levels observed in resistant isolates resulted in an increase in fluconazole MIC, and disruption in clinical isolate Cp29 reduced the MIC from 32 to 8 μg/mL. We were surprised to observe a role for CpCDR1B, as Mrr1-mediated resistance in other Candida species appears to be driven primarily by overexpression of MDR1. CpMrr1-mediated fluconazole resistance in C. parapsilosis instead appears to be driven by overexpression of both CpMDR1B and CpCDR1B, with both making an equal contribution. Disruption of these transporter genes had similar effects on susceptibility to voriconazole, while isavuconazole and posaconazole resistance appeared only minimally affected by the disruption of CpCDR1B with a decrease in observed MIC of a single dilution. No change in itraconazole susceptibility was observed, suggesting that these latter three agents may represent useful alternatives to fluconazole for the treatment of C. parapsilosis exhibiting CpMrr1-mediated resistance.
We have shown that, in addition to mutations in CpERG11, CpMRR1 mutations represent an important mechanisms of fluconazole resistance in C. parapsilosis clinical isolates. However, it is important to note that 8 out of our 35 clinical isolates have no sufficient explanation for the fluconazole resistance observed. It is therefore apparent that additional mechanisms of fluconazole resistance are operative in these isolates, and as such, the discovery and characterization of these remaining mechanisms of fluconazole resistance in C. parapsilosis are needed.
MATERIALS AND METHODS
Strains and media.
All C. parapsilosis isolates used in this study have been previously described (22). Isolates and derived strains were kept at −80°C in 40% glycerol stock. All strains and isolates were maintained on YPD (1% yeast extract, 2% peptone, and 2% dextrose) agar plates at 30°C or in YPD liquid medium at 30°C in a 220-rpm shaking incubator. RPMI with morpholinepropanesulfonic acid (MOPS) and 2% glucose, pH 7.0, was used for both drug susceptibility testing growth prior to RNA isolation techniques. Chemically competent DH5α cells were utilized for plasmid construction and grown in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin.
CpMRR1 sequence analysis.
Genomic DNA was isolated as previously described (31), and the coding sequence of C. parapsilosis CpMRR1 (CPAR2_807270) was amplified via PCR with CpMrr1-AmpF and CpMrr1-AmpR primers. PCR products were purified by QIAquick PCR purification kit (Qiagen). Single nucleotide polymorphisms were determined using evenly spaced primers for multiple Sanger sequencing reactions (Applied Biosystems; Veriti). Sequencing primers and all other primers used in this study can be found in Table S1 in the supplemental material.
Drug susceptibility determination.
Antifungal susceptibilities for triazoles were determined by broth microdilution for all clinical isolates and strains according to the Clinical and Laboratory Standards Institute document M27, 4th edition (32). Triazoles were obtained from Sigma-Aldrich, and concentrated stocks were prepared in dimethyl sulfoxide. Drug stocks were diluted 1:50 for serial inoculation of RPMI medium on 96-well plates. MICs were recorded after 24 h of incubation at 35°C. All drug susceptibility testing was performed in biological triplicates. Graphical representation was made using GraphPad Prism version 9.2.0.
C. parapsilosis transformation.
Transformation of C. parapsilosis isolates was performed using electroporation methods previously described with alteration (22). Cells were grown for 6 h at 30°C in 2 mL YPD liquid medium. After incubation, 25 to 400 μL of cell suspension was used to inoculate 25 mL fresh YPD liquid medium, depending on C. parapsilosis isolate growth, for overnight growth at 30°C. Optical density was read at 600 nm (OD600) for a minimum of 2.0, and competent cells were prepared as previously described (22). Electroporation was performed on competent cells using the C. albicans protocol on a Gene Pulsar Xcell (Bio-Rad). After recovery for 6 h at 30°C in a 50-50 YPD and 1 M d-Sorbitol medium, transformed cells were plated on YPD plates containing 200 μg/mL of nourseothricin for selection.
