Lipid composition and cell surface hydrophobicity of Candida albicans influence the efficacy of fluconazole–gentamicin treatment

Abstract Adherence of the fungus, Candida albicans , to biotic (e.g. human tissues) and abiotic (e.g. catheters) surfaces can lead to emergence of opportunistic infections in humans. The process of adhesion and further biofilm development depends, in part, on cell surface hydrophobicity (CSH). In this study, we compared the resistance of C. albicans strains with different CSH to the most commonly prescribed antifungal drug, fluconazole, and the newly described synergistic combination, fluconazole and gentamicin. The hydrophobic strain was more resistant to fluconazole due to, among others, overexpression of the ERG11 gene encoding the fluconazole target protein (CYP51A1, Erg11p), which leads to overproduction of ergosterol in this strain. Additionally, the hydrophobic strain displayed high efflux activity of the multidrug resistance Cdr1 pump due to high expression of the CDR1 gene. On the other hand, the hydrophobic C. albicans strain was more susceptible to fluconazole–gentamicin combination because of its different effect on lipid content in the two strains. The combination resulted in ergosterol depletion with subsequent Cdr1p mislocalization and loss of activity in the hydrophobic strain. We propose that C. albicans strains with different CSH may possess altered lipid metabolism and consequently may differ in their response to treatment.


| Strains and growth conditions
The C. albicans strains used in the present study are listed in Table 1. CAF2-1 and CAF4-2 were kind gifts from Professor D. Sanglard (Lausanne, Switzerland). KS052, KS063, KS068 and KS069 were constructed during our study. Strains were pregrown at 28 C on yeast extract peptone dextrose (YPD) medium (2% glucose, 1% soy peptone, 1% yeast extract) in a shaking incubator (120 rpm). Agar in a final concentration of 2% was used for medium solidification.
For the experiments, cells were grown in 20 ml of YPD medium (28 C; shaking: 120 rpm; starting A 600 = 0.1; with or without fluconazole, gentamicin, or the combination of both drugs added at t = 0 hr) until they reached the stationary phase (24 hr). Cells were then centrifuged (4.5 k rpm, 5 min), washed twice (4.5 k rpm, 5 min) with either phosphate-buffered saline (PBS) or 50 mM HEPES-NaOH buffer (pH 7.0), and resuspended in either PBS or HEPES-NaOH to the indicated A 600 .
C. albicans strains were transformed by electroporation with the linear gel-purified CDR1-GFP-NAT1 or CDR2-GFP-NAT1 cassettes according to the protocol by Sasse et al. (2011). The presence of the NAT1 marker was verified using the primer pair, NAT1_F and NAT1_R

| Cell surface hydrophobicity
This assay was performed according to Biniarz et al. (2015), with modifications. Briefly, 1 ml of hexadecane was added to the C. albicans suspensions (PBS, A 600 = 0.5, 4 ml). The samples were shaken for 3 min, and the phases were allowed to separate for 45 min. The A 600 of the aqueous phase was then measured, and CSH was calculated according to Biniarz et al. (2015).

| Sterol analysis in PMs
Sterol analysis was performed as described previously (Singh, MacKenzie, Girnun, & Del Poeta, 2017 The column was heated at 100 C over 0.5 min; then, the temperature was increased to 240 C at a rate of 25 C min −1 and finally to 300 C (for 5 min) at a rate of 3 C min −1 with helium gas as a carrier (flow rate = 1 ml min −1 ; Singh et al., 2017). The temperature of the injection port was 250 C. CHOL was used as an internal standard.
Trimethylsilyl-derived ergosterol and lanosterol were analysed according to retention times and fragmentation spectra for standards. Trimethylsilyl ethers of the other sterol metabolites were identified by comparison with the NIST MS database or literature data and quantitated using a standard curve for lanosterol.

| Phospholipid analysis in PMs
Phospholipid concentrations were determined using an Agilent 1200 High-Performance
Briefly, at least 5 g of CAF2-1 or CAF4-2 cells were harvested and vigorous stirring at 4 C for 4 h, the CHCl 3 layer was concentrated using nitrogen gas.

