ABSTRACT
We determined the echinocandin susceptibility and FKS1 genotypes of 13 clinical isolates of Candida auris that were recovered from 4 patients at a tertiary care center in Salvador, Brazil. Three isolates were categorized as echinocandin-resistant, and they harbored a novel FKS1 mutation that led to an amino acid change W691L located downstream from hot spot 1. When introduced to echinocandin-susceptible C. auris strains by CRISPR/Cas9, Fks1 W691L induced elevated MIC values to all echinocandins (anidulafungin, 16 to 32×; caspofungin, >64×; micafungin, >64×).
KEYWORDS: Candida, Candida auris, FKS1, anidulafungin, antifungal resistance, antifungal susceptibility testing, caspofungin, echinocandin resistance, echinocandins, micafungin
TEXT
Echinocandins are antifungal lipopeptides that noncompetitively inhibit β-1,3-d-glucan synthase, which catalyzes the synthesis of the 1,3-β-glucan polymers of the fungal cell wall. In Candida spp., pharmacodynamic resistance to echinocandin class drugs occurs predominantly when a fungal strain develops mutations in two highly conserved hot spot (HS) regions of the FKS genes that encode the catalytic subunits of β-1,3-d-glucan synthase. In vitro, the presence of amino acid substitutions in Fks subunits manifests as elevated MIC values (1).
Candida auris is an emerging fungal pathogen that exhibits elevated rates of antifungal drug resistance (including resistance to echinocandin class drugs) and causes hospital outbreaks (2). In C. auris, Fks1 HS regions are located at amino acid positions F635 to P643 (HS1), and D1350 to L1357 (HS2), and echinocandin resistance-conferring mutations were reported at several HS positions in clinical isolates (Table 1). Moreover, C. auris FKS1 mutants harboring F635Δ (HS1) and M690I (downstream from HS1) were obtained through the serial in vitro caspofungin exposure of the strain B112020 (clade II, Japan, type strain) (3). Additionally, S639F, S639Y, D642Y, R1354H, and R1354Y FKS1 mutants, in the NCPF 8971 strain background (clade I), were isolated after echinocandin treatment in a murine gastrointestinal colonization model (4).
TABLE 1.
Echinocandin resistance-conferring alterations found in the Fks1 of Candida auris clinical isolates
| Hot spot | Phenotype (Fks1) | Genotype (FKS1) | References | |
|---|---|---|---|---|
| HS1 | WT | F L T L S L R D P | TTCTTGACTTTGTCCTTGAGAGATCCT | Reference gene: B9J08_000964 |
| F635Δ | - L T L S L R D P | - - - TTGACTTTGTCCTTGAGAGATCCT | 15 | |
| F635Y | Y L T L S L R D P | TACTTGACTTTGTCCTTGAGAGATCCT | 16 | |
| F635L | L L T L S L R D P | CaTCTTGACTTTGTCCTTGAGAGATCCT | 16 | |
| S639F | F L T L F L R D P | TTCTTGACTTTGTTCTTGAGAGATCCT | 8, 15–17 | |
| S639P | F L T L P L R D P | TTCTTGACTTTGCCCTTGAGAGATCCT | 17 – 19 | |
| S639T | F L T L T L R D P | TTCTTGACTTTGACCTTGAGAGATCCT | 20 | |
| S639Y | F L T L Y L R D P | TTCTTGACTTTGTACTTGAGAGATCCT | 19, 21 | |
| D642Y | F L T L S L R Y P | TTCTTGACTTTGTCCTTGAGATATCCT | 17 | |
| HS2 | WT | D W I R R Y T L | GACTGGATTAGACGTTATACCTTG | Reference gene: B9J08_000964 |
| R1354S | D W I R S Y T L | GACTGGATTAGAAGTTATACCTTG | 16 | |
| R1354H | D W I R H Y T L | GACTGGATTAGACATTATACCTTG | 17 |
Bold, modified amino acids or nucleotides; underlined, codons where modification occured.