Modification of putative drug transporter genes.
For overexpression of the genes of interest, the 1,000-bp upstream region of CpTEF1 was amplified utilizing CpTEF1-1 and CpTEF1-2 primers, and a nourseothricin resistance marker, originally derived from pV1200 (33), was amplified using pJMR_forward_p1 and pJMR_reverse_p3. The two amplified templates were then combined via fusion PCR (annealing temperature, 58°C) with the primers pJMR5_p5_NOT1 and pJMR5_p2_ApaI (see Table S1). This fusion product was then digested with ApaI and NotI-HF restriction enzymes, and products were ligated into a plasmid backbone containing an f1 bacteriophage origin of replication, an ampicillin resistance marker, and an origin of replication sequence to make pJMR5. Repair templates for overexpression were amplified from pJMR5 using 5′ and 3′ primers, which introduced 50 to 70 bases of homology to the sequence immediately upstream of the gene of interest (GOI) and were purified using the Gene Clean II kit (MP Biomedicals). Using the Cas9-RNP method previously described (27) with some modification, approximately 1 μg of the purified repair template was mixed with 4 μM Cas9-RNP complexes targeting the immediate upstream sequence for CpMDR1 (CPAR2_301760), CpMDR1B (CPAR2_603010), and CpCDR1B (CPAR2_304370) and added to the electrocompetent C. parapsilosis cells. Promoter insertion was confirmed via PCR screening.
pCP-tRNA-guided transformation.
For the precise manipulation of CpMRR1 and the disruption of putative efflux pump genes, the pCP-tRNA plasmid was used as previously described (24) with minor modification. Briefly, two 23-bp oligonucleotides with SapI restriction sites (Integrated DNA Technologies) were annealed to generate site-specific guide sequences (see Table S1). SapI-digested pCP-tRNA plasmid was purified with the QIAquick PCR purification kit (Qiagen) and subsequently cloned with the designated guide-RNA. Repair templates were generated by primer extension using 50- to 70-base microhomology from the gene of interest and contained either the desired point mutations for CpMRR1 investigation or the addition of a 22-base STOP-codon sequence for GOI disruption (Integrated DNA Technologies). Representative mutation strains were identified by Sanger sequencing (Applied Biosystems; Veriti). Plasmid ejection was induced by overnight growth in liquid YPD followed by isolation on YPD-agar plates and subsequent replicative patching on YPD agar plates containing 200 μg/mL nourseothricin.
RNA sequencing.
C. parapsilosis cultures were grown similar to those described for MIC preparation with some modification. C. parapsilosis strains were grown in biological triplicate at 30°C overnight in YPD liquid medium and subsequently plated onto Sabouraud dextrose (BD) minimal medium agar for 24 h growth at 30°C. Sterile loops were used to transfer cells into 20 mL RPMI for inoculums with an OD600 of 0.1. Cultures were incubated at 35°C with shaking at 110 rpm for 8 h, after which the cells were centrifuged at 4,000 rpm for 5 min. Supernatants were removed, and the pellets were stored at −80°C for a minimum of 24 h. RNA isolation was performed using the RiboPure yeast (Invitrogen) system per manufacturer’s instructions. RNA sequencing was performed using Illumina NextSeq for stranded mRNA. Libraries were prepared with paired-end adapters using Illumina chemistries per manufacturer’s instructions, with read lengths of approximately 150 bp with at least 50 million raw reads per sample. RNA-seq data were analyzed using CLC Genomics Workbench version 20.0 (Qiagen), and reads were trimmed using default settings for failed reads and adaptor sequences and then subsequently mapped to the C. parapsilosis genome (GenBank accession number GCA_000182765.2) with paired reads counted as one and expression values set to RPKM. Principal-component analysis was utilized for assessment of the clustering of biological replicates. Whole-transcriptome differential gene expression analysis was performed with the prescribed algorithm of CLC Genomics Workbench version 20.0. Mismatch, insertion, and deletion costs were set to default parameters, and the Wald test was used for all group pairs against the matched parent control strain. Genes were considered differentially regulated when a fold change of ≥2 or ≤−2 was observed accompanied by a false-discovery rate (FDR) P value of ≤0.05.
