Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

Background Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. Results Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. Conclusion This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-021-00197-0.


Background
Candida albicans is a diploid polymorphic fungus that grows as yeast with pseudohyphae and true hyphae [1,2]. It resides as a harmless commensal on the skin and in the mucosal lining of the gastrointestinal and genitourinary tracts in humans and is the most prevalent fungal pathogen that can cause both superficial (such as oral and vaginal candidiasis) and systemic infections [3,4]. Systemic infections are life-threatening and common in immunologically weak individuals, such as HIV patients, neonates with low birth weight, transplant recipients, and chemotherapy patients [5]. Several other species of Candida, including C. glabrata, C. tropicalis, C. parapsilosis, C. haemulonii, and C. krusei were also isolated from clinical samples; however, C. albicans remains the most common fungal pathogen [6]. Several potential antifungals are extensively used in clinical settings for the treatment and management of Candida infections. Based on the chemical compositions, the antifungals are classified into several groups: polyenes, pyrimidine analogs, echinocandins, thiocarbamates, allylamines, azoles, and morpholines [7]. These drugs inhibit the biosynthesis of crucial molecules, such as ergosterol and β-1,3-glucan, which are essential components of the fungal cell. Some antifungals can perforate the cell wall leading to the death of the fungi [8,9]. Intriguingly, the clinical isolates are becoming increasingly resistant to the available antifungals, which pose a threat to the treatment and management of Candida infections, especially bloodstream infections [10,11].
The drug resistance in C. albicans has been studied extensively in the last two decades to comprehend the underlying mechanism. It seems to have developed drug resistance via overexpression of drug transporters, alterations of drug targets, utilization of compensatory and catabolic pathways, and biofilm formation [11][12][13]. However, the ABC (ATP-binding cassette) (Cdr1p, Cdr2p) and major facilitator superfamily (MFS) (Mdr1p) transporters are considered the major contributors to drug resistance in C. albicans [13,14].
Moreover, the modifications or alterations of biosynthetic pathways significantly contribute to drug resistance in this fungal pathogen. For example, the azole drugs (including fluconazole) inhibit the enzyme 14alpha lanosterol demethylase (encoded by the ERG11 gene) required for the biosynthesis of ergosterol. However, multiple mutations in the ERG11 gene (encoding lanosterol 14α-demethylase) make inactive these drugs against C. albicans [15]. In addition, mutations in ERG3 (encoding sterol Δ 5,6 desaturase) render resistance to Candida cells as the mutated enzyme fails to convert 14α-methylated sterols into toxic 3,6-diol derivatives [16]. Moreover, the effect of the toxic compound, 5fluorocytosine (5-FC), is nullified by incorporating the mutations in the FUR1 gene encoding uracil phosphoribosyltransferase [17]. The mutations in the FSK1 gene encoding a subunit of β-1,3-glucan synthase complex render the Candida cells resistant to echinocandins [18]. In addition, many azole-resistant clinical isolates of C. albicans acquired gain-of-function mutations in the transcription factors: Tac1p, Mrr1p, and Upc2p [19,20], which can counteract environmental stress and survive in adverse conditions. Furthermore, pathogenic yeast C. albicans chromosomal nondisjunction causes genetic changes in response to environmental cues [21,22]. This phenomenon generates aneuploidies that can adapt to stressful conditions for survival. In the presence of fluconazole, nondisjunction is observed in C. albicans, giving rise to Chr4 monosomy and Chr3 trisomy. The strains bearing these alterations become resistant to fluconazole [23]. Interestingly, in the absence of a complete sexual cycle, C. albicans resorts to a parasexual cycle in which two diploid cells generate a tetraploid. Subsequently, the tetraploids undergo a concerted chromosome loss and generate multiple aneuploidies, i.e., it can generate numerous changes in the genome, including but not limited to genome organization and variations in chromosome copy number. The Candida cells grown under high concentrations of fluconazole can incorporate numerous changes in the whole genome [23]. Therefore, the presence of Chr4 monosomy in fluconazole-resistant mutants might not confer fluconazole resistance.
Hence, a systematic chromosomal truncation approach should be applied to validate Chr4 monosomy and its relation to fluconazole resistance. This method was successfully applied to understand the regulation of Lsorbose utilization in C. albicans [24,25]. Therefore, in this study, we adopted the telomere-mediated chromosomal truncation method [24] to truncate Chr4 and assess the response of the isoform against antifungals. Herein, we have carried out eight systematic truncations in one of the homologs of Chr4, whereas the second homolog remains intact. Next, the strains carrying the truncated Chr4 were tested against three classes of commonly used antifungals: azoles, polyenes, and echinocandins. This systematic study suggested that Chr4 may not be involved in drug resistance. However, it could play an essential role in the drug tolerance of this pathogen.
Yeast extract/peptone/dextrose (YPD) and synthetic media were prepared as described previously [28]. Uridine was added at a concentration of 50 μg/mL to the media as required. All the strains were routinely grown at 30°C. Fluconazole (Sigma, USA) solution (10 mg/mL) was prepared in dimethyl sulfoxide (DMSO). The bacterial strains were grown in YT media (0.5% sodium chloride, 0.5% yeast extract, and 1% tryptone) at 37°C. The E. coli strains containing plasmids were grown in YT media containing 100 μg/mL ampicillin. The stock solution of zymolyase (Sigma) (1 mg/mL) was prepared in 15% glycerol and stored at − 20°C. Mueller-Hinton agar plates (Himedia, Mumbai, India) were used for minimum inhibition concentration (MIC) and disc diffusion assays.

