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Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: Emergence of clinically important mutations in sphingolipid biosynthesis
Sphingolipid biosynthesis mutations emerge in gut-colonizing C. glabrata during caspofungin therapy
Hassoun Yasmine Investigation Methodology Writing – original draft 1
Aptekmann Ariel A. Data curation Formal analysis Methodology 1
Keniya Mikhail V. Investigation Methodology Validation 1
Gomez Rosa Y. Investigation 1
Alayo Nicole Investigation 1
Novi Giovanna Investigation 1
Quinteros Christopher Investigation 1
Kaya Firat Investigation Methodology 1
Zimmerman Matthew Investigation Methodology Supervision 1
Caceres Diego H. Data curation 2 3 4
Chow Nancy A. Data curation 2
Perlin David S. Funding acquisition Supervision Writing – review & editing 1 5 6 *
https://orcid.org/0000-0003-3655-0963
Shor Erika Conceptualization Funding acquisition Supervision Writing – original draft Writing – review & editing 1 5 *
1 Hackensack Meridian Health Center for Discovery and Innovation, Nutley, New Jersey, United States of America
2 Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
3 Center of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands
4 Studies in Translational Microbiology and Emerging Diseases (MICROS) Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Bogota, Colombia
5 Hackensack Meridian School of Medicine, Nutley, New Jersey, United States of America
6 Georgetown University Lombardi Comprehensive Cancer Center, Washington, D.C., United States of America
Konopka James B. Editor
Stony Brook University, UNITED STATES OF AMERICA
DSP received an honorarium from N8 Biomedical, a mutual fund with more than $5,000 Merck stock, and an unlicensed patent for echinocandin resistance.

* E-mail: david.perlin@hmh-cdi.org (DSP); erika.shor@hmh-cdi.org (ES)
9 9 2024
9 2024
20 9 e101252120 11 2023
19 8 2024
https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting β-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.

Author summary

Invasive fungal infections cause high mortality due to our limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal class with increasing resistance in Candida glabrata, an opportunistic pathogen colonizing host mucosal surfaces. It is known that echinocandin-resistant C. glabrata isolates emerge from drug-sensitive gut-colonizing strains, but there are large knowledge gaps regarding how this resistance develops and whether it involves mutations in genes other than those encoding the echinocandin drug target. We studied the evolutionary dynamics in C. glabrata colonizing the mouse gut during treatment with caspofungin, a widely used echinocandin. In addition to well-described drug target mutations, we identified mutations in sphingolipid biosynthesis gene FEN1 rapidly and frequently emerging in the gut-colonizing fungus during caspofungin therapy. We also identified multiple loss-of-function fen1 mutations in clinical C. glabrata isolates and showed that they contribute to reduced caspofungin sensitivity of these strains. Together, this work illuminates the rich evolutionary dynamics of gut-colonizing fungi during antifungal therapy and identifies a new genetic determinant of reduced clinical caspofungin susceptibility.

http://dx.doi.org/10.13039/100000060 National Institute of Allergy and Infectious Diseases 5R01AI109025 Perlin David S. This work was supported by grant 5R01AI109025 from the National Institute for Allergy and Infectious Diseases to DSP and ES. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS Publication Stagevor-update-to-uncorrected-proof
Publication Update2024-09-19
Data AvailabilityAll next generation sequencing data associated with this study have been deposited at NCBI under accession number PRJNA1010358. The link to the dataset is https://www.ncbi.nlm.nih.gov/bioproject/1010358.
Data Availability

All next generation sequencing data associated with this study have been deposited at NCBI under accession number PRJNA1010358. The link to the dataset is https://www.ncbi.nlm.nih.gov/bioproject/1010358.
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pmcIntroduction

Invasive fungal infections cause a significant health burden worldwide, with associated mortality equaling or surpassing that of malaria and TB [1–4]. Invasive Candida infections are associated with a mortality of >40% even with antifungal treatment [3,5,6]. The epidemiology of invasive candidiasis has been changing globally, with formerly predominant C. albicans decreasing in prevalence, while non-albicans Candida species, such as C. glabrata (recently reclassified as Nakaseomyces glabratus), C. parapsilosis, and C. tropicalis now account for ever greater fractions of invasive Candida infections [5,7,8]. Alarmingly, the non-albicans Candida species are characterized by reduced susceptibility to the azole antifungal drug class, which has been a global staple of antifungal therapy for several decades [9]. It has been proposed that the extensive use of azoles, both in medicine and in agriculture, has driven this epidemiological shift [10]. Due to this changing epidemiology, the other frontline antifungal drug class, the echinocandins, is becoming the treatment of choice for invasive candidiasis. Echinocandins have an excellent safety profile and are highly efficacious against Candida infections [11,12]. However, as echinocandins have been used clinically for over 20 years, resistance to this antifungal drug is also rising, most prominently in C. glabrata [13–18], where many cases of rapid emergence of echinocandin resistance during therapy have been documented [19–24].

Echinocandins, which are fungicidal in Candida, target the biosynthesis of the fungal cell wall by inhibiting the enzyme β-glucan synthase [25]. The best understood echinocandin resistance mechanism is via mutations in genes encoding β-glucan synthase (FKS1 and FKS2 in C. glabrata) that alter the structure of the enzyme, rendering it less susceptible to inhibition [14,26,27]. While fks mutations have been firmly linked to C. glabrata echinocandin resistance leading to clinical failure, there is also evidence for a contribution of additional, as yet unidentified genetic mechanisms. For instance, in many epidemiological studies a significant proportion (up to 40%) of resistant C. glabrata isolates do not contain fks mutations [16–18,28]. Furthermore, although the mechanism of action of different echinocandins (caspofungin, micafungin, and anidulafungin) is thought to be identical, it is not uncommon for C. glabrata strains to show resistance to one or two but not all three of these drugs [15–18,29]. Thus, it is clear that there exist additional, as yet unknown, important determinants of susceptibility of C. glabrata to all echinocandins as well as to individual drugs of this class.

A number of studies have attempted to elucidate the genetic underpinnings of echinocandin resistance in C. glabrata by comparing whole genome sequences of resistant and susceptible clinical isolates [24,30–34]. This approach is complicated by the high genetic diversity of C. glabrata, where thousands or even tens of thousands of SNPs and indels can separate different clinical isolates [35]. Thus, genome comparisons typically identify many putative genetic changes regulating echinocandin sensitivity, but validating the role of each individual SNP is extremely labor-intensive and usually not carried out, nor are the mechanisms by which these SNPs may alter echinocandin sensitivity elucidated. In another approach to identify the missing genetic basis for echinocandin resistance, in vitro evolution has been used to show that C. glabrata passaged in rich laboratory medium containing increasing concentrations of anidulafungin acquires mutations in FKS1 and FKS2 as well as mutations in ergosterol biosynthesis gene ERG3, implicating the latter in echinocandin resistance via as yet unknown mechanisms [36]. However, it is not yet clear how relevant this narrow mutational signature of resistance is to the mutations driving the evolution of echinocandin resistance in the context of the host. Importantly, the gastrointestinal (GI) tract and the abdominal cavity were identified as key host reservoirs in which C. glabrata drug resistance emerges [37–39] and in vitro growth in rich medium may not recapitulate the evolutionary dynamics of C. glabrata existing within these host niches.

In this study, we examined the evolution of resistance to the echinocandin caspofungin in C. glabrata colonizing the mouse gut. We found that both fks and non-fks mutations arose in GI-colonizing C. glabrata during treatment with caspofungin. The majority of non-fks mutations were in the FEN1 gene, which encodes a fatty acid synthase and functions in sphingolipid biosynthesis. Further analysis demonstrated that fen1 mutants have a fitness advantage in the gut specifically during caspofungin treatment, that these mutants accumulate phytosphingosine (PHS, a sphingolipid biosynthesis intermediate), and that genetic depletion of PHS by deletion of alkaline ceramidase YPC1 sensitizes both the wild type strain and the fen1 mutant to caspofungin. Interestingly, in-depth analysis of the emergence and abundance of both drug target (fks) and non-drug target (fen1) mutations during caspofungin treatment by amplicon sequencing revealed a rapid emergence and diversification of caspofungin-adapted strains in the GI tract. Importantly, we also identified three different point mutations in FEN1 in C. glabrata clinical isolates and showed that all of these clinical fen1 mutations cause loss of protein function and reduced susceptibility to caspofungin. In summary, our study uncovered mutations in FEN1 as a novel genetic mechanism reducing the susceptibility of C. glabrata to the widely used echinocandin caspofungin in the clinical setting and showed that this effect is due to an increase in the membrane level of PHS. Beyond its clinical implications, our work also sheds light on the complex evolutionary dynamics of C. glabrata within the host GI reservoir, comprising both drug target and non-target mutations, which collectively undermine the effectiveness of antifungal treatment in patients colonized with C. glabrata.

Results

Non-drug target mutations arise in C. glabrata colonizing the gut during caspofungin treatment

In a previous study, which focused on the role of cell wall integrity pathways in echinocandin tolerance, we examined the evolution of echinocandin resistance during caspofungin (CSF) treatment in the mouse gut C. glabrata colonization model [40]. Approximately half of the evolved C. glabrata strains showed increased MICs for both CSF and micafungin and had FKS2 hot-spot mutations, but the rest of the strains showed increased MICs for CSF only and lacked mutations in either FKS1 or FKS2 [40]. We also observed the development of such non-fks mutants in the mouse gut during CSF treatment of mice colonized with C. glabrata strains lacking error-prone DNA polymerases Rev1 and Rev3. To identify the causes of reduced CSF susceptibility in these strains, we sequenced the genomes of seven non-fks mutants with increased CSF MICs, as well as four strains with FKS2 hot-spot mutations that had also evolved in the mouse gut during CSF treatment (Fig 1A and S1 Table). Interestingly, we found that six out of 11 sequenced strains–five strains without fks2 mutations and one strain with an fks2 mutation–contained mutations in the FEN1 gene (ELO2 in Saccharomyces cerevisiae) (Fig 1A). FEN1 encodes a fatty acid elongase that functions in sphingolipid biosynthesis [41,42] (Fig 1B). This finding was consistent with previous reports that mutations in genes of the sphingolipid biosynthesis pathway, including FEN1, reduce C. glabrata and C. albicans sensitivity to CSF [43,44]. Also consistent with those reports, the fen1 mutant strains had increased susceptibility to micafungin (MCF), as well as increased or unaltered susceptibility to anidulafungin (ANF) (Fig 1A).