Reverse transcriptase quantitative PCR.
C. parapsilosis overexpression strains were grown in YPD medium, and RNA was isolated using methods as previously described (22). cDNA was synthesized from isolated RNA using SuperScript first-strand synthesis system according to the manufacturer’s protocol. CpACT1 (CPAR2_201570), CpMDR1, CpMDR1B, and CpCDR1B were amplified from synthesized cDNA by PCR utilizing SYBR green master mixes in accordance with the manufacturer’s instructions. Gene-specific primers were used in PCR at 95°C for 10 min for AmpliTaq Gold activation and then 40 cycles of denaturation at 95°C for 15 s followed by annealing/extension at 60°C. The dissociation curve was determined using the 7500 detection real-time PCR system (Applied Biosystems). Changes in expression among isolates and transformants were calculated using the 2−ΔΔCT method and performed in triplicate. Data representation was made using GraphPad Prism version 9.2.0.
Data availability.
Study data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE196409.
ACKNOWLEDGMENTS
This work was supported by NIH NIAID grants R01 A1058145 and R01 AI131620 awarded to P.D.R.
Footnotes
Supplemental material is available online only.
REFERENCES
- 1.Vallabhaneni S, Baggs J, Tsay S, Srinivasan AR, Jernigan JA, Jackson BR. 2018. Trends in antifungal use in US hospitals, 2006–12. J Antimicrob Chemother 73:2867–2875. 10.1093/jac/dky270. [DOI] [PubMed] [Google Scholar]
- 2.Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. 10.1126/scitranslmed.3004404. [DOI] [PubMed] [Google Scholar]
- 3.Tóth R, Nosek J, Mora-Montes HM, Gabaldon T, Bliss JM, Nosanchuk JD, Turner SA, Butler G, Vágvölgyi C, Gácser A. 2019. Candida parapsilosis: from genes to the bedside. Clin Microbiol Rev 32:e00111-18. 10.1128/CMR.00111-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Toda M, Williams SR, Berkow EL, Farley MM, Harrison LH, Bonner L, Marceaux KM, Hollick R, Zhang AY, Schaffner W, Lockhart SR, Jackson BR, Vallabhaneni S. 2019. Population-based active surveillance for culture-confirmed candidemia - four sites, United States, 2012–2016. MMWR Surveill Summ 68:1–15. (Author Correction, MMWR Morb Mortal Wkly Rep 68:934.) 10.15585/mmwr.ss6808a1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. 2017. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol 7:2173. 10.3389/fmicb.2016.02173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Flowers SA, Barker KS, Berkow EL, Toner G, Chadwick SG, Gygax SE, Morschhäuser J, Rogers PD. 2012. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryot Cell 11:1289–1299. 10.1128/EC.00215-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Morio F, Pagniez F, Lacroix C, Miegeville M, Le Pape P. 2012. Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence. J Antimicrob Chemother 67:2131–2138. 10.1093/jac/dks186. [DOI] [PubMed] [Google Scholar]
- 8.Rybak JM, Dickens CM, Parker JE, Caudle KE, Manigaba K, Whaley SG, Nishimoto AT, Luna-Tapia A, Roy S, Zhang Q, Barker KS, Palmer GE, Sutter TR, Homayouni R, Wiederhold NP, Kelly SL, Rogers PD. 2017. Loss of C-5 sterol desaturase activity results in increased resistance to azole and echinocandin antifungals in a clinical isolate of Candida parapsilosis. Antimicrob Agents Chemother 61:e00651-17. 10.1128/AAC.00651-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Castanheira M, Deshpande LM, Messer SA, Rhomberg PR, Pfaller MA. 2020. Analysis of global antifungal surveillance results reveals predominance of Erg11 Y132F alteration among azole-resistant Candida parapsilosis and Candida tropicalis and country-specific isolate dissemination. Int J Antimicrob Agents 55:105799. 10.1016/j.ijantimicag.2019.09.003. [DOI] [PubMed] [Google Scholar]