Molecular biology methods
Molecular biology methods, including restriction digestion of plasmids, gel elution of DNA fragments, ligation of DNA fragments into vectors, and polymerase chain reaction (PCR), were carried out as described previously [29]. Plasmids from E. coli were isolated using the alkaline lysis method [30]. E. coli transformation was carried out using the calcium chloride method [29], and C. albicans transformation was performed using the spheroplast method [31,32].

Vectors and plasmid constructions
The pSFU1 plasmid containing a URA3 flipper was a kind gift from J. Morshhauser [33]. The plasmid "BSA" containing C. albicans telomere was obtained from McEachern and Hicks [34]. The plasmids pRC3915 (integrative plasmid) and pRC2312 (replicative plasmid) were obtained from Cannon et al. [35]. The plasmid pKA05 was constructed by subcloning a 0.75-kbp SalI-SacI fragment containing Candida telomere from the plasmid "BSA" into pSFU1 at the NotI-SacI site as described previously [25] and used as the basic plasmid for generating truncation constructs ( Supplementary Fig.  S1A). For truncating Chr4 at the intended sites, the DNA sequences were retrieved from the Candida Genome Database (CGD) [36], and the primers were designed (Table 1) to amplify the mapping sequence (MS). The PCR products were first cloned into pTZ57R/T vector using InsTA PCR Cloning kit (Thermo Fisher Scientific, Vilnius, Lithuania) and subsequently into pKA05 to generate the truncation constructs. The plasmid pKA484 was constructed by inserting a 2.2-kbp PstI-BamHI fragment (containing the open reading frame Orf19.3120) into the vector pRC2312. This construct was used for overexpression of Orf19.3120. The plasmids used in this study are listed in Table 2.

Chromosomal preparation, separation, and band analysis
In order to verify the karyotypes of the generated Candida strains, intact chromosomes were prepared as described previously [32]. Briefly, fresh C. albicans cells were grown in YPD liquid media to the cell density of 10 7 cells/mL and harvested by centrifugation. Cells were washed with 50 mM ethylenediaminetetraacetic acid (EDTA) pH 7.5 and resuspended at 1 × 10 9 cells/mL. For making the plugs, 200 μL cells, 20 μL zymolyase, and 250 μL of 1.2% low-melting agarose were mixed vigorously in a 1.5-mL tube and poured into the molds (Bio-Rad Laboratories, USA) as 100 μL aliquots/well and solidified at room temperature for 20 min. First incubation was at 37°C for 18 h in solution containing 0.5 M EDTA (pH 9.0), 25 mM Tris-HCl (pH 8.0), and 4% βmercaptoethanol, and the second incubation was at 55°C in 0.5 M EDTA (pH 9.0), 25 mM Tris-HCl (pH 8.0), 1% N-lauroylsarcosine, and 0.01% proteinase K for 48 h.
The pulse-field gel electrophoresis system CHEF-DR II (Bio-Rad) was used in separating the chromosomes as described previously [32]. Agarose gels were stained using SYBR Green I nucleic acid gel stain (Sigma) for 30 min. The images of the gels were analyzed using Quantity One Software, and the intensity of the chromosome bands was analyzed by Image Lab Software Version 5.1 (Bio-Rad).