10.1371/journal.ppat.1012521.g001 Fig 1 Mutations in fatty acid elongase Fen1, which functions in sphingolipid biosynthesis, frequently evolved in gut-colonizing C. glabrata during caspofungin treatment.

A. Genetic and echinocandin susceptibility information for 11 gut-evolved mutants. All parental strains used to colonize the mice belonged to the DPL1021 (ATCC90030) background. Some of the parental strains carried the gene deletions shown in the “Genotype” column; “WT” indicates the absence of such gene deletions. These gene deletions had been made as part of our investigation of the roles of the cell wall integrity pathway (slt2Δ and yps1Δ [40]) and error-prone DNA replication (rev1Δ and rev3Δ) in the evolution of CSF-resistant mutants in gut-colonizing C. glabrata. B. Diagram of the yeast sphingolipid biosynthesis pathway. FEN1, which encodes a fatty acid elongase, is outlined in red. * = stop codon; CSF = caspofungin; MCF = micafungin; ANF = anidulafungin.

All gut-evolved strains also contained multiple other coding SNPs relative to the parental strain DPL1021 (ATCC90030) and each other, suggesting that these SNPs had arisen during evolution in the mouse gut (S1 Table). Among genes previously implicated in echinocandin resistance, GI-63 carried a mutation in the ERG3 gene, which encodes an enzyme involved in the biosynthesis of ergosterol [45]. Mutations in ERG3 gene can lead to alterations in sterol composition and confer resistance to azoles, as well as echinocandins [45–47] and have also been reported to arise during in vitro evolution of echinocandin resistance [36].

Deletion or mutation of FEN1 reduces CSF susceptibility in C. glabrata colonizing the gut but not C. glabrata infecting the kidney

To directly analyze the role of FEN1 during host colonization and infection, we deleted the FEN1 ORF and colonized mice with fen1Δ and fen1-W145* (GI-240, Fig 1A). We found that daily CSF treatment (20 mg/kg) resulted in a stronger reduction in fungal burdens in mice colonized with the wild type strain than in mice colonized with the fen1Δ or fen1-W145* mutants (Fig 2A). We also directly compared in vivo fitness of the wild type strain with that of a fen1 mutant by colonizing the mouse gut with a mix of both strains and following each strain’s abundance over seven days, with or without daily CSF treatment. We found that whereas in the absence of CSF treatment, the wild type strain was significantly more fit than either fen1Δ or fen1-W145* strains, in the presence of CSF treatment the two fen1 mutants were more fit, fully taking over the gut population by day 5 (Fig 2B). This result was consistent with our initial observation that fen1 mutants were selected in the gut during CSF treatment and confirmed that loss of Fen1 function improved C. glabrata fitness in the presence of CSF.

10.1371/journal.ppat.1012521.g002 Fig 2 Deletion or mutation of FEN1 improves fitness of gut-colonizing C. glabrata during caspofungin (CSF) treatment.

A. Gut-colonizing fen1 mutants survive during CSF therapy better than the isogenic wild type strain. The mice were colonized with the indicated C. glabrata strains and treated daily with CSF (20 mg/kg) starting at day 3 post-colonization. Fecal samples were obtained every other day and plated to obtain GI fungal CFU counts. Multivariate Analysis of Variance (MANOVA) was used to assess the relationship between treatments over time on gut microbiota with a significant threshold set at 0.05. Tukey’s Post Hoc analysis was performed to look for pairwise significance. “Fungal burden depletion” refers to C. glabrata gut levels decreasing to undetectable levels during CSF treatment and not rebounding during the timeframe of the experiment. B. In direct competition experiments, gut-colonizing fen1 mutants outcompete the wild type strain specifically during CSF treatment. The mice were colonized with an equal mix of two strains: wild type and an isogenic fen1 mutant (fen1Δ or fen1-W145*). Starting at day 1 post-colonization, the mice were treated daily with CSF (20 mg/kg). Fecal samples were plated every other day, and the resulting colonies were analyzed for their FEN1 status (wild type or mutant). To identify fen1Δ colonies, the plates were replica-plated on hygromycin-containing medium. To identify fen1-W145* colonies, a specific molecular beacon was designed and used in colony PCR (see Materials and Methods). C. In a model of systemic infection, the fen1Δ mutant was as sensitive to CSF treatment as the wild type strain. Neutropenic mice were infected via the retro-orbital route with the indicated strains of C. glabrata, and treatment with CSF (1.25, 2.5 or 5 mg/kg) or vehicle (PBS) was initiated at day 1 post-infection. The kidneys were harvested on day 3, homogenized and plated on YPD for CFU counts. Each data point corresponds to a single mouse. *** p < 0.001 (ordinary one-way ANOVA).

We also analyzed the effect of fen1 mutation on C. glabrata CSF susceptibility in the context of a bloodstream infection. Mice were immunosuppressed and infected via the retro-orbital route with either wild type, fen1Δ, or fks2-S663P mutant strains, the latter of which is a prominent cause of clinical echinocandin resistance and served as a control. Kidney fungal burdens were assessed as a measure of CSF efficacy as described before [48]. We used a range of CSF doses (1.25, 2.5, and 5 mg/kg), the highest of which corresponds to the humanized dose. Consistent with the documented resistance of the fks2-S663P mutant [26], we found that its burden in the kidney was not reduced as much as those of the wild type strain at all three concentrations, with the difference being statistically significant at 5 mg/kg (Fig 2C). In contrast, there was no difference in kidney burdens between the fen1Δ and the wild type strain at any of the caspofungin concentrations (Fig 2C), showing that in the context of bloodstream infection, unlike during gut colonization, the fen1Δ mutant remained susceptible to CSF.

Deletion of FEN1 results in reduced caspofungin association with the fungal plasma membrane

Sphingolipid biosynthesis has been previously implicated in regulating fungal susceptibility in vitro to CSF but not to other echinocandins [43,44]. However, the molecular basis for the reduced CSF susceptibility of sphingolipid mutants, such as fen1Δ, is not known. It has been previously proposed that the fatty acid-like tail of CSF (Fig 3A), which is not present in other echinocandins, may modulate its interaction with the plasma membrane and membrane-bound glucan synthase [43]. To test this hypothesis, we first analyzed the CSF-plasma membrane interaction using fluorescent CSF. This compound (custom synthesized and purified at WuXi Biologicals, China) was obtained by reacting a free amine group on CSF with Alexa Fluor 647 (AF647) modified with an amine-reactive NHS ester (Fig 3A). The WT and fen1Δ strains were incubated with CSF-AF647 and analyzed by high resolution imaging (ONi Nanoimager). The imaging showed that CSF-AF647 showed a strong association with the cellular periphery in the WT cells (Fig 3B), consistent with its association with the plasma membrane. Importantly, this association was fully dependent on the CSF moiety of the molecule, as AF647 alone (in which the amino-reactive ester had been quenched with TRIS) did not bind to C. glabrata cells (S1C Fig). Interestingly, the binding of CSF-AF647 to the fungal cell surface was significantly reduced in the fen1Δ cells, and this reduction observed when FEN1 was deleted in strains CBS138 (Fig 3B) and ATCC90030 (S1A Fig), showing that it was independent of C. glabrata strain background. Thus, these observations suggested that a key difference between WT and fen1Δ cells is a decreased association between CSF and the plasma membrane.

10.1371/journal.ppat.1012521.g003 Fig 3 Deletion of FEN1 reduces caspofungin (CSF) binding to C. glabrata cells.

A. Alexa Fluor 647 was conjugated to the indicated primary amine group of CSF (red circle). The synthesis was carried out at WuXi Biologics (China). The product was confirmed by nuclear magnetic resonance and high-resolution mass spectrometry and determined to be 98.67% pure. CSF’s fatty acid-like tail is indicated by the dashed blue box. B. High-resolution imaging showed that CSF-AF647 strongly associated with the fungal cell surface in WT cells, but that this association was greatly reduced in the fen1Δ mutant. C. CSF interaction with cellular membranes was significantly reduced in the fen1Δ mutant relative to the WT strain. Cells were incubated with CSF, disrupted, and crude membranes were extracted and analyzed by LC-MS/MS. The detected CSF levels were internally normalized to each sample’s protein levels. Each data point corresponds to a single biological replicate. D. Protein composition of the crude membrane preparations (Coomassie-stained 8% TRIS-Glycine SDS PAGE) was unaltered by fen1Δ. BF = bright-field; AF = Alexa Fluor. *** p<0.001 (two-tailed unpaired T-test).

Unfortunately, the addition of AF647 significantly reduced the antifungal activity of CSF-AF647 relative to CSF: the MIC of CSF-AF647 was ~0.85% of that of CSF in both WT and fen1Δ strains (S1B Fig). Furthermore, our mass spectrometry (MS) analysis of the CSF-AF647 preparation showed that it contained 0.6% unlabeled CSF, which may have accounted for some of the observed antifungal activity of CSF-AF647. Thus, the reduced association of CSF with plasma membrane in the fen1Δ mutant needed to be verified using a different approach not reliant on the CSF-AF647 conjugate. To this end, we used MS to directly measure the binding of unlabeled CSF to cellular membranes in WT and fen1Δ strains. C. glabrata cells were incubated with 0.25 μg/ml CSF for 20 minutes, followed by immediate cell harvesting and membrane extraction. 10% the membrane preparation was saved for protein quantification, and the rest was analyzed by LC-MS/MS. The detected CSF levels were internally normalized to protein levels in the same sample. Consistent with the imaging data (Fig 3B), we observed a 50% reduction in CSF association with the membranes derived from the fen1Δ mutant relative to the membranes derived from the WT strain (Fig 3C). On the other hand, the membrane protein band patterns of the WT and fen1Δ strains were very similar (Fig 3D), consistent with the conclusion that the reduced CSF association in the fen1Δ mutant was likely due to its altered membrane lipid composition. In summary, both imaging and MS-based analyses have shown that the interaction of CSF with the plasma membrane is significantly reduced in the fen1Δ mutant, potentially explaining the reduced sensitivity of this mutant to CSF.