| Sterol and steryl esters analysis in LDs
Lipid samples from LDs were diluted in 1ml CHCl 3 -MetOH (1:4, vol/vol). Then, 10 μl of lipid extract was measured using an Agilent 1200 High-Performance Liquid Chromatography system and a 4500 Q-TRAP mass spectrometer equipped with an ESI source and Kinetex

| Di-4-ANEPPS assay
The PM potential (Δψ) of CAF2-1 and CAF4-2 was measured using di-4-ANEPPS fluorescent dye, according to the protocol of . For data analysis, the red-blue signal ratio was calculated by dividing the sum of fluorescence intensities (IFs) between 580 and 620 nm by the sum of IFs between 540 and 580 nm, as described previously ).

| Real time polymerase chain reaction
RNA was isolated from the CAF2-1 and CAF4-2 suspensions (PBS; A 600 = 10) using the Total RNA Mini Kit (A&A Biotechnology; Gdynia, Poland). Synthesis of cDNA and calculation of gene expression levels were performed according to Szczepaniak, Łukaszewicz, and Krasowska (2015). The following gene-specific primers were used: RDN18F and RDN18R, ERG11F and ERG11R, and CDR1F and CDR1R (Table 2).

| Microscopic studies
The strains, KS052 and KS068, were suspended in PBS, concentrated, and observed under a Zeiss Axio Imager A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp.

| Statistical analysis
At least three independent replicates were performed for each experiment. Statistical significance was determined using Student's t test (binomial, unpaired).

| C. albicans susceptibility to the fluconazolegentamicin combination depends on CSH
CSH is an important feature in the adhesion of pathogenic microorganisms to abiotic and biotic surfaces (Krasowska & Sigler, 2014). In the case of C. albicans, higher CSH causes greater tolerance towards the antiadhesive properties of biosurfactants (Biniarz et al., 2015). As clinical strains of Candida spp. differ in CSH (Silva-Dias et al.,

2015)
, we aimed to assess the effect of fluconazole with and without gentamicin on C. albicans strains with different CSH (Figure 1).
At a fluconazole concentration range of 0.5-2 μg/ml, the hydrophobic C. albicans CAF4-2 strain had increased resistance F I G U R E 1 (a) Percentage of viability of the Candida albicans CAF2-1 and CAF4-2 strains in the presence of fluconazole (0-16 μg/ml) after culture in yeast extract peptone dextrose (YPD) medium for 24 hr (mean ± SD, n = 6). Statistical analysis was performed by comparing the percentage viability of both strains at the same concentrations; (b) Percentage of cell surface hydrophobicity (CSH, presented as mean ± SD, n = 3) of the C. albicans CAF2-1 and CAF4-2 strains grown in the following conditions: C-control without antimicrobial agents, F-treated with fluconazole 4 μg/mL, G-treated with gentamicin 256 μg/ml, FG-simultaneously treated with fluconazole 4 μg/ml and gentamicin 256 μg/ml. Statistical analysis was performed by comparing either untreated CAF4-2 with untreated CAF2-1 or treated strains with untreated strains; (c) Percentage of viability of the C. albicans CAF2-1 and CAF4-2 strains in the presence of fluconazole (1-8 μg/ml, chart legend) and in presence of gentamicin 128 or 256 μg/ml (G 128 and G 256, respectively; mean ± SD, n = 6). Statistical analysis was performed by comparing viability at the same fluconazole concentrations between samples treated and untreated with gentamicin. Statistical significance in all cases is presented as follows: *p < .05; **p < .01; ***p < .001 compared with the hydrophilic CAF2-1 strain (Figure 1a). At higher fluconazole concentrations, the vulnerability of both strains was similar. This effect might have occurred due to prolonged fluconazole treatment, which can induce a resistant C. albicans phenotype (Morschhäuser, 2016). To overcome this effect, we simultaneously tested the effect of the antibacterial drug, gentamicin, and the antifungal drug, fluconazole (Figure 1b-c). We observed that treatment with fluconazole and fluconazole-gentamicin combination had different effects on the hydrophobicity of both strains (Figure 1b).
Under control conditions (Figure 1b, trial: C), the CAF4-2 strain was over threefold more hydrophobic than CAF2-1, which is similar to what was shown in our previous studies (Biniarz et al., 2015). greater viability reduction. At a higher gentamicin concentration of 256 μg/ml, a decrease of viability was present for both strains, but was greater for the CAF4-2 strain (Figure 1c).