Minimum inhibitory concentration (MIC) assay
The MIC for the generated Candida strains was determined according to the protocol of the Clinical and Laboratory Standards Institute (CLSI) [37]. Briefly, fresh cells were collected from YPD plates and counted under a light microscope. Approximately 2 × 10 6 cells were mixed with 0.7% molten agar and poured onto Mueller-Hinton agar plates. The MIC strips were placed in the middle of the plate and incubated at 30°C for 24 h. The images were captured using Quantity One Software. Ezy MIC TM strips were used for three classes of antifungals: fluconazole (0.016-256 μg/mL), amphotericin B (0.002-32 μg/mL), and caspofungin (0.002-32 μg/mL) (Himedia, Mumbai, India).

Spot assay against fluconazole
For spot assay on fluconazole plate, young cells were collected from YPD plates streaked directly from − 80°C . The cells were counted under a light microscope and spotted on synthetic dextrose (SD) plates containing fluconazole as 10-fold serial dilutions.

Microdilution assay against fluconazole
We carried out broth microdilution assay against fluconazole as described previously [37,38]. The drug sensitivity test was carried out for the strains carrying truncations in one of the homologs of chromosome 4. Briefly, young cells were collected from the YPD plate, washed, and counted under a light microscope. Then, approximately 200 cells/well were inoculated into 96well flat-bottomed microtiter plate. Subsequently, the antifungal drug fluconazole was added to the wells in increasing concentration from 0 to 128 μg/mL. The wells containing only media and media plus cells (without drug) were considered negative and positive controls. All the samples were plated in triplicate and incubated at 30°C for 24 h, and optical densities (OD) were measured at 600 nm using a multi-detection microplate reader (Thermo Fisher Scientific, USA). Then, the graph of OD 600 vs. fluconazole concentrations.