Manipulating phytosphingosine metabolism can sensitize C. glabrata to CSF

Previous studies have shown that in C. albicans some sphingolipid biosynthesis mutants accumulated phytosphingosine (PHS), a sphingolipid biosynthesis intermediate [44], and that increased PHS can render GS less susceptible to inhibition by echinocandins [49,50]. Thus, we performed a sphingolipid-focused lipidomic analysis of two fen1 mutants (fen1Δ and GI-299) and one non-fen1 gut-evolved strain (GI-113) both in the absence and presence of 1 μg/ml CSF. The results showed that all three gut evolved mutants (both fen1 and non-fen1) had elevated levels of PHS, which were increased further during CSF exposure (Fig 4A and S2 Table). To further probe the importance of PHS accumulation in regulating CSF susceptibility, we deleted YPC1, the gene encoding alkaline ceramidase, which in S. cerevisiae catalyzes the conversion of phytoceramide to PHS [51,52] (Fig 1B). Interestingly, we observed that ypc1Δ had the opposite effect of fen1Δ, hypersensitizing C. glabrata to CSF, which was evident on agar plates containing CSF (Fig 4B). Furthermore, ypc1Δ was epistatic to fen1Δ, as the double mutant was as sensitive as the ypc1Δ alone (Fig 4B). Consistent with these results, ypc1Δ also restored CSF-AF647 association with the fungal cells in the fen1Δ mutant (Fig 4C). We also attempted to delete YPC1 in gut-evolved strain GI-113, but despite multiple attempts, no deletants were recovered, suggesting a possible synthetic lethality with one of the mutations in that strain (S1 Table). Together, these results show that accumulation of PHS is associated with reduced CSF sensitivity and reduced CSF binding to the plasma membrane, whereas genetic depletion of PHS, conversely, sensitizes C. glabrata to CSF and enhances CSF association with the plasma membrane.

10.1371/journal.ppat.1012521.g004 Fig 4 Manipulating phytosphingosine levels can sensitize C. glabrata to caspofungin (CSF).

A. Lipidomic analysis showed that phytosphingosine (PHS, red arrowhead) levels are increased in gut-evolved CSF-adapted C. glabrata mutants. The indicated strains were either left untreated (No CSF) or exposed to CSF (0.5 μg/ml) for one hour. The samples were analyzed at the Lipidomics Shared Resource at the Medical University of South Carolina. All samples were normalized against inorganic phosphate and the untreated WT control. B. Deletion of alkaline ceramidase YPC1, which breaks down phytoceramide to produce PHS [51, 52], sensitized wild type and fen1Δ C. glabrata to CSF. This sensitization was evident when C. glabrata was grown on solid CSF-containing YPD medium (0.125 μg/ml and 0.25 μg/ml CSF) but not in liquid RPMI, where ypc1Δ did not alter the CSF MIC. Both growth on solid medium and the MIC measurement showed that ypc1Δ was epistatic to fen1Δ, sensitizing it to CSF. MCF = micafungin; ANF = anidulafungin C. Deletion of YPC1 also restored CSF binding to the fungal cell surface in the fen1Δ strain. BF = bright-field; AF = Alexa Fluor.

Evolutionary dynamics of fen1 and fks mutations in gut-colonizing C. glabrata during CSF treatment

To check whether the results obtained for the strains chosen for whole genome sequencing (Fig 1A) were representative of general C. glabrata evolution in the gut (i.e., that fen1 mutations arose frequently in this population during caspofungin treatment), we used yeast samples obtained and stocked during previous gut colonization experiments (Fig 5A). Because this analysis was done post hoc, it was done on a limited number of colonies: on those occasions, two colonies per mouse per day had been cultured, FKS1 and FKS2 hot spots sequenced, and frozen stocks made. We now sequenced FEN1 as well (Fig 5A). This analysis confirmed that a variety of fen1 mutations had arisen in the different mice, including two different fen1 mutations co-existing in mouse 4 on day 7. Interestingly, in mice 1, 4, and 5 where both fen1 and fks2 mutations were observed, the fen1 mutations had appeared earlier than fks2 mutations (Fig 5A; no fks1 mutations had been detected). In mice 2, 3 and 6 only fen1 mutations were detected, and these also appeared early–on days 7 and 9, i.e., 4 and 6 days after the initiation of CSF treatment. These results suggested that the gut-colonizing population of C. glabrata during CSF treatment is highly dynamic and diverse, rapidly acquiring both drug target and non-drug target mutations that improve fitness in the presence of CSF.

10.1371/journal.ppat.1012521.g005 Fig 5 Dynamics of drug target (fks) and non-drug target (fen1) mutations arising in gut-colonizing C. glabrata during caspofungin (CSF) treatment.

A. Sanger sequencing of FKS1 and FKS2 hot spots and FEN1 ORF in individual colonies (two per mouse per day) derived from gut-colonizing C. glabrata reveals the wide variety of fen1 mutations, some detected as early as day 7 (i.e., 4 days after initiation of CSF treatment). No FKS1 hot spot mutations were found. B. Amplicon sequencing of FKS1 and FKS2 hot spots and FEN1 ORF revealed the time of appearance and relative abundance of fks1, fks2, and fen1 mutant alleles in gut-colonizing C. glabrata during CSF treatment. Fraction of reads containing the indicated allele relative to all reads containing that position was used as a proxy for the relative abundance of the mutant allele in the gut-colonizing C. glabrata population. Parts of Panels A and B of this figure were made using Biorender.com (lab license).

To gain an even deeper insight into the timing and spread of FKS and FEN1 mutations in C. glabrata colonizing the gut during CSF treatment, we used amplicon sequencing (Fig 5B). Four mice were colonized with C. glabrata and treated with CSF as in our previous studies [37, 40] (S2A Fig). Feces were collected on the indicated days and plated on YPD agar plates supplemented with antibiotics, as before, but instead of picking individual yeast colonies, ~1000 colonies were pooled for DNA isolation, followed by amplicon PCR and next generation sequencing. The resulting reads were mapped to the amplicon sequences to identify SNPs appearing in the gut-colonizing C. glabrata (Fig 5B). First, we observed that the original inoculum contained several SNPs in FKS1 HS1 present at low frequencies (S2B Fig), suggesting that these mutations arose in the C. glabrata culture used for mouse oral gavage. These mutations tended to persist in the mouse gut at similar frequencies throughout the two-week CSF treatment, indicating that they did not affect fitness under these conditions (S2B Fig). In contrast, mutations in FEN1 and FKS2 appeared and rapidly increased in abundance in mice 1, 3 and 4 (Fig 5B), indicating that they increased fitness. The earliest fen1 mutation (W39*), in mouse 3, was detected on day 7, whereas the earliest fks2 mutation (R1378H), in mouse 1, was detected on day 9 (and in this mouse no fen1 mutation was ever detected). Interestingly, although both of these mutations arose relatively early during the treatment, neither of them swept the population but remained at a <50% abundance (Fig 5B), suggesting that they co-existed with other fitness-promoting mutations that had arisen in other cells. Furthermore, the fen1-W39* mutation in mouse 3 started decreasing in abundance by day 13 and was undetected on day 15, at which point the fks2-F659I mutation, which is associated with clinical echinocandin resistance [53], had appeared in the same population (Fig 5B). Finally, although a few FKS1 HS mutations had appeared in mice 3 and 4, none of them significantly increased in abundance or were detected on multiple days, indicating that they had marginal effects on fitness during CSF treatment. Thus, this experiment confirmed that GI-colonizing C. glabrata was highly genetically dynamic during CSF treatment, with caspofungin-adapted mutations promoting fitness arising, increasing in relative abundance, and co-existing with other such mutations.

Clinical C. glabrata isolates contain loss-of-function fen1 mutations that cause reduced susceptibility to CSF

To investigate whether fen1 mutations also occur in C. glabrata isolates infecting humans, we used two approaches: mining publicly available genomes and sequencing FEN1 in strains from our lab collection. We used two sources of clinical isolate genome data: those in Biswas et al. (52 strains) [30] and those collected by the Centers for Disease Control (CDC) as part of the Emerging Infections Program (EIP) for candidemia surveillance and made publicly available on NCBI [67]. Because strains of different sequence types (ST) are expected to contain ST-specific polymorphisms in FEN1, we also determined the ST of each strain, either from the whole genome sequence or by Sanger sequencing using the standard C. glabrata multi-locus sequence typing scheme [54]. These analyses identified three clinical C. glabrata isolates carrying unique mutations in FEN1 that were not ST-associated polymorphisms: G143V in strain WM_18.26 [30], Y298* in strain DPL239 (Perlin lab collection), and G52A in strain CAS14-5989 (CDC collection) (Fig 6A and S3 and S4 Tables). WM_18.26 also had the fks2-S663P mutation and a high reported CSF MIC (16 μg/ml), while CAS14-5989 also contained the fks1-S595T mutation but a low CSF MIC (0.03 μg/ml, according to CDC records) (Fig 6A). DPL239 with fen1-Y298* contained wild type FKS1 and FKS2 sequences but an elevated CSF MIC (1 μg/ml). The combination of fen1-G143V with fks2-S663P in the genome of a differently named clinical isolate (CMRL4) has previously been reported [34]. Because both WM_18.26 and CMRL4 were originally reported by the same group within one year of each other [30,55], it is likely that they are either the same strain or two closely related strains. Interestingly, the CMRL4 strain was obtained from a patient who had had 30 days of CSF treatment [55], consistent with emergence of the fen1-G143V mutation during CSF therapy. No history of antifungal treatment was available for strains DPL239 and CAS14-5989, but because the latter also contained an fks1 hot-spot mutation, it may be hypothesized that the fen1 and fks1 mutations may have arisen under a similar environmental pressure.

10.1371/journal.ppat.1012521.g006 Fig 6 Clinical C. glabrata mutations in FEN1 result in loss of protein function and reduced susceptibility to caspofungin (CSF).

A. Analysis of publicly available C. glabrata genomes and Sanger sequencing of FEN1 in the Perlin lab strain collection identified three unique (non-ST-associated) fen1 point mutations in clinical strains. Two of the strains also contained mutations in either FKS1 or FKS2. The MIC values for CSF and micafungin (MCF) for WM_18.26 were obtained from [30], for DPL239 from our measurements, and for CAS14-5989 from CDC records. B. All three identified clinical fen1 mutations phenocopy fen1Δ and reduce C. glabrata CSF sensitivity. Strains containing wild type FEN1 do not grow in the presence of CSF, whereas strains lacking either functional chromosomal or plasmid FEN1 grow in the presence of the drug. When introduced into C. glabrata on a low-copy plasmid, neither fen1G143V nor fen1G52A were able to complement fen1Δ CSF-resistant phenotype. Conversely, the CSF-resistant phenotype of clinical and gut-evolved strains carrying chromosomal fen1 mutations was complemented by WT FEN1 introduced on a low-copy plasmid.