| Lipid metabolism is affected differently in hydrophilic and hydrophobic C. albicans strains after treatment with the fluconazole-gentamicin combination
The fluconazole resistance that is acquired in clinical C. albicans isolates by over-or down-expression of the ERG11 gene leads to alterations in the fungal sterol profile (Alizadeh et al., 2017;Mukherjee, Chandra, Kuhn, & Ghannoum, 2003). In order to understand the different response of the hydrophobic (CAF4-2) and hydrophilic (CAF2-1) strains towards the fluconazole-gentamicin combination (Figure 1), we evaluated ERG11 gene expression ( Figure 2) and the sterol profile (Table 3)  To compare the differences in response of both strains to treatment with the antimicrobial agents, we separately calculated 2 -ΔΔ CT values for ERG11 expression by normalizing both control conditions (strains untreated) to the value = 1 (Figure 2b). Both the CAF2-1 and the CAF4-2 strains responded similarly to fluconazole treatment in terms of ERG11 gene expression, with both increasing about fourfold.
However, Erg11p activity was inhibited by fluconazole in both cases, as indicated by the accumulation of lanosterol and the appearance of the atypical sterol metabolites, 24-methyl-lanosterol and eburicol (Table 3). Martel et al. (2010) reported previously that blocking the activity of lanosterol 14α-demethylase (Erg11p) resulted in accumulation of lanosterol and its methylated derivatives. In the hydrophilic CAF2-1 strain, despite the increased level of the ERG11 transcript, synthesis of F I G U R E 2 (a) Relative ERG11 gene expression in the Candida albicans CAF4-2 strain compared with C. albicans CAF2-1. Statistical analysis was performed by comparing both experiments. (b) Relative ERG11 gene expression in the C. albicans CAF2-1 and CAF4-2 strains grown in the following conditions: control without antimicrobial agents, Flc-treated with fluconazole 4 μg/ml, Gent-treated with gentamicin 256 μg/ml, Flc + Gent-simultaneously treated with fluconazole 4 μg/ml and gentamicin 256 μg/ml. Statistical analysis was performed by comparing the ERG11 expression level of treated with untreated strains, separately. Gene expression levels are reported as mean ± SD of 2 −ΔΔCT values (n = 6); normalized to 1 for CAF2-1 in (a) or separately to CAF2-1 and CAF4-2 in (b). Statistical significance in all cases is presented as follows: *p < .05; **p < .01; ***p < .001 the pathway product, ergosterol, was fully blocked by fluconazole (Table 3). In the hydrophobic CAF4-2 strain, Erg11p activity was not fully inhibited by fluconazole due to the residual presence of ergosterol.
The higher ERG11 expression in this strain may be one of the reasons for higher tolerance of CAF4-2 towards fluconazole (Figure 1a).
Expression of the ERG11 gene after treatment with gentamicin alone was about fivefold higher for the CAF2-1 strain (Figure 2b). This resulted in about 10-fold higher accumulation of ergosterol, a high level of lanosterol and the presence of 24-methyl-lanosterol in this strain (Table 3). Prokhorova et al. (2017) reported that aminoglycosides including gentamicin not only target bacterial ribosomes but also interact with eukaryotic 80S ribosomes leading to inhibition of nearly every aspect of protein synthesis, which most likely may include biosynthesis, degradation, and targeting of ergosterol. These findings may indicate that in CAF2-1, despite higher expression of the ERG11 gene and a higher level of ergosterol, demethylation of lanosterol is partially inhibited ( Table 3). The hydrophobic CAF4-2 strain responded differently to gentamicin.
Despite a twofold higher expression of the ERG11 gene (Figure 2b), the level of ergosterol was reduced by about 70% compared with the untreated hydrophilic CAF4-2 strain (Table 3). We did not observe an increase in the concentration of lanosterol or its methylated derivatives (Table 3). This indicates that gentamicin may have inhibited ergosterol biosynthesis in the CAF4-2 strain but at a different step than where demethylation of lanosterol occurs.