Truncations of Chr4 in C. albicans
In order to elucidate the role of Chr 4 concerning drug resistance, we adopted a well-established telomeremediated chromosomal truncation approach that analyzes L-sorbose utilization in this pathogen [24,25]. To truncate Chr4, approximately 1 kbp PCR product (MS) was amplified from the intended truncation site using genomic DNA of parental strain CAF4-2, and then the truncation cassette was constructed. The exogenous construct consisted of three essential components: MS, selection marker (URA3), and Candida telomere (plasmid-borne telomere, PBT). After transformation into Candida strain, the truncation cassette replaces the entire portion of one homolog of Chr4 from the intended site to the telomere (Fig. 1). Furthermore, Chr4 is 1603 kbp long in which the centromere is located at 992-996.2 kbp position according to assembly 22 of the Candida Genome Database [36]. Since the left portion of Chr4 is longer (~992 kbp) than the right portion (~606 kbp), we performed the first truncation (truncation 1) at 969.925 kbp position (left side of the centromere) to remove approximately 970 kbp of the left portion as shown in Fig. 1a. The plasmid pKA52 was digested with KpnI-SacI to release the truncation cassette, which was transformed with the wild type strain CAF4-2. The ura + transformants were screened by PCR using specific  1B). Subsequently, we verified the truncated homolog of Chr4 by running a contour-clamped homogenous electric field (CHEF) program; nine positive candidates were obtained ( Table 3). The chromosomal separation of one representative candidate of truncation 1 (969.925 kbp position) is shown in Fig. 2a. The size of the intact homolog of Chr4 is 1603 kbp, which is slightly above the 1532 kbp size marker of S. cerevisiae. However, the expected size of the truncated homolog of Chr4 was approximately 638 kbp (including URA3 flipper) and between 577 kbp and 784 kbp of S. cerevisiae markers (Fig. 2a). The PCR verification results for truncation 1 are shown in Fig. 2b. The primers KC49/KC102 could amplify the 1.5 kbp PCR product if truncation occurs at 969.925 kbp position (truncation1). The Candida strain carrying truncated Chr4 (truncation 1) showed a 1.5 kbp band on agarose gel electrophoreses, whereas wild type  strain (no truncation) did not produce any PCR product (Fig. 2b).
To determine whether the Candida strain carrying truncation 1 becomes resistant, we conducted the MIC test with three antifungal classes: fluconazole (azole), amphotericin B (polyene), and caspofungin (echinocandin). The MIC values for truncation 1 (at 969.925 kbp position) against fluconazole, amphotericin B, and caspofungin were 0.25, 1.0, and 0.125 μg/mL, respectively, whereas those for the wild type strain were 0.19, 0.75, and 0.064 μg/mL, respectively. These findings suggested that the strain carrying the truncated version of Chr4 (truncation 1) is sensitive to these antifungals, similar to that observed for the wild type strain (truncation 1 in Fig. 3, Supplementary Table S1).
As the removal of the left portion of Chr4 failed to produce any resistant phenotype, we carried out seven additional truncations (2-8) on the right portion of Chr4. These seven truncations were performed at 1002.852, 1102.087, 1201.989, 1301.783, 1369.883, 1529.969, and 1542.907 kbp positons on the right portion of Chr4 (Fig. 4). The karyotype of one   Fig. 5a. The truncated homolog of Chr4 (approximately 1305 kbp) runs just above Chr5 (size of Chr5 is 1191 kbp) and below the 1532 kbp size marker of S. cerevisiae; this truncation was further validated by PCR (Fig. 5b). Moreover, we attempted to truncate both left and right portions of Chr4 in the same strain to mimic Chr4 monosomy. However, we did not obtain any candidate after screening approximately 100 transformants. This failure could be attributed to two compelling reasons: First, the generation of monosomy of Chr4 may be a low-frequency event in C. albicans, and second, the deletion of both the left and right portions of Chr4 could be lethal due to the presence of recessive mutations or allelic differences. In summary, we performed eight systematic chromosomal truncations in one of the two homologs of Chr4 (Table 3). Truncations 1 and 2 removed the left and  .3120 in drug resistance; it encodes a half-size PDR-subfamily ABC (ATP-binding cassette) transporter [36,40].

Determination of MIC values for the generated strains
We determined the MIC values for all the eight Candida strains carrying a truncated version of one homolog of Chr4. These strains were tested using MIC strips for fluconazole, amphotericin B, and caspofungin (Fig. 3

Microdilution assay against fluconazole
We carried out a microdilution assay against fluconazole for all the eight strains carrying truncations in one homolog of Chr4. The Candida strain CAF4-2 transformed with the integrative plasmid pRC3915 served as a control. Readings were recorded 24 h after inoculation, and graphs were plotted against fluconazole concentrations (Fig. 6). These results demonstrated that the generated strains remain sensitive to fluconazole.

Spot assay against fluconazole
We spotted Candida strains carrying truncated homolog of Chr4 on SD plates containing various concentrations of fluconazole: 8, 16, 32, and 64 μg/mL (starting with 10 7 cells/mL). We considered high concentrations of fluconazole due to its fungistatic nature. The plates were incubated at 30°C, and images were captured every 24 h for 3 days. The spotting assay of the generated strains on fluconazole showed distinct phenotypes when incubated for a prolonged period. The strain-sensitivity to fluconazole in 24 h was similar to that in the wild type strain; however, differential sensitivity towards fluconazole was detected in 48 h, which became prominent in 72 h (Fig. 7). The strain carrying truncation 5 was most sensitive among the strains. However, the strain carrying truncation 4 showed the least sensitivity to fluconazole, followed by truncation 8, while the other five stains did not differ markedly compared to the wild type strain. This analysis suggested that some truncated homologs of Chr4 were tolerant to fluconazole, although the MIC values were in the sensitive range.