Although the fen1-G143V mutation was previously reported, it was not known whether it affected Fen1 function, and the same was true for the newly discovered fen1-G52A mutation. Because strains WM_18.26 and CAS14-5989 were not readily available, we introduced the G143V and G52A mutations, separately, into FEN1 carried on a low copy plasmid and transformed these plasmids into the fen1Δ strain. Unlike the plasmid carrying wild type FEN1, which fully complemented the loss of Fen1 by restoring wild type CSF sensitivity, the plasmids carrying fen1-G143V or fen1-G52A behaved like the empty vector, strongly suggesting that the fen1-G143V mutation caused a loss of Fen1 function (Fig 6B). To determine whether the reduced CSF sensitivity of strain DPL239 was due to the fen1-Y298* mutation, we transformed it either with the FEN1 plasmid or an empty vector. Whereas the empty vector had no effect, the FEN1 plasmid sensitized the strain to CSF (Fig 6B), strongly suggesting that the fen1-Y298* mutation contributes to the reduced CSF susceptibility of this clinical C. glabrata isolate. The same result was obtained with gut-evolved strain GI-240 (Fig 6B), confirming that it contained a loss-of-function fen1 mutation (Fig 1A). Together, these experiments showed that loss-of-function fen1 mutations occur in clinical C. glabrata strains and contribute to the strains’ reduced susceptibility to CSF.

Discussion

In this study, we investigated the evolution of resistance to the widely used echinocandin class drug caspofungin in C. glabrata colonizing the GI tract of immunocompetent mice. We found that, in addition to the well-described mutations in echinocandin target β-glucan synthase, other mutations, predominantly in the FEN1 gene involved in sphingolipid biosynthesis, arose rapidly after the start of caspofungin treatment and increased in abundance in the gut due to their strong fitness advantage over the wild-type strain in the presence of the drug. Our lipidomic, genetic, high-resolution imaging, and MS analyses revealed that the reduced caspofungin sensitivity of fen1 mutant strains is due to elevated intracellular levels of phytosphingosine (PHS), a sphingolipid biosynthesis intermediate, and is associated with reduced caspofungin binding to C. glabrata plasma membrane. Importantly, we also identified several different fen1 mutations in clinical C. glabrata isolates, and our analyses of these mutations showed that they result in loss of protein function and reduced susceptibility to caspofungin. Together, these results identify a new genetic determinant of clinical caspofungin susceptibility in C. glabrata and illuminate the rich dynamics of evolutionary trajectories in gut-colonizing C. glabrata during caspofungin treatment.

Our study illuminates the rapid and complex evolutionary dynamics taking place in the gut C. glabrata reservoir during echinocandin therapy. Both Sanger sequencing of individual colonies and amplicon sequencing of pooled colonies have revealed that mutations in FEN1 emerge and rapidly increase in relative abundance as early as 4 days after the initiation of caspofungin treatment. These results also provide evidence of mixed mutant populations of gut-commensal C. glabrata during caspofungin treatment, where different sub-populations carry different mutations in FEN1, FKS2, and other genes affecting caspofungin sensitivity, and where the relative abundance of these mutant sub-populations fluctuates according to their relative fitness in the presence of caspofungin. Indeed, our amplicon sequencing analysis demonstrated that fen1-W39* and fks2-R1378H alleles arose early during treatment but did not sweep the gut population, indicating that other genetic mutations reducing caspofungin susceptibility had also arisen in the same populations, improving their relative fitness to make them competitive with fen1-W39* or fks2-R1378H. We also provide evidence that in both clinical and mouse gut-evolved C. glabrata strains, fks and fen1 mutations can be acquired sequentially in the same cells (e.g., in GI-297, WM_18.26, and CAS14-5989). Although clinical histories are not always available, the identification of clinical strains containing both mutations in FEN1 and in FKS1 or FKS2 strongly suggests that those mutations had arisen under the same environmental pressure. Together, our results are consistent with a model for evolution of caspofungin resistance in the gut where caspofungin treatment rapidly kills the majority of cells in the gut (which can be seen as the several-log drop in fungal burdens, Fig 2A), but rare drug-tolerant cells survive (Fig 7). These cells may be caspofungin-tolerant either due to pre-existing mutations or to non-genetic mechanisms [56]. Such tolerant cells preferentially proliferate in the gut, and additional mutations further improving fitness in the presence of caspofungin may arise during this proliferation, perhaps stimulated by stress-induced mutagenesis [57,58]. Weak, incremental improvements in fitness can be achieved by a number of genetic alterations (e.g., any loss-of-function mutation in FEN1 or a weak mutation in FKS2) and can therefore occur at a relatively high frequency. The occurrence of these early mutations allows sufficient numbers of C. glabrata cells to survive and proliferate in the gut during caspofungin treatment to eventually develop a rare, clinically echinocandin-resistant mutation in FKS2 (e.g., S663P), which may then take over the population and eventually cause an echinocandin-resistant systemic infection.

10.1371/journal.ppat.1012521.g007 Fig 7 Model for evolution of caspofungin (CSF)-resistant C. glabrata in the gut reservoir during CSF treatment.

Our results support the model where, upon the initiation of CSF treatment, CSF-sensitive cells are killed but rare CSF-tolerant cells expand in the gut. This CSF-tolerant cells may be due to pre-existing rare mutations or to non-genetic mechanisms. Such CSF-tolerant cells preferentially expand in the gut, and additional mutations with increased fitness during CSF treatment are further selected and expanded. At this stage the gut is a genetically heterogeneous population of cells with increased fitness in the presence of CSF, such as those with mutations in FEN1 or weak mutation in FKS2. Ultimately, this survival and proliferation of the gut C. glabrata reservoir during CSF treatment may culminate in the formation and preferential expansion of a clinically CSF-resistant mutation, such as fks2S663P, which has the highest fitness in the presence of the drug and may eventually cause an echinocandin-resistant systemic infection.

There are several potential reasons why the gut may provide a permissive environment for the evolution of echinocandin resistance. First, drug concentrations reached in the GI tract during treatment are several times lower than those reached in the plasma and various organs [37]. Second, fungi form prominent biofilms in the GI tract, which are more tolerant to antifungal treatments [59]. Either of these possibilities is consistent with our observation that fen1 mutants have a fitness advantage over wild-type cells in the gut but not in the kidney, where caspofungin concentrations are high enough to eliminate both fen1 and wild-type strains equally well, whereas a fks2 hot-spot mutation with a stronger resistance phenotype is killed less efficiently. Thus, a paradigm emerges wherein in the context of disseminated infection, echinocandin treatment effectively eliminates infecting circulating strains with moderately reduced drug susceptibility (such as fen1), whereas in the context of GI colonization, the same strains survive during treatment, maintaining a continuous viable C. glabrata reservoir in which stronger fks mutations can then emerge (Fig 7), leading to therapy failure. This scenario may be different for the new β-glucan synthase inhibitor ibrexafungerp, which is orally bioavailable and therefore may be present in the GI tract at concentrations high enough to eliminate the gut-colonizing C. glabrata population, but this remains to be experimentally verified.

Several previous studies have reported that mutations in the sphingolipid pathway genes, including FEN1, reduce fungal susceptibility to caspofungin but not to other echinocandins in vitro and that these mutations cause an increase in cellular PHS levels [41,43,49,50]. Our study builds on those results and reveals several key new mechanistic insights. First, we demonstrate that genetic depletion of PHS by deletion of alkaline ceramidase-encoding gene YPC1 sensitizes C. glabrata to caspofungin, strengthening the conclusion that phytosphingosine levels are a key determinant of caspofungin susceptibility. Furthermore, ypc1Δ was fully epistatic to fen1Δ, confirming that increased PHS is the cause of reduced caspofungin sensitivity in the fen1Δ strain. Second, we show that caspofungin association with the cellular surface is significantly reduced in the fen1Δ mutant but is restored in the fen1Δ ypc1Δ mutant, correlating with their reduced and restored caspofungin sensitivity, respectively. This result, as well as our MS analysis of membrane-associated CSF, suggests that the reduced caspofungin efficacy in the fen1Δ mutant is underpinned by its reduced capacity to bind to the plasma membrane due to the membrane’s altered lipid composition (i.e., higher phytosphingosine content), which in turn likely decreases caspofungin ability to inhibit membrane-embedded glucan synthase. A similar hypothesis was proposed by Healey et al. [43], suggesting that the accumulation of long chain bases, such as phytosphingosine, in the plasma membrane reduced the association of caspofungin, but not other echinocandins, with glucan synthase. Further studies are necessary to fully elucidate the effect of sphingolipid pathway mutations on glucan synthase and its interaction with different echinocandin class drugs.

Our results provide one potential explanation for frequent observations of clinical strains showing differential susceptibility to different echinocandin class drugs [15–18,29]. Specifically, based on our results and previous in vitro studies [43], it is likely that strains showing non-susceptibility only to caspofungin are likely to carry mutations in the sphingolipid biosynthesis pathway or, perhaps, mutations in other pathways that cause the accumulation of PHS. We further speculate that other, as yet unknown, pathways differentially affect C. glabrata susceptibility to micafungin and anidulafungin, and that studying the evolution of resistance to these drugs in the mouse gut colonization model can help identify clinically-relevant mutations that reduce C. glabrata susceptibility to one echinocandin while not affecting, or increasing, its susceptibility to the other echinocandins. If such mutants were identified, this information could have important therapeutic implications. For instance, the fact that the fen1 mutations arising in gut-colonizing C. glabrata are highly susceptible to micafungin suggests that, contrary to the conventional wisdom of not combining drugs belonging to the same class, using a combination of caspofungin and micafungin may be of value because it would constrict the pathogen’s evolutionary trajectory towards pan-echinocandin-resistant fks mutations.

Our conclusions about diverse C. glabrata strains coexisting in the gut are highly consistent with genomic analyses of sequential clinical isolates from human patients, which show that a single individual can carry multiple closely related strains, including multiple drug-resistant variants that emerge during drug therapy [24,60]. Together, these observations in the mouse model and in human candidiasis patients underscore the capacity of C. glabrata to undergo micro-evolution in the host and may explain the very high genetic diversity among clinical strains [30, 35]. This capacity for evolution makes C. glabrata a formidable clinical challenge; nevertheless, understanding the mechanisms promoting its evolvability within relevant host niches may help develop effective measures to reduce the evolution of drug-resistant strains.

Materials and methods

Ethics statement

All animal study protocols used in this work have been approved by the Hackensack Meridian Health Center for Discovery and Innovation Institutional Animal Care and Use Committee under protocol number 262.