For both strains treated with the fluconazole-gentamicin combination, the presence of lanosterol and methylated lanosterol derivatives indicated partial inhibition of Erg11p activity (Table 3).
However, this combination of drugs resulted in much higher expression of ERG11 in the CAF2-1 strain (almost 25-fold higher) than in CAF4-2 (5-fold higher; Figure 2b). This in turn resulted in ergosterol overproduction in CAF2-1. In the CAF4-2 strain, the investigated combination of antimicrobial agents resulted in ergosterol production that was even less than after treatment with fluconazole alone (Table 3).
This may lead to different responses in the two strains to gentamicin alone and could be one of the reasons why CAF4-2 is more sensitive than CAF2-1 to the fluconazole-gentamicin combination (Figure 1c).
The proper ratio of sterols to other lipids in PMs is necessary to maintain physiological structure and fluidity of the PM (Simons & Lkonen, 2000). In Eukaryota, the overproduction and elevated levels of sterols were reported to exhibit toxic effects towards the cells (Shimada et al., 2019;Tabas, 2002). In yeast cells, excessive ergosterol is either secreted into the extracellular environment or esterified and stored in lipid droplets (LDs) (Hu et al., 2017;Spanova et al., 2012).
Interruption with ergosterol biosynthesis by inhibiting squalene synthase was already reported to affect accumulation LDs in C. albicans (Ishida et al., 2011). However, the effect of azole drugs on  (Table 4).
In the hydrophobic CAF4-2 strain, we have observed a similar trend of ergosterol and steryl esters accumulation as in the case of ergosterol in the PM (Table 3). The highest levels of either ergosterol and steryl esters were observed in untreated CAF4-2 cells and the lowest after treatment with fluconazole-gentamicin combination (Table 4). On the other hand, the hydrophilic CAF2-1 strain excessively accumulated both ergosterol and steryl esters after fluconazole treatment (Table 4). Kim et al. (2004) reported that a homologue of ergosterol O-acyltransferase gene, ARE2, which controls the storage and decomposition of sterols in lipid droplets, is induced in C. albicans treated with ketoconazole. Ergosterol was not detected in the CAF2-1 PM, treated with fluconazole (Table 3), so it can be speculated that fluconazole impairs ergosterol transport to PM and promotes its deposition in LDs. The opposite situation was observed treating CAF2-1 cells with gentamicin, where a decrease of ergosterol and steryl esters was observed in LD fraction (Table 4) and an increase in PM (Table 3). Gentamicin was reported to affect lipid T A B L E 3 Sterols (μg/mg dry mass of isolated plasma membrane lipids, mean ± SD, n = 3) in Candida albicans CAF2-1 and CAF4-2 strains grown in the following conditions: control without antimicrobial agents, Flc-treated with fluconazole 4 μg/ml, Gent-treated with gentamicin 256 μg/ml, Flc + Gent-simultaneously treated with fluconazole 4 μg/ml and gentamicin 256 μg/ml Note. Statistical analysis was performed by comparing the sterols of untreated CAF4-2 with untreated CAF2-1 (included in the CAF4-2 control row) or by comparing a separately treated strain with an untreated strain (included in Flc, Gent, and Flc + Gent rows). Abbreviation: ND, not detected.  (Table 4). Singh, Mahto, and Prasad (2013) reported that fluconazole- The untreated CAF4-2 strain had 5% more PE and 5% less PI than the CAF2-1 strain (Figure 3a). Only slight differences in composition of PA, PG, or PS were present after treating both strains with the antimicrobial agents. We observed an increase in the PC concentration and a decrease in the PE concentration in the CAF2-1 strain treated with fluconazole, which is in agreement with previous findings (Singh et al., 2013). However, we saw an approximate 17% increase in the PI concentration in CAF4-2 treated with fluconazole and about an 11% increase when the strain was treated with the fluconazole-gentamicin combination (Figure 3a). Gentamicin interacts with PM by specific binding with PI and other negatively charged PLs (Kovács et al., 2012;Lesniak, Pecoraro, & Schacht, 2005)