Disc diffusion assay
We conducted a disc diffusion assay for eight strains against seven antifungals, including five azoles and two polyenes. All the strains showed sensitivity to these drugs, forming a clear zone of inhibition (Table 4,  Overexpression of Orf19.3120 The overexpression of many genes renders the Candida strains resistant to multiple antifungals. Since ORF Orf19.3120 was predicted to encode a half-size PDRsubfamily ABC transporter [40], we overexpressed the molecule in the CAF4-2 Candida strain to test its response to fluconazole. Thus, we transformed CAF4-2 with the plasmid pKA484 (Orf19.3120 cloned in pRC2312). The transformants were detected on a fluconazole plate incubated at 30°C for 2 days. Consequently, we found that the overexpression of Orf19.3120 does not render the strain resistant to fluconazole.
However, it grew slightly more than the control strain, indicating a putative role in fluconazole resistance (Fig.  8).

Discussion
In the history of C. albicans, the role of aneuploidy formation exhibited an efficient and effective means to generate critical genome modifications in response to environmental cues [41]. These changes are critical, such as altered chromosome copy number and translocation of a segment or truncation of a chromosome that exhibits multiple phenotypes, including drug resistance. In this study, we adopted a systematic chromosomal truncation approach to truncate Chr4 and assessed the strains for antifungals responses. The eight chromosomal truncations on one homolog of Chr4 generated Candida strains that were tested against antifungals. Either of the two homologs of Chr4 has the same probability of truncation as both are the same except for single nucleotide polymorphisms (SNPs) [42]. Truncation in one homolog generates monosomy of Chr4 with respect to the portion removed. For example, truncation at 969.925 kbp position on the left portion leaves only one left portion of the second homolog giving rise to the strain monosomy of Chr4 with respect to the left portion. The removal of the left portions of both homologs is not possible as there is one essential gene in approximately every 16 kbp of chromosomal stretch [36]. The generated Candida strains carrying the truncated homolog of Chr4 were assessed against multiple antifungals using different methods, such as determination of (echinocandin) for these strains were similar to those for the wild type. These results suggested that strains carrying truncated Chr4 remain sensitive to these drugs. Furthermore, microdilution and disc diffusion assays were carried out for all eight strains to assess their responses to antifungals. The disc diffusion assay results showed slight variation in the responses to antifungals. Most of the generated strains showed slightly less sensitivity to the drugs compared to the wild type (Table 4). Interestingly, when strains were spotted on the plates containing increasingly higher concentrations of fluconazole, the Candida strain carrying truncation 4 (1201.989 kbp position on Chr4) showed better growth at fluconazole 8-64 μg/mL. However, the MIC value of this strain was the same as that of wild type (0.19 μg/mL for fluconazole). These findings indicated that the MIC values cannot predict the tolerance of a specific strain to antifungals. Previous observations revealed that antifungal tolerance operates at different pathways in this fungal pathogen. For example, the Rim pathway participates in antifungal tolerance through Hsp90p and Ipt1p [43]. However, tolerance to caspofungin can be mediated through the regulation of FSK gene expression and cell wall remodeling [44]. The antifungal tolerance could also be a subpopulation effect in which clinical isolates of C. albicans grow beyond the MIC. This extended growth is often associated with persistent candidemia [45]. Fluconazole is commonly used as the first-line drug for the treatment and management of Candida infections. Therefore, fluconazole tolerance can adversely affect the treatment of C. albicans bloodstream infections, and the patients could be at a high risk of morbidity and mortality [46]. Chr4 does not contain any drug resistance gene except the ORF Orf19.3120 (coordinates: 1538056-1539795 on Chr4), which encodes a half-size PDR-subfamily ABC transporter. To assess the role of Orf19.3120 in drug resistance, truncations 7 and 8 were performed on either side of this ORF at 1529.969 kbp and 1542.907 kb positions, respectively. The strain carrying truncation 8 grew slightly better than the strain carrying truncation 7 (Fig.  7). The overexpression of specific drug transporter genes, such as CDR1 and CDR2 produced a drugresistant phenotype [47]. Hence, we overexpressed Orf19.3120 in Candida strain CAF4-2 and assessed its phenotype on the plate containing fluconazole.
Interestingly, the Candida strain with overexpressed Orf19.3120 showed an optimal growth in the presence of fluconazole (Fig. 8), thereby suggesting that Orf19.3120 may have a minor role in drug resistance. Conversely, the strain lacking this ORF was sensitive to fluconazole in the presence of silver nanoparticles [48].
Genomic plasticity is one of the major characteristics of C. albicans [49]. Rapid unusual genome changes in this pathogen might occur when mitotic cells are propagated in vitro as well as in vivo. Also, aneuploidy is detected in some pathogenic fungi. These phenomena suggested that variations in chromosome organization and copy number are common, rapid, and efficient means to generate diversity in response to stressful conditions, including the presence of drugs [49]. Moreover, Chr4 trisomy has been reported in a clinical isolate with a putative role in elevated fluconazole resistance. However, Chr4 trisomy failed to increase fluconazole resistance in the background of standard Candida strain SC5314 [50] used for sequencing the Candida genome. Furthermore, some clinical isolates harbor trisomy of Chr4 and Chr7 but their roles in drug resistance are not yet ascertained [51]. In addition, the presence of Chr4 monosomy in any clinical isolates of C. albicans and its association with drug resistance also has not been reported. Therefore, Chr4 monosomy or trisomy cannot be considered responsible for drug resistance. However,   in specific genetic backgrounds with predisposing mutations or genomic changes, these conditions could be implicated in drug resistance.