Yeast strains and media

The C. glabrata strain used in the mouse conization experiments was DPL1021 (ATCC90030) and the described deletion mutants constructed in the DPL1021 background. Cells were cultured in standard yeast extract-peptone-dextrose (YPD) medium at 37°C, which is the optimum growth temperature for this species. Deletion mutants were generated in-house using a CRISPR-CAS9 targeted integration replacing the desired ORF by a nourseothricin (NAT)- or hygromycin (HYG)-resistance cassette. The deletion construct containing the NAT-resistance or HYG-resistance cassette flanked by regions homologous to the locus of interest was amplified from genomic DNA using primers listed in S5 Table. Integration or the deletion cassettes was performed using CRISPR as described previously [61]. Transformants were selected on nourseothricin (NAT)- or hygromycin (HYG)-containing plates and validated by PCR amplification and sequencing of the targeted locus using external primers (S5 Table). At least two independent transformants were generated and analyzed for every deletion mutant. Primers were ordered from Integrated DNA Technologies (Coralville, IA, United States) and Azenta (South Plainfield, NJ, United States), and all Sanger sequencing of the above-described constructs was done by Azenta (South Plainfield, NJ, United States).

Murine model of Candida glabrata GI colonization

The GI model of C. glabrata colonization was performed as described in [40] with some modifications. 6-week-old female CF-1 immunocompetent mice (Charles River Laboratories) treated subcutaneously, daily from day-2 to 15, with 320 mg/kg of piperacillin-tazobactam (PTZ, 8:1 ratio, AuroMedics Pharma LLC, East Windsor, NJ, United States) to clear native intestinal bacterial microbiota. On day 0, mice were inoculated via oral gavage with approximately 1.5x108 CFU of C. glabrata in 0.1 ml of PBS. In this manner mice were colonized with strain DPL1021 (a.k.a. ATCC90030) or its derivative mutants. Daily intraperitoneal administration of 20 mg/kg of caspofungin (Selleck chemicals) or PBS was initiated on day 3 post inoculation and continued through day 15. Fresh fecal samples were collected every other day throughout the experiment to assess fungal burden in the GI tract. One mouse was included as an untreated control (treated with PBS alone). All mice in the PBS alone control groups maintained a constant fungal burden of 106−108 CFU/g of stool. The mice were housed individually to avoid horizontal transfer of C. glabrata strains.

Competitive fitness in the GI tract

For the mixed colonization fitness study, mice were gavaged with an equal mix of C. glabrata DPL1021 (ATCC90030) wild type and isogenic fen1Δ or fen1-W145* mutants totaling 1.5x108 CFU in 0.1 ml of PBS. In each group, four mice were treated daily with caspofungin as described above and four other mice were used as an untreated control (treated with PBS alone). Fecal samples were plated on the indicated days and the FEN1 status of the colonies determined as follows. To distinguish fen1Δ from WT colonies, the plates were replica-plated to YPD plates containing 250 μg/ml nourseothricin. To distinguish fen1-W145* from WT colonies, colony PCR using a molecular beacon was performed as described below.

Asymmetric PCR and molecular beacon-based melting curve analysis

One set of primers was designed to amplify the W145* region of fen1 on C. glabrata. The fen1 region was amplified by using excess primer fen1-W145-RV (5’-GAGTTTATTGACACCCTCTTCTTGG-3’) and limiting primer fen1-W145-FW (5’-GATCTACCATCACGGTTTATTCTATGCC-3’). The molecular beacon (MB) was designed by adding two artificial arm sequences to both ends of the target sequence. The MB was labeled with the fluorophores 5-carboxyfluorescein (FAM) at 5’ and tetramethylrhodamine (TAMRA) at 3’ end, targeting the W145* allele sequence of fen1, and it’s: (5’-FAM-TTGCAAATTGGCTTGCCCCCACT-TAMSp-3’). The secondary structure of the MB was evaluated by the software OligoAlanyzer 3.0 (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/). Asymmetric PCR was carried out on the AriaMx real-time PCR system (Agilent Technology, CA) in 20 μl reaction volume using SensiFAST probe no-ROX mix (Bioline, London, UK). The fen1 duplex assay contained 5 μM fen1-145-FW, 20 μM fen1-145-RV and 5 μM the MB. Instead of using genomic DNA, we used a single colony of C. glabrata to discriminate between the DPL1021 (ATCC90030) wild type strain and the fen1-W145Stop mutant. The PCR conditions were 95°C for 3 min; 45 cycles of 10 s at 95°C, 20 s at 60°C, and 30 s at 72°C; and 2 min at 72°C. Immediately after amplification, melting curve analysis was initiated as a minute incubation at 95°C, after which it was melted from 53°C to 64°C with a ramp rate of 0.1°C/s.

Systemic infection

The systemic infection study was performed essentially as described [56] with some modifications using outbred CD-1 mice weighing 22–24 g (Charles River Laboratories). Neutropenia was induced and maintained through administration of 150, 100, and 100 mg/kg cyclophosphamide via IP injection on days -4, -1, and 2 post infection, respectively. Groups of mice were randomized into 3 inoculation arms infected with WT (DPL1021/ATCC90030) and fks2-S663P and fen1Δ mutants in the same strain background. On Day 0, mice were inoculated with 0.1 ml of a cell suspension containing 1x107 CFU/mouse via Retro-Orbital 50 μl. Within each inoculation arm, mice were randomized into the antifungal treatment sub-groups (caspofungin 1.25, 2.5, and 5 mg/kg i.p.) and into the vehicle treatment (PBS i.p.) sub-group. The numbers of mice used are shown in Fig 2C (each dot indicates one mouse). Antifungal therapy was started at 24h post-infection and continued through day 2 (2 days total), during which caspofungin and vehicle control were administered once daily. On Day 3, the mice were euthanized via CO2 narcosis and kidneys were aseptically harvested for CFU count. Both kidneys were placed in M tubes containing 2.5 ml PBS, homogenized and plated onto YPD plates for kidney burden counts.

Echinocandin susceptibility testing

Echinocandin susceptibility testing was performed using a broth microdilution method following CLSI standards (Clinical Laboratory Standards Institute (CLSI), 2017) with some modifications. The media used was 2X RPMI and the final concentrations tested ranged from 0.015 to 2 μg/ml in two-fold increasing concentrations. Minimum inhibitory concentrations (MICs) were visually read after 48 h of incubation at 37°C and at least three biological replicates were performed. CSF and CSF-AF647 susceptibilities (S1B Fig) were measured side by side using the concentrations shown in the figure. In that experiment all wells contained 2% DMSO to match the well with the highest CSF-AF647 concentration (205 μg/ml) and growth was assessed after 48h by measuring optical density at 800 nm because CSF-AF647 does not absorb at this wavelength.

Lipidomic analysis

Yeast were precultured, diluted to 0.3 OD600 unit/ml and cultured to 1 OD unit/ml, then treated with caspofungin (0.5 μg/ml) for 1 hour. The cells were washed and stored at -20°C. The lipidomic analysis was done at the lipidomic resource of the Medical University of South Carolina. Inorganic phosphate (Pi) levels were measured in the same samples and all lipids were normalized to Pi.

Multi-locus Sequence Typing of C. glabrata

STs were assigned by using the MLST scheme based on 6 loci (FKS2, LEU2, NMT1, TRP1, UGP1, URA3) according to Dodgson et al., 2003 [54].

Spot assays

C. glabrata cells grown overnight were washed twice with phosphate-buffer saline (PBS) and adjusted to 5x105 CFU/ml in PBS. An equal volume (3 μl) of 10-fold serial dilutions of each strain were spotted onto YPD plates either lacking drug or containing the indicated concentrations of caspofungin. The plates were incubated at 37°C for 2 days.

Super-resolution microscopy

Cells were cultured overnight in YPD broth, then washed twice and resuspended in Synthetic Defined (SD) Medium to OD600 = 0.3 and allowed to grow to approximately OD600 = 1. Subsequently, the cells were incubated with 0.26 μM CSF-AF647 (WuXi Biologics) for 30 minutes in the dark at 37°C. The cells were then washed twice with PBS, fixed with 2% paraformaldehyde, and washed twice more with PBS. For imaging, the cells were dispersed on u-Slide 8 well high glass bottom (iBidi, Cat.No: 80807) that had been pre-treated with Poly-L-Lysine (ScienceCell, Cat.No: 0403). The imaging chamber was overlaid with 100 ul of BCubed A and 1ul of BCubed B dSTORM buffers (Oxford Nanoimager, ONi). Visualization of the cells was performed using the Nanoimager (Oxford Nanoimaging, ONi) using the 640 nm laser configuration for CSF-AF647, utilizing a 100x oil-objective lens. The images were analyzed using CODI software.

Whole genome sequencing of gut-evolved C. glabrata strains

Genomic DNA of gut-evolved C. glabrata strains was extracted using the Quick-DNA Miniprep Kit (Zymo Research). Library preparation and next-generation sequencing was conducted by Azenta (Plainfield, NJ). Preliminary sample quality check was performed using FastQC version 0.12.0 [62], only runs with a median Q > 30 where further analyzed. Reads were aligned to DPL1021 (ATCC 90030) reference genome using STAR version 2.7.1 [63], aligned reads where sorted and then indexed using samtools version 1.18 [2], alignment metrics where also generated using samtools using flagstat command, variant calling was done using bcftools version 1.18 [64] combining mpileup and call commands under standard parameters, variants where deduplicated using Picard version 3.1 f (Picard Toolkit, Broad Institute, 2019) using MarkDuplicates command and the alignment metrics previously generated, reads where further annotated using snpsift version 5.1 [65] and filtered by effect severity using snpeff version 5.1 [66]. The genome sequencing data have been deposited as NCBI PRJNA1010358.

Identification of unique SNPs in FEN1, FKS1, and FKS2 in publicly available genomes

488 paired end read files from 244 runs were downloaded from NCBI PRJNA524686 [67] and 118 paired end read files from 59 runs were downloaded from PRJNA480138. Multi Locus Sequence Typing (MLST) was done by matching sequences of ’FKS2’, ’LEU2’, ’NMT1’, ’TRP1’, ’UGP1’, ’URA3’ loci to reference alleles [54] (https://pubmlst.org/organisms/candida-glabrata). SNPs in FEN1, FKS1, and FKS2 were identified using the variant calling method described above.