- 10.Asadzadeh M, Ahmad S, Al-Sweih N, Khan Z. 2017. Epidemiology and molecular basis of resistance to fluconazole among clinical Candida parapsilosis isolates in Kuwait. Microb Drug Resist 23:966–972. 10.1089/mdr.2016.0336. [DOI] [PubMed] [Google Scholar]
- 11.Grossman NT, Pham CD, Cleveland AA, Lockhart SR. 2015. Molecular mechanisms of fluconazole resistance in Candida parapsilosis isolates from a U.S. surveillance system. Antimicrob Agents Chemother 59:1030–1037. 10.1128/AAC.04613-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Thomaz DY, de Almeida JN, Jr, Lima GME, de Oliveira Nunes M, Camargo CH, Grenfell RC, Benard G, Del Negro GMB. 2018. An azole-resistant Candida parapsilosis outbreak: clonal persistence in the intensive care unit of a Brazilian teaching hospital. Front Microbiol 9:2997. 10.3389/fmicb.2018.02997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Choi YJ, Kim Y-J, Yong D, Byun J-H, Kim TS, Chang YS, Choi MJ, Byeon SA, Won EJ, Kim SH, Shin MG, Shin JH. 2018. Fluconazole-resistant Candida parapsilosis bloodstream isolates with Y132F mutation in ERG11 gene, South Korea. Emerg Infect Dis 24:1768–1770. 10.3201/eid2409.180625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Singh A, Singh PK, de Groot T, Kumar A, Mathur P, Tarai B, Sachdeva N, Upadhyaya G, Sarma S, Meis JF, Chowdhary A. 2019. Emergence of clonal fluconazole-resistant Candida parapsilosis clinical isolates in a multicentre laboratory-based surveillance study in India. J Antimicrob Chemother 74:1260–1268. 10.1093/jac/dkz029. [DOI] [PubMed] [Google Scholar]
- 15.Arastehfar A, Daneshnia F, Hilmio˘glu-Polat S, Fang W, Yasar M, Polat F, Metin DY, Rigole P, Coenye T, Ilkit M, Pan W, Liao W, Hagen F, Kostrzewa M, Perlin DS, Lass-Flörl C, Boekhout T. 2020. First report of candidemia clonal outbreak caused by emerging fluconazole-resistant Candida parapsilosis isolates harboring Y132F and/or Y132F+K143R in Turkey. Antimicrob Agents Chemother 64:e01001-20. 10.1128/AAC.01001-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Coste AT, Karababa M, Ischer F, Bille J, Sanglard D. 2004. 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 3:1639–1652. 10.1128/EC.3.6.1639-1652.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Morschhäuser J, Barker KS, Liu TT, BlaB-Warmuth J, Homayouni R, Rogers PD. 2007. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 3:e164. 10.1371/journal.ppat.0030164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Neji S, Hadrich I, Trabelsi H, Abbes S, Cheikhrouhou F, Sellami H, Makni F, Ayadi A. 2017. Virulence factors, antifungal susceptibility and molecular mechanisms of azole resistance among Candida parapsilosis complex isolates recovered from clinical specimens. J Biomed Sci 24:67. 10.1186/s12929-017-0376-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Zhang L, Xiao M, Watts MR, Wang H, Fan X, Kong F, Xu YC. 2015. Development of fluconazole resistance in a series of Candida parapsilosis isolates from a persistent candidemia patient with prolonged antifungal therapy. BMC Infect Dis 15:340. 10.1186/s12879-015-1086-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Souza ACR, Fuchs BB, Pinhati HMS, Siqueira RA, Hagen F, Meis JF, Mylonakis E, Colombo AL. 2015. Candida parapsilosis resistance to fluconazole: molecular mechanisms and in vivo impact in infected Galleria mellonella larvae. Antimicrob Agents Chemother 59:6581–6587. 