Conclusion
In summary, we performed systematic chromosomal truncations of Chr4 of Candida albicans and assessed their responses to antifungals. The partial or segmental aneuploidies generated were challenged against three classes of antifungals, such as azoles, polyenes, and echinocandin. All the strains carrying truncated Chr4 were sensitive to these antifungals, similar to that for the wild type strain. However, some truncations exhibited a highly tolerant phenotype against fluconazole, a frontline antifungal drug. Drug tolerance was also observed when the strains were incubated in fluconazole for > 2 days. Therefore, C. albicans uses Chr4 as a drug-tolerant arsenal, which would benefit its propagation and colonization at different niches, posing a threat to candidiosis treatment and management. Finally, we concluded that it is unlikely that Chr4 is involved in drug resistance in this fungal pathogen. However, it may participate in developing resistance to antifungals in specific genetic backgrounds. Nevertheless, truncations 4 and 8 would be explored in future studies to unveil the genes responsible for fluconazole tolerance. The strains will also be tested against other commonly used antifungals to deduce their efficacy with respect to these two truncations. In addition, we aim for truncations in the left portion of Chr4 to identify the chromosomal segments involved in drug resistance/tolerance. Further studies would be required to compare the genes' expression in the strains carrying truncated Chr4, which would be valuable in understanding the Candida biology.
Additional file 1: Supplementary Table S1. Minimum inhibitory concentration (MIC) for the strains carrying truncated chromosome 4. Supplementary Fig. S1. (A) Schematic diagram of plasmid pKA05 [25]. This plasmid was used as a backbone plasmid for generating truncation constructs. Mapping sequences (MS) from truncation sites of chromosome 4 were inserted at K-Xh (KpnI-XhoI) and the resulting plasmids were digested with KpnI-SacI and transformed into Candida. (B) Schematic diagram of PCR verification for chromosomal truncation. Primers P1 and P2 were designed from upstream of MS of chromosome 4 and URA3 flipper, respectively. The primers amplify PCR product of expected size only when truncation occurs at the intended site on Chr4. PBT, plasmid-borne Candida telomere. Supplementary Fig. S2.