Amplicon sequencing

Fecal samples were plated on YPD plates containing piperacillin and tazobactam to reduce bacterial contamination. Approximately 1000 colonies were collected and pooled together for each sample. In cases where only a limited number of colonies grew from the first plating (e.g., mouse 3 day 9, see S2A Fig) the remainder of the fecal sample was plated to obtain the desired number of colonies. Samples from mouse 2 day 7 and mouse 4 day 15 were not obtained because of extensive mold and bacterial contamination on the YPD plates. Genomic DNA was extracted from the pooled colonies using phenol-chloroform extraction. For the sequencing of FKS1 and FKS2 hotspot regions (HS1 and HS2), primer pairs were designed to encompass each of the four hotspots, generating amplicons of 200 to 300 bp. To sequence the entire FEN1 ORF, 6 overlapping amplicons, ~300bp each, were also generated by PCR using the Q5 High-Fidelity MasterMix (New England Biolabs), followed by purification with a DNA sequencing clean-up kit (Zymo Research). All primers are listed in S5 Table. DNA concentrations was quantified using the Qubit system (ThermoFisher). The 10 amplicons from each sample were mixed together in approximately equimolar concentrations and amplicon sequencing was performed at CD Genomics (Shirley, NY 11967, USA). SNPs were identified using the variant calling method described above. The percentage of cells containing a mutation was approximated as the number of reads containing a SNP divided by the number of reads aligning to that region on the deduplicated, aligned and sorted read file for each run. The amplicon sequence reads have been deposited as NCBI PRJNA1010358.

Analytical method for CSF quantification in CSF-AF647 by LC-MS/MS

DMSO stocks of 10.27 mg/ml CSF-AF647 conjugate were diluted in extraction solution (1:1, MeCN:MeOH) containing verapamil (internal standard). 1 mg/ml DMSO stock of CSF (Selleck chemicals) was used to create calibration standards for quantification of CSF in the CSF-AF647 conjugate stock. Working solutions covering the desired concentration range were prepared by diluting the stock solutions in extraction solution. Samples were vortexed for 5 minutes and 150 μl was transferred to a 96 well plate for analysis LC-MS/MS analysis was performed on a SCIEX QTRAP 6500+ triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera UHPLC system. Chromatography was performed on an Agilent SB-C8 (2.1 x 30mm; particle size 3.5 μm) using a reverse phase gradient with a flow rate of 0.6 mL/min. MQW deionized water with 0.1% formic acid (FA) was used for the aqueous mobile phase and 0.1% FA in MeCN for the organic mobile phase. Multiple-reaction monitoring (MRM) of precursor/fragment transitions in electrospray positive-ionization mode was used to trace CSF and verapamil. MRM transitions of 1093.70/1033.50 and 455.40/165.00 were used for CSF and verapamil respectively. Data processing was performed using the Analyst software (version 1.7.2 Applied Biosystems Sciex).

Crude membrane preparation and CSF quantification by LC-MS/MS

Exponentially growing C. glabrata cells were incubated with 0.25 μg/ml CSF for 20 min at 37°C, then harvested by centrifugation, washed twice with 1 ml cold PBS, and resuspended in 0.5ml of disruption buffer (50 mM Tris-HCl, pH 7.6, 150mM sodium chloride, 1 mM dithiothreitol, 20% glycerol, and protease inhibitors (Roche Complete Ultra)). The cells were homogenized in Lysing matrix E tubes (MP Bio 116914100) by BeadBlaster 24 Microtube Homogenizer (Benchmark Scientific). Beads and debris were removed by centrifugation at 3500g for 5 min, then the supernatants were centrifuged at 17,000g for 15 min at 4°C to obtain membrane pellets. The membranes were resuspended in 100 μl disruption buffer, 10 μl was saved for protein measurement via Bradford assay, and 90 μl was pelleted again and snap frozen. Prior to LC-MS/MS analysis, 200 μl of extraction solution (1:1, MeCN:MeOH) containing verapamil (internal standard) was added to membrane pellets. Extracts were sonicated for 5 minutes, vortexed for 5 minutes with beads and centrifuged at 4,000 rpm for 5 minutes. 150 μl of supernatant was transferred to a 96 well plate for analysis. The LC-MS/MS analysis was performed as described above.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software, applying the appropriate statistical tests described in the corresponding figure legends.

Disclaimer

The findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control (CDC).

Supporting information

S1 Fig Structure and synthesis scheme of Alexa Fluor 647-conjugated caspofungin (CSF).

A. CSF-AF647 association with the fungal cells was significantly reduced in fen1Δ cells in ATCC90030 strain background. B. Alexa Fluor 647-conjugated CSF (CSF-AF647) showed reduced antifungal activity, which was further significantly reduced in strains carrying the echinocandin-resistant mutation fks2-S663P and fen1Δ. Growth was measured as OD800 to avoid interference from absorbance by CSF-AF647. C. AF647 alone does not bind to C. glabrata cells, unlike CSF-AF647. The amine-reactive NHS ester of AF647 (Lumiprobe) was quenched in 10X excess TRIS and the molecule was incubated with C. glabrata cells at the same molar concentration and for the same amount of time as CSF-AF647, followed by ONi imaging (see Methods).

(EPS)

S2 Fig C. glabrata gut colonization for amplicon sequencing.

A. Fungal burdens per gram of stool in the GI tracts of four mice used in the amplicon sequencing experiments. B. Mutations in FKS1 hot-spots 1 and 2 present at low levels in the original culture used to gavage the mice in the amplicon sequencing experiment. The same mutations were recovered at approximately the same levels from the colonized mice during CSF treatment. Samples from mouse 2, day 7 and mouse 4, day 15 were not obtained due to heavy bacterial and mold contamination of the plates.

(EPS)

S1 Table Table of coding SNPs identified in the gut-evolved C. glabrata strains.

(XLSX)

S2 Table Levels of lipids analyzed as part of the ceramide-sphingolipid panel in wild type and fen1 mutants.

These results were used to make the heatmap in Fig 4A. The levels were normalized to inorganic phosphate measured in the same samples (arbitrary units).

(XLSX)

S3 Table FKS1, FKS2, and FEN1 coding SNPs found in the genomes of clinical C. glabrata isolates sequenced and made publicly available by the CDC (https://www.ncbi.nlm.nih.gov/biosample?Db=biosample&DbFrom=bioproject&Cmd=Link&LinkName=bioproject_biosample&LinkReadableName=BioSample&ordinalpos=1&IdsFromResult=524686).

(XLSX)

S4 Table FKS1, FKS2, and FEN1 coding SNPs found in the clinical C. glabrata genomes from Biswas et al., 2018 [30].

(XLSX)

S5 Table Primers used in this study.

(XLSX)

We thank research and administrative staff at the Centers for Disease Control for providing information on clinical C. glabrata strains [67]. We also thank Candidemia staff from the Georgia Emerging Infections Program, Maryland Emerging Infections Program, and Tennessee Emerging Infections Program; and participating Emerging Infections Program Laboratories. Finally, we thank Dr. Milena Kordalewska for help with designing the fen1-W145* molecular beacon, Tara Lozy for assistance with statistical analysis, and the CDI shared flow cytometry and microscopy cores for assistance with the corresponding experiments.

10.1371/journal.ppat.1012521.r001
Decision Letter 0
Olszewski Michal A Section Editor
Konopka James B. Academic Editor
© 2024 Olszewski, Konopka
2024
Olszewski, Konopka
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version0
21 Dec 2023

Dear Dr. Shor,

Thank you very much for submitting your manuscript "Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: emergence of clinically important mutations in sphingolipid biosynthesis" (PPATHOGENS-D-23-02041) for consideration at PLOS Pathogens. As with all papers peer reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent peer reviewers. Based on the reports, we regret to inform you that we will not be pursuing this manuscript for publication at PLOS Pathogens.

The reviewers thought that the topic was interesting and noted the importance of studies on the evolution of drug resistance in Candida glabrata. However, they also had significant concerns that limited their enthusiasm for the manuscript. In particular, Reviewers 1 and 2 thought that the previous reports that a fen1 mutation causes Caspofungin resistance in C. glabrata and other fungi undercut aspects of the novelty and significance. Both of these reviewers stated that further research would be needed to advance the understanding of the mechanisms by which altered PHS affects Caspofungin susceptibility in order to increase the significance of these studies. Reviewer 1 also had several concerns about the limitations of the data supporting the interpretations of the dynamics of the evolution of Caspofungin resistance. For one, they thought that there were too few mice and too few colonies studied to make significant conclusions. Furthermore, they thought that the emergence of the fen1 mutant strains at early times but then disappearance at later times raised further questions. Both Reviewers 1 and 2 also had concerns about some experiments involving the use of the evolved strains, which may carry additional mutations. In light of these serious concerns, additional studies would need to be done to make the manuscript acceptable for publication.

The reviews are attached below this email, and we hope you will find them helpful if you decide to revise the manuscript for submission elsewhere. We are sorry that we cannot be more positive on this occasion. We very much appreciate your wish to present your work in one of PLOS's Open Access publications.

Thank you for your support, and we hope that you will consider PLOS Pathogens for other submissions in the future.

Sincerely,

Michal Olszewski

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

**************************************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this study, the authors investigated the evolutionary dynamics of C. glabrata colonizing the gut of mice during caspofungin (CSF) treatment. Utilizing whole genome and amplicon sequencing, they observed a rapid emergence of both drug target (FKS2) and non-drug target mutations, resulting in reduced susceptibility to CSF. About half of the mutants carry mutations in the FEN1 gene, which encodes a fatty acid elongase involved in sphingolipid biosynthesis. The authors further demonstrated that the fen1∆ and fen1-W145* mutants completely outcompeted the wild-type strain in the gut during CSF treatment. Lipidomics analysis detected significantly increased intracellular levels of phytosphingosine, a sphingolipid biosynthesis intermediate. Blocking phytosphingosine synthesis by deleting the YPC1 gene, which encodes an alkaline ceramidase, hypersensitized the wild-type strain to caspofungin and was epistatic to fen1Δ. Additionally, several different fen1 mutations were identified in clinical C. glabrata isolates, and all of them were shown to phenocopy the fen1Δ mutant, causing reduced CSH susceptibility.

The data presented do not support the authors’ claim that ‘These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.’. First, various aspects of the main findings presented in this manuscript have been reported in other fungi including S. cerevisiae, Aspergilus, C. albicans, and C. glabrata. Second, the experiments investigating the evolution dynamics was not well-designed.

Previous findings include:

1. Disruption of FEN1 reduces cellular sphingolipid levels and results in the accumulation of the long chain base, phytosphingosine. (JBC 272: 17376; 1997).