| Activity, localization, and expression of the Cdr1 transporter are altered in the hydrophobic strain treated with the fluconazole-gentamicin combination
In Saccharomyces cerevisiae cells, reduction in Δψ and PM depolarization causes mislocalization of ergosterol and PM proteins from charged membrane domains (Grossmann, Opekarová, Malinsky, Weig-Meckl, & Tanner, 2007). On the other hand, ergosterol depletion causes mislocalization of C. albicans' Cdr1p from PM when expressed in S. cerevisiae deficient in ergosterol (Pasrija, Panwar, & Prasad, 2008). The fluconazole-gentamicin combination differentially affected ergosterol content (Table 3) and Δψ (Figure 3b) in hydrophobic and hydrophilic C. albicans strains, and so, we checked the effect of this composition on the expression, localization, and activity of Cdr1p in both strains (Figure 4).
No differences in CDR1 gene expression or Cdr1p localization or activity were present after treating CAF2-1 cells with gentamicin. In the case of the hydrophobic CAF4-2 strain, higher CDR1 gene expression ( Figure 4b) and transporter activity (Figure 4d) occurred.
Treatment with the fluconazole-gentamicin combination resulted in almost 25-fold higher CDR1 expression in the CAF2-1 strain ( Figure 4b), leading to higher fluorescence of the Cdr1p-GFP protein ( Figure 4e). Previously, we observed Cdr1p-GFP mislocalization in stationary phase cells (Szczepaniak et al., 2015), which also occurred here ( Figure 4e). However, after treatment with the fluconazolegentamicin combination, we also noticed dispersion of the protein inside some CAF2-1 cells. Cdr1p-GFP localized properly into PMs in most CAF2-1 cells, most likely because fluconazole-gentamicin treatment leads to overproduction of ergosterol in the CAF2-1 strain (Table 3), which is crucial for proper Cdr1p localization (Pasrija et al., 2008). The efflux activity of Cdr1p in CAF2-1 cells treated with the fluconazole-gentamicin combination was similar to the activity in untreated cells. This is probably due to two effects, being F I G U R E 3 (a) Percentage distribution of phospholipids (phosphatidic acid, PA; phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylglycerol, PG; phosphatidylinositol, PI and phosphatidylserine, PS) in PMs isolated from CAF2-1 and CAF4-2 strains grown in the following conditions: control without antimicrobial agents, Flc-treated with fluconazole 4 μg/ml, Gent-treated with gentamicin 256 μg/ml, Flc + Gent-simultaneously treated with fluconazole 4 μg/ml and gentamicin 256 μg/ml. Included values are means of three independent experiments, SD is not shown but was ≤5% in all cases. (b) PM potential (Δψ) expressed as RB values (mean ±SD, n = 4) calculated from the fluorescence spectra of di-4-ANEPPS incorporated into the PMs of the Candida albicans CAF2-1 and CAF4-2 strains grown in the following conditions: control without antimicrobial agents, Flc-treated with fluconazole 4 μg/ml, Gent-treated with gentamicin 256 μg/ml, Flc + Gentsimultaneously treated with fluconazole 4 μg/ml and gentamicin 256 μg/ml. Statistical analysis was performed by comparing cells treated with antimicrobial agent(s) with the corresponding untreated control (*p < .05; **p < .01; ***p < .001) delocalization of the transporter in some cells and Cdr1p overproduction (visible as more intense Cdr1p-GFP fluorescence) in the remainder of the cells (Figure 4).

| CONCLUSIONS
Differences in the CSH of C. albicans may be associated with changes in lipid metabolism and Cdr1 transporter activity and result in resistance to fluconazole or the synergistic combination of fluconazole with other drugs. In our study, the hydrophobic CAF4-2 strain was more resistant to fluconazole due to ergosterol overproduction and ERG11 gene overexpression, as well as overproduction and higher activity of the Cdr1 transporter. However, this strain was more susceptible to the synergistic effect of fluconazole with gentamicin, which resulted from substantial ergosterol depletion with treatment, as well as the mislocalization and loss of activity of the Cdr1 efflux pump.