Here, 13 isolates of C. auris (B61 to B73; clade I) that were recovered from 4 patients (A, n = 4; B, n = 7; C, n = 1; X, n = 1) at a tertiary care center in Salvador, Brazil, were evaluated for their echinocandin susceptibility and FKS1 sequences. We performed antifungal susceptibility testing (AFST) with echinocandins (ANF, anidulafungin; CAS, caspofungin; MCF, micafungin), according to the CLSI methodology (5, 6), using C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 as quality control strains. The MICs were interpreted by using tentative breakpoints, as suggested by the CDC (7). The full FKS1 gene was amplified by PCR and sequenced by Sanger sequencing, and its sequence was analyzed (reference gene: B9J08_000964), as we described previously (8). 3 isolates that were recovered from the urine of patient A were categorized as echinocandin-resistant (ANF, 2 to 16 mg/L; CAS, >16 mg/L; MCF, >16 mg/L) and harbored a novel FKS1 mutation, G2072T, which led to an amino acid change, namely, W691L (TGG → TTG). The remaining 10 isolates were echinocandin-susceptible (ANF MIC, 0.125 to 0.25 mg/L; CAS MIC, 0.125 to 0.25 mg/L; MCF MIC, 0.125 to 0.25 mg/L) and presented a WT FKS1 genotype (Table 2). The full Fks1 sequences of clinical isolates B61 to B73 were deposited into the NCBI GenBank under the accession numbers OQ632632 to OQ632644.
TABLE 2.
Antifungal drug susceptibility and Fks1 phenotypes of clinical isolates, parental strains, and transformants
| Clinical isolate |
Patient | Specimen | Fks1 | GenBank accession number | MIC [mg/L] |
|||
|---|---|---|---|---|---|---|---|---|
| Study number | Strain code | ANF | CAS | MCF | ||||
| B61 | 1923/2021 | A | Urine | W691L | OQ632632 | 2 | >16 | >16 |
| B62 | 1985/2021 | A | Urine | W691L | OQ632633 | 16 | >16 | >16 |
| B63 | 1854/2021 | B | Urine | WT | OQ632634 | 0.125 | 0.125 | 0.125 |
| B64 | 1858/2021 | B | CVC tip | WT | OQ632635 | 0.125 | 0.125 | 0.125 |
| B65 | 2075/2021 | B | CVC tip | WT | OQ632636 | 0.25 | 0.25 | 0.25 |
| B66 | 2076/2021 | B | Blood | WT | OQ632637 | 0.25 | 0.25 | 0.25 |
| B67 | 2077/2021 | B | CVC tip | WT | OQ632638 | 0.125 | 0.125 | 0.125 |
| B68 | 1537/2020 | X | CVC tip | WT | OQ632639 | 0.125 | 0.125 | 0.125 |
| B69 | 1982/2021 | A | CVC tip | WT | OQ632640 | 0.25 | 0.25 | 0.125 |
| B70 | 1980/2021 | A | Urine | W691L | OQ632641 | 2 | >16 | >16 |
| B71 | 1861/2021 | C | NA | WT | OQ632642 | 0.25 | 0.25 | 0.25 |
| B72 | 1921/2021 | B | CVC tip | WT | OQ632643 | 0.125 | 0.125 | 0.125 |
| B73 | 1922/2021 | B | CVC tip | WT | OQ632644 | 0.125 | 0.125 | 0.125 |
| DPL1384 (clinical isolate, clade I) | WT | 0.125 | 0.5 | 0.25 | ||||
| DPL1384 + B61 FKS1 (7 independent transformants) | W691L | 2 to 4 | >16 | >16 | ||||
| CBS 10913 (type strain, clade II) | WT | 0.06 | 0.25 | 0.125 | ||||
| CBS 10913 + B61 FKS1 (3 independent transformants) | W691L | 2 | >16 | >16 | ||||
W691 in the Fks1 of C. auris is located 48 amino acids downstream from the FKS1 HS1, and it is equivalent to W695 and W715 in the Fks1 of Saccharomyces cerevisiae and the Fks2 of C. glabrata, respectively (9, 10). According to Johnson and Edlind, the Fks1 W695 in S. cereviasiae is located in hot spot 3 (amino acids 690 to 700), which is predicted to be embedded within the outer leaflet of the membrane and may interact with the extended aliphatic tails of echinocandins (9). In C. glabrata, a W715L amino acid substitution in Fks2 was shown to lead to elevated echinocandin MIC values (10, 11).