2. Sharma S, et al. (2014) Sphingolipid biosynthetic pathway genes FEN1 and SUR4 modulate amphotericin B resistance. Antimicrob Agents Chemother 58(4):2409-14 (In budding yeast).

3. Increased resistance to caspofungin was first reported in C. glabrata fen1Δ by Healey et al. more than 10 years ago, which was attributed to increased cellular levels of some sphingolipid biosynthesis intermediates, including phytosphingosine (Mol Microbiol 86:303; 20120). Similar results were also reported for Candida albicans fen1Δ fen12Δ mutant and Aspergillus nidulans (basA mutant) in another paper by Healey et al. (Antimicrob Agents Chemother 59:3377; 2015). While the authors cited these papers, they failed to explicitly describe the previous findings in the Introduction.

4. Furthermore, a recent paper by Gao et al. demonstrated significant increase in the cellular levels of sphingolipid biosynthesis intermediates in Candida albicans fen1Δ fen12Δ mutants (Chew et al. Nat Commun 9:4495; 2019), significantly reducing susceptibility to fluconazole, and deleting the transcription factor that promote key genes in the synthesis pathways abolished the resistant phenotype.

Reviewer #2: This manuscript examines the appearance of echinocandin resistance in Candida glabrata (Cg). The primary finding is of a strong link between biosynthesis of sphingolipids and resistance to echinocandin antifungal drugs. Loss of function mutations in a gene called FEN1 leads to the accumulation of a precursor to ceramide production (PHS) with an accompanying decrease in echinocandin susceptibility. The authors also demonstrate that loss of the ceramidase enzyme Ypc1 (breaks down ceramide into PHS and very long chain fatty acid constituents) both increased susceptibility to caspofungin and restored susceptibility to a fen1 null strain. A gut colonization model was used to demonstrate that fen1 mutants could be selected during in vivo growth of Cg during caspofungin treatment. Additionally, sequencing of clinical mutants revealed that fen1 mutations could be found in these isolates. The authors also used plasmid complementation to analyze the functionality of several of these clinical mutant alleles of FEN1 and provided an argument that these all represented loss of function forms of Fen1.

This is nice work and succinctly presented. The dynamic analysis of caspofungin treatment on Cg resistance in a mouse exposed to caspofungin using amplicon sequencing is well done.

Reviewer #3: This is a well-written manuscript that describes mutations in FEN1 in Candida glabrata that impart increased fitness in the setting of gut colonization and caspofungin treatment. Echinocandin resistance has emerged in candida species, including C. glabrata and in this species is typically attributed to mutations in the gene encoding the drug target glucan synthase (FKS2). However, resistance that cannot be explained by such mutations has been observed. Moreover, it is well established that the gut serves a reservoir for the emergence of echinocandin resistance. This group previously evolved echinocandin resistant strains of C. glabrata using the mouse gut colonization model during caspofungin treatment and observed FKS2 mutations in strains exhibiting increased MICs to both caspofungin and micafungin. However, there were several strains that exhibited increased MICs to only caspofungin and that had no FKS2 mutations. These are the focus of the current study where after WGS, mutations in other genes were observed, most commonly mutations in FEN1 encoding fatty acid elongase such as that leading to the W145* amino acid substitution that should result in a loss of function. Introduction of this mutation or deletion of FEN1 resulted in strains with enhanced fitness in the mouse gut under caspofungin treatment. As opposed to the gut where caspofungin concentrations are lower, this was not observed for kidney fungal burden in a systemic model of infection where caspofungin concentrations are higher. Elevated phytosphingosine was observed in the fen1 KO mutant as well as one gut evolved strain with a FEN1 mutation and one without, suggesting this accumulation may drive this phenotype. This was supported by hypersusceptibility observed when YPC1 (alkaline ceramidase) was deleted. Experimental evidence further indicated that the FEN1 mutations arise before FKS2 mutations suggesting the increased fitness conferred by FEN1 mutations may provide a jumping off point for the evolution of fulminant resistance conferred by FKS2 mutations. This was supported by the observation of FEN1 mutations in clinical isolates with reduced susceptibility to caspofungin. In short, these data support a role for FEN1 as a genetic determinant of reduced susceptibility to caspofungin in C. glabrata.

This paper is significant in my opinion for two reasons. First, it gets at the question of factors that contribute to the emergence of FKS2 mutations and caspofungin resistance specifically as only such mouse experiments could. Secondly, it underscores the need to understand more than just classical genetic determinants of antifungal resistance, but also those genetic determinants of more subtle changes in susceptibility that might represent stepping stones for the emergence of fulminant resistance. Such information might ultimately lead to strategies to mitigate the emergence of resistance.

The introduction is clear and concise with the needed background, the results are clearly presented with good use of figures and tables, and the discussion is a sound treatment of the findings. Methods are clearly presented and appropriate in my opinion.

--------------------

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The manuscript extensively discusses the evolution dynamics of CSF-resistant mutants based on the results shown in Figure 4. However, the small sample sizes, i.e., two colonies per day and a total of six mice, are not sufficient to produce any credible evolution patterns. The detection of various mutations in the population on different days appears random, raising more questions than answers. For example, while panel A shows that all fen1 mutations appeared before fks2 mutations, panel B shows the opposite. There is no evidence that the earlier appearance of tolerance or resistance due to fen1 mutations has allowed time for FKS2 resistance to evolve. Although mutations were detected in both fen1 and fks2 in the two colonies from mouse #1 on day 15, the fen1 mutation differs from the one detected on day 9. The fen1-D166N mutant in mouse #2 must be extremely dominant, as it was detected in both colonies from day 7 to day 15; however, no mutations were detected in FKS2. In mouse #4, the two fen1 mutations detected on day 7 have nothing to do with the FKS2 mutations detected from day 9 to day 15. The fen1-Q10stop mutant found on day 7 in the two colonies from mouse #5 is intriguing. The evolved stop codon at the tenth position of the amino acid sequence makes it almost like the fen∆ mutant, which was shown to completely outcompete the WT strain under CSF treatment (Figure 2B). Yet, the fen1-Q10stop mutation was not detected again from day 7 to day 15. While one can argue that unknown mutations can influence the competitiveness of different mutants, significantly larger sample sizes are required to reveal credible evolutionary patterns of various drug-resistant mutations.

2. In Figure 2C, it is not clear why only the strain FEN1-W145stop (correct nomenclature should be fen1-W145stop) was used in this experiment but not fen1∆. These two mutants are not equal because fen1-W145stop carries several other mutations which may have unknown influence on this strain’s fitness. The result shows that the fen1-W145stop mutant exhibits similar sensitivity to CSF as the WT strain during systemic infection in mice. However, only one CSF concentration, 5 mg/kg, was used in this experiment. This is a very high concentration compared with the dose used in humans (50-70 mg per day for adults; assuming the average weight of a person is 60 kg). It is likely that this concentration is significantly higher than the MIC of both strains, masking the difference in susceptibility to CSF between them. The experiment should be repeated using lower CSF concentrations, such as 1 mg / kg. Also, is there an estimate of the CSF concentration in the gut where CSF is IP-administered at 20 mg / kg?

3. The authors used different gut-evolved fen mutants in different experiments. For example, they used GI-240 to generate the data in Figure 2, but GI-299 and GI-240 for Figure 3 and Figure 5, respectively. This raises the question whether different strains exhibit significantly different phenotypes, particularly fitness in the gut under CSF treatment. This is worrisome because many of the evolved strains carry multiple mutations.

4. Lines 188-189. It is described that ‘one non-fen1 gut-evolved strain (GI-63)’ was included in the lipidomics analysis. However, the strain was not seen in Figure 3A.

Reviewer #2: The problem is that it has already been shown in two other fungi that changes leading to defects in sphingolipid biosynthesis and even specifically loss of FEN1 function leads to decreased echinocandin susceptibility. The work described here does not take things too far beyond this fairly well-established observation. If insight could be provided into the basis of the effect of sphingolipid deficiency on echinocandin resistance, then the impact of this work would be enhanced. For example, is the endocytosis of the Fks proteins influenced by defects in normal sphingolipid production? Is the activity of the Fks proteins enhanced or the ability of echinocandins to inhibit these enzymes reduced?

Reviewer #3: (No Response)

--------------------

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. Figure 1. Column 2: What does ‘genotype’ refer to? I thought all the GI strains are evolved strains and most of them carry mutations. Thus, what does WT mean? Also, the four gene deletion mutants were not explained, and they are not mentioned anywhere else in the text.

2. Figure 2A. I figured that sterilization and no sterilization mean if mice were treated with the antibiotic or not. If so, it’s better to use + or – antibiotic treatment. The use of a single antibiotic cannot sterilize the gut of mice. Antibiotic cocktails are often used but still cannot ensure sterilization.

3. Figure 2A. The near zero CFU on day 7 in three mice inoculated with WT C. glabrata are strange. Excluding these outliers, gut colonization of the WT strain would not be significantly different from that of the mutants.

4. Line 160. Nomenclature: is W145* the same as W145stop? If so, please use W145* and explain it when it first appears.

5. Line 399, italicize C. glabrata.

6. Line 400, citation format for Healey et al. is different from the rest.

Reviewer #2: (No Response)

Reviewer #3: I have only minor points:

Aside from the original evolved strains, I didn’t see any MICs for other echinocandins. One would expect no change or increased susceptibility to micafungin and anidulafungin in the mutants. Is this observed and does this represent a therapeutically exploitable phenotype?

Additional discussion of the clinical relevance would be of value in my opinion. While this is interesting and informative biology, there may be ways in which this information might inform how echinocandins might be used to reduce the emergence of resistance.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

10.1371/journal.ppat.1012521.r002
Author response to Decision Letter 0
Submission Version1
10 May 2024

Attachment Submitted filename: Hassoun et al Response to Reviewers.docx

10.1371/journal.ppat.1012521.r003
Decision Letter 1
Olszewski Michal A Section Editor
Konopka James B. Academic Editor
© 2024 Olszewski, Konopka
2024
Olszewski, Konopka
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version1
7 Jun 2024

Dear Dr. Shor,

Thank you very much for submitting your manuscript "Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: emergence of clinically important mutations in sphingolipid biosynthesis" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

The reviewers agree that the manuscript has been significantly improved by the revisions. However, there are still some concerns, especially regarding the new data in Figure 3. 

One concern is that the Halo-tagged Fks1 does not show expected plasma membrane localization. This raises questions about whether the Halo-tagged Fks1 is reporting the true localization of Fks1. In addition to comments from raised by the Reviewer 1, a Western blot should be done to see if there is free Halo that could account for cytoplasmic localization.