10.1128/AAC.01177-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Magobo RE, Lockhart SR, Govender NP. 2020. Fluconazole-resistant Candida parapsilosis strains with a Y132F substitution in the ERG11 gene causing invasive infections in a neonatal unit, South Africa. Mycoses 63:471–477. 10.1111/myc.13070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Berkow EL, Manigaba K, Parker JE, Barker KS, Kelly SL, Rogers PD. 2015. Multidrug transporters and alterations in sterol biosynthesis contribute to azole antifungal resistance in Candida parapsilosis. Antimicrob Agents Chemother 59:5942–5950. 10.1128/AAC.01358-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Branco J, Silva AP, Silva RM, Silva-Dias A, Pina-Vaz C, Butler G, Rodrigues AG, Miranda IM. 2015. Fluconazole and voriconazole resistance in Candida parapsilosis is conferred by gain-of-function mutations in MRR1 transcription factor gene. Antimicrob Agents Chemother 59:6629–6633. 10.1128/AAC.00842-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Lombardi L, Oliveira-Pacheco J, Butler G. 2019. Plasmid-based CRISPR-Cas9 gene editing in multiple Candida species. mSphere 4:e00125-19. (Author Correction, 53:e00494-20, 2020.) 10.1128/mSphere.00125-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Schubert S, Barker KS, Znaidi S, Schneider S, Dierolf F, Dunkel N, Aïd M, Boucher G, Rogers PD, Raymond M, Morschhäuser J. 2011. Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans. Antimicrob Agents Chemother 55:2212–2223. 10.1128/AAC.01343-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Prasad R, Banerjee A, Khandelwal NK, Dhamgaye S. 2015. The ABCs of Candida albicans multidrug transporter Cdr1. Eukaryot Cell 14:1154–1164. 10.1128/EC.00137-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Rybak JM, Doorley LA, Nishimoto AT, Barker KS, Palmer GE, Rogers PD. 2019. Abrogation of triazole resistance upon deletion of CDR1 in a clinical isolate of Candida auris. Antimicrob Agents Chemother 63:e00057-19. 10.1128/AAC.00057-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Silva AP, Miranda IM, Guida A, Synnott J, Rocha R, Silva R, Amorim A, Pina-Vaz C, Butler G, Rodrigues AG. 2011. Transcriptional profiling of azole-resistant Candida parapsilosis strains. Antimicrob Agents Chemother 55:3546–3556. 10.1128/AAC.01127-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Borgeat V, Brandalise D, Grenouillet F, Sanglard D. 2021. Participation of the ABC transporter CDR1 in azole resistance of Candida lusitaniae. J Fungi 7:760. 10.3390/jof7090760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Biermann AR, Demers EG, Hogan DA. 2021. Mrr1 regulation of methylglyoxal catabolism and methylglyoxal-induced fluconazole resistance in Candida lusitaniae. Mol Microbiol 115:116–130. 10.1111/mmi.14604. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Amberg DC, Burke DJ, Strathern JN. 2006. Preparation of genomic DNA from yeast using glass beads. Cold Spring Harb Protoc 2006:pdb.prot4151. 10.1101/pdb.prot4151. [DOI] [PubMed] [Google Scholar]
- 32.CLSI. 2017. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard 4th ed. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 33.Vyas VK, Barrasa MI, Fink GR. 2015. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1:e1500248. 10.1126/sciadv.1500248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Pfaller MA, Diekema DJ. 2012. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 50:2846–2856. 10.1128/JCM.00937-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Download aac.00289-22-s0001.pdf, PDF file, 0.1 MB (104.8KB, pdf)
Data Availability Statement
Study data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE196409.