To confirm that the Fks1 W691L variant confers echinocandin resistance in C. auris, a wild-type (WT) FKS1 gene fragment in echinocandin-susceptible C. auris DPL1384 (clinical isolate, clade I) and CBS 10913 (type strain, clade II) was replaced with a 465 bp FKS1 fragment (C1899 to C2364) harboring W691L (amplified from isolate B61) by using a CRISPR/Cas9 system, as described previously (12), except that the cells were made electrocompetent with a Frozen-EZ Yeast Transformation Kit (Zymo Research, Irvine, CA, USA) (13). The repair template was PCR-generated with Q5 High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA) and Cau_FKS1_CRISPR_F1 (5′-CTTCTTCTTGACTTTGTCCTTGAGAGATCCT ATTAGAAACTTGTCCACC-3′), and Cau_FKS1_CRISPR_R1 (5′-GGTGCTCTCAAAGTTCTCTTGCCCTCGATTTCAGAAGGAACCTGGTGATAC-3′) primers. The crRNA target sequence was CGATTTCAGAAGGAACCTGGTGG, and the Cau_FKS1_CRISPR_R1 primer introduced a modified PAM site (without an amino acid change) into the PCR product to prevent the Cas9 digestion of the repair template. The transformants were selected on YPD plates containing 4 mg/L MCF. We previously used a similar selection approach to introduce the non-hot spot modification E655K in the Fks2 of C. glabrata (13). The correctness of the transformation was validated by PCR and sequencing of the entire FKS1 gene (8). After that, the echinocandin MIC values of the correct transformants were determined according to the CLSI methodology (5, 6). When introduced to echinocandin-susceptible strains, FKS1 W691L induced elevated MIC values to all echinocandins (ANF, 16 to 32×; CAS, >64×; MCF, >64×) (Table 2).
The interactions between Fks1 enzyme variants and echinocandin drugs are not well-understood. However, a recent study presenting the cryo-electron microscopy structures of the Fks1 of Saccharomyces cerevisiae revealed active enzyme-membrane interactions and altered lipid arrangements around the mutated enzymes (14). Although limited in its scope (only two mutated Fks1 positions were analyzed) the results provide insights into possible binding sites for echinocandin drugs and improve our understanding of why certain enzyme regions (hot spots versus non-hot spots) have the propensity to impact echinocandin drug susceptibilities.
In conclusion, we report Fks1 W691L as a novel, echinocandin resistance-conferring, non-hot spot modification in C. auris. Our discovery emphasizes that, despite the significant progress that has been made in recent years in understanding echinocandin resistance in C. auris, the diversity of the FKS1 mutations that contribute to C. auris echinocandin resistance may be underestimated. Ultimately, improving the knowledge of the resistance mechanism will help diagnose and treat C. auris-infected patients.
ACKNOWLEDGMENTS
Partial results of this study were presented at ASM Microbe 2022 in Washington, D.C.
The study received Institutional Review Board approval (CEP8260200319).
J.N.A.J. and A.L.C. were awarded research grants (J.N.A.J., FAPESP 2018/19347; A.L.C., FAPESP 2017/02203-7) by Fundação de Amparo à Pesquisa do Estado de São Paulo. A.L.C. serves on the advisory boards and educational programs of Amgen, Ache, Biotoscana-United Medical, Eurofarma, Mundipharma, Gilead, and Pfizer. D.S.P. receives funding from the U.S. National Institutes of Health (NIH) and contracts with Merck, Regeneron, and Pfizer. He serves on advisory boards for N8 Medical and Scynexis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
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