There are also questions about the studies using caspofungin covalently conjugated to Alexa Fluor 647 (CSF- AF647). This modified form of caspofungin appears to be ~120-fold reduced in activity, which raises the concern that this level of activity could be due to a small amount of contaminating untagged caspofungin. In addition to addressing comments of the reviewers, it would help if you could determine if there is a significant amount of untagged CSF in the labeled prep. Perhaps this could be done by Mass spectrometry.

Some other controls for the CSF- AF647 need to be performed that are standard for validating this type of labeling experiment. As mentioned by Reviewer 1, one control is to treat cells with AF647 alone to determine if AF647 binds cells independently of caspofungin. Another control is to determine whether the observed binding can be competed with a slight excess of unlabeled caspofungin to determine whether they bind the same target on the cell surface.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

Please ensure that your revisions are complete to ensure the publication.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

James B. Konopka

Academic Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

The reviewers agree that the manuscript has been significantly improved by the revisions. However, there are still some concerns, especially regarding the new data in Figure 3.

One concern is that the Halo-tagged Fks1 does not show expected plasma membrane localization. This raises questions about whether the Halo-tagged Fks1 is reporting the true localization of Fks1. In addition to comments from raised by the Reviewer 1, a Western blot should be done to see if there is free Halo that could account for cytoplasmic localization.

There are also questions about the studies using caspofungin covalently conjugated to Alexa Fluor 647 (CSF- AF647). This modified form of caspofungin appears to be ~120-fold reduced in activity, which raises the concern that this level of activity could be due to a small amount of contaminating untagged caspofungin. In addition to addressing comments of the reviewers, it would help if you could determine if there is a significant amount of untagged CSF in the labeled prep. Perhaps this could be done by Mass spectrometry.

Some other controls for the CSF- AF647 need to be performed that are standard for validating this type of labeling experiment. As mentioned by Reviewer 1, one control is to treat cells with AF647 alone to determine if AF647 binds cells independently of caspofungin. Another control is to determine whether the observed binding can be competed with a slight excess of unlabeled caspofungin to determine whether they bind the same target on the cell surface.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This is a revised manuscript. I thank the authors for their efforts in addressing the issues I raised previously.

The authors added new experiments, where they used fluorescence-labeled CSF to assess CSF’s interaction with the cell surface in the wild type versus the fen1∆ mutant. However, the new data raised some important concerns, which should be addressed.

Reviewer #3: As this manuscript has undergone initial review by three additional reviewers I will keep my assessment brief. This is a well-written manuscript that describes important experiments assessing the in vivo evolution of caspofungin resistance in a murine GI colonization model. These experiments implicate mutations in FEN1 as contributing to the resistant phenotype. Importantly, while much is known about the effect of loss of FEN1 function on echinocandin susceptibility (as pointed out by reviewer 1), this work shows that such loss of function through development of mutations in this gene can be selected for in vivo under caspofungin pressure. This is a key finding. I believe the authors have thoroughly responded to the concerns of reviewer 1 (as well as the other reviewers) and I am supportive of this paper and its importance for the two reasons outlined by reviewer 3. I would also add that the authors then used their experimental observations to explore publicly available WGS data for echinocandin resistant C. glabrata isolates, identified similar mutations in FEN1, and experimentally confirmed that they influence echinocandin susceptibility. This is an important and impactful capstone to this paper in my opinion. I have no additional critique or concerns.

Reviewer #4: This revised manuscript describes experiments that examine the genotypes and phenotypes behind the emergence of caspofungin resistance in gut colonizing Candida glabrata. This is then related to mutations detected in clinical isolates. As earlier reviewer comments state the importance of sphingolipid synthesis pathway and antifungal resistance has been previously elucidated. Here the study goes a step further to unpick the mechanism to explain why sphingolipid levels, in particular phytosphingosine, influence drug susceptibility. The manuscript is substantially strengthened by the inclusion of new tools and the application of high resolution microscopy to analyse the binding of fluorescently-tagged caspofungin to the plasma membrane in gut-evolved and fen1 mutated strains. The microscopy clearly demonstrate that there is less caspofungin binding when PHS levels are increased. The authors include a neat control, the ypc1 mutant, which lowers the PHS levels thus increasing caspofungin binding. This association between caspofungin binding and PHS levels is clear. What is less clear is how these changes in the membrane lipids alter Fks protein abundance at the plasma membrane and/or Fks enzyme activity leading to changes in cell wall glucan levels. The use of the Halo-tagged Fks1 shows clear differences in Fks1 levels and potentially localization between WT and fen1 mutant. In Figure 3D are the zoomed in panels at the bottom overlays of JF549 and AF647 stains? If so would you have expected to see some overlap in their localisation?

The finding that is hard to reconcile is that fen1 mutant doesn’t confer caspofungin resistance in the bloodstream immunocompromised mouse model where kidney fungal burdens were reduced as much as WT cells when mice were treated with range of caspofungin doses (up to 5mg/kg). In the discussion the authors suggest that this may be due to concentrations of bioavailable caspofungin in bloodstream versus the gut. Yet FEN1 mutations do emerge in clinical isolates so there is selection pressure to evolve these mutations in potentially diverse host niches (difficult to ascertain this without detailed information on the clinical isolates). This will require further studies to dissect the impact of host niche on the importance of fen1 mutations.

Minor comments:

Line 123, in summary rather than sum

Line 126 states intracellular levels of PHS, do you mean membrane levels?

Line 148, specify Candida species

Line 155 ergosterol

Line 209 Figure 3D

Figure 2A it is unconventional to use the term sterilization, you explain its meaning in the legend but as you are monitoring fungal counts in faeces you cannot be completely sure that no fungi remain in the GI tract. Why not state caspofungin treatment or plus caspofungin?

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (1) Figure 3C. Firstly, the results clearly demonstrate that the activity of CSF was reduced by approximately 120-fold (MIC of 0.11 µM for CSF vs. 13 µm for CSF-AF647 against WT) after conjugation with AF647, indicating a significant change in the structure of CSF and loss of activity. Therefore, in contrast to the authors’ statement that ‘it retained significant antifungal activity’ (Line 201), the results show that the conjugate retained very low activity. Secondly, the CSF-AF647 is 98.67% pure. Is the observed low activity of CSF-AF647 due to the presence of a small amount of free CSF molecules or other active intermediates in the sample, i.e., ~1%? Are the 1% impurity and 100-fold decrease of activity a coincidence? Thirdly, there appears to be an issue with the assay of CSF-AF647 activity, as there is no clear concentration dependence in the growth gradient, in contrast to the CSF assay result that shows good gradients. If labeling reduces the activity of a molecule, it is not suitable for studying the molecule’s interaction with a cellular target.

(2) In Figure 3D (CSF-AF647), firstly, the control using AF647 alone is missing. Secondly, a competition experiment should be conducted by adding different concentrations of unlabeled CSF to see whether it reduces the signal of CSF-AF647 on the cell surface in a concentration-dependent manner. Secondly, while Figure 3A shows that HT-FKS1 is functional, which was tested at one SCF concentration, the JF540 staining patterns are very different between the WT and the fen1∆ strain. There are many more bright dots in fen1∆ cells compared to WT cells, and these dots do not seem localized to the plasma membrane where Fsk proteins are expected to be (There is no colocalization between HT-Fks1 and CSF-F647). The data suggest possible defects in either protein folding or transportation, although a fraction of HT-Fks1 can still confer resistance to CSF. Also, how is Fks2’s cellular localization affected in the fen1∆ mutant? These issues complicate the interpretation of the results, and the data are not sufficient to conclude that the observed reduced association of CSF-AF647 with the cell surface is responsible for the CSF resistance of the fen1∆ mutant. It is merely a correlation.

(3) Lines 265-270. SNPs in FKS1 HS1 did not affect the fitness of C. glabrata in the gut of mice treated with CSF for two weeks, while mutations in FKS2 increased the fitness. Does Fks1 and Fks2 respond differently to CSF? Why? A relevant question is why Fks2 cellular localization was not examined in Figure 3?

Reviewer #3: None

Reviewer #4: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: N.A.

Reviewer #3: None

Reviewer #4: Minor comments:

Line 123, in summary rather than sum

Line 126 states intracellular levels of PHS, do you mean membrane levels?

Line 148, specify Candida species

Line 155 ergosterol

Line 209 Figure 3D

Figure 2A it is unconventional to use the term sterilization, you explain its meaning in the legend but as you are monitoring fungal counts in faeces you cannot be completely sure that no fungi remain in the GI tract. Why not state caspofungin treatment or plus caspofungin?

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

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10.1371/journal.ppat.1012521.r004
Author response to Decision Letter 1
Submission Version2
25 Jul 2024

Attachment Submitted filename: Response to reviewers.pdf

10.1371/journal.ppat.1012521.r005
Decision Letter 2
Olszewski Michal A Section Editor
Konopka James B. Academic Editor
© 2024 Olszewski, Konopka
2024
Olszewski, Konopka
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version2
19 Aug 2024

Dear Dr. Shor,

We are pleased to inform you that your manuscript 'Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: emergence of clinically important mutations in sphingolipid biosynthesis' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

James B. Konopka

Academic Editor

PLOS Pathogens

Michal Olszewski

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

I am happy to say that the reviewers all agree that your manuscript makes an important contribution and is now acceptable for publication.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Please see my previous review.

Reviewer #3: My initial comments (Reviewer 3) still stand for this iteration of this manuscript.

In my opinion, the authors have been greatly responsive to reviewer critiques and have presented an improved manuscript. They have embraced most of the comments offered by reviewers 1 and 3, and where possible have undertaken additional experimentation as suggested.

Reviewer #4: Thank you for addressing comments of all reviewers. The revised manuscript has been strengthened by the changes made and other points are out of scope of this study as are major challenges in the field.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The authors have conducted experiments in response to my comments, and the results and conclusions made are satisfactory.

Reviewer #3: None

Reviewer #4: Nothing further acquired.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: N.A.

Reviewer #3: None

Reviewer #4: None

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Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

10.1371/journal.ppat.1012521.r006
Acceptance letter
Olszewski Michal A Section Editor
Konopka James B. Academic Editor
© 2024 Olszewski, Konopka
2024
Olszewski, Konopka
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
3 Sep 2024

Dear Dr. Shor,

We are delighted to inform you that your manuscript, "Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: emergence of clinically important mutations in sphingolipid biosynthesis," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064
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