The Role of ERG11 Point Mutations in the Resistance of Candida albicans to Fluconazole in the Presence of Lactate

Candidiasis refers to both superficial and deep-tissue fungal infections often caused by Candida albicans. The treatment of choice for these infections is the use of azoles, such as fluconazole (FLC). However, the increased use of antifungal agents has led to the emergence of azole-resistant isolates of C. albicans. Thus, the development of alternative drugs that are more efficient and with a better toxicological profile is necessary. This study aimed to determine the susceptibility profile of C. albicans CAF2-1 strain to FLC in the presence of glucose or lactate. The research was also focused on single nucleotide polymorphism (SNP) and the determination of the effect of the identified point mutations on the amino acid sequence of the Erg11 protein. The results show the growth of C. albicans CAF2-1 in the presence of FLC was significantly lower in the presence of lactate than in glucose. As a result, among recorded 45 amino acid mutations, the following mutations may be associated with the reduced susceptibility of C. albicans to FLC: G10D, G10V, I11M, I11R, Y13N, F31V, L35F, A249D, Q250H, E266G, R267G, N273K, D275C, D275G, D275R. Moreover, a twice higher number of hot-spot mutations was found in the presence of glucose as a sole carbon source compared to cells grown on lactate.


Introduction
Candida albicans is an important opportunistic yeast that can cause various infections in humans, including superficial infections, such as oral or vaginal diseases, and life-threatening systemic infections [1]. Candidal infections are called candidiasis and have emerged as important public health problems. In particular, invasive candidiasis is associated with high rates of morbidity and mortality in humans [2,3].
Unfortunately, the emergence of antifungal-resistant isolates constitutes a significant problem for treatment strategies [7]. In particular, the broad usage of azoles, such as FLC, has given rise to concerns regarding the emergence of resistance to this class of antifungal

The Carbon Source Influences the Viability of C. albicans at Increasing Concentrations of Fluconazole
First, we examined the growth curve of C. albicans CAF2-1 on different carbon sources: lactate and glucose. As it turned out, the growth of C. albicans exhibited a diminished rate in the presence of lactate in the YPL medium compared to the control condition (YPD medium) ( Figure 1A). The logarithmic growth phase was much longer, and the growth curve showed a flatter profile.
Subsequently, we monitored the growth of C. albicans CAF2-1 in YPD or YPL medium supplemented with increasing concentrations of FLC (in the range of 0-16 µg/mL). The obtained results expressed by optical density (OD 600 ) indicated a significantly weaker growth of C. albicans on the YPL medium both in the presence and without FLC ( Figure 1B). In turn, the viability analysis (expressed in % of viability) gave detailed information on the effect of FLC on C. albicans' CAF2-1 growth ( Figure 1C). We observed that lactate exerted a moderate impact on C. albicans CAF2-1 in the presence of FLC starting from 0.5 µg/mL (82% of viability), whereas the growth inhibition on glucose started at 1.0 µg/mL of FLC (87.6% of viability). Along with the increasing concentration of the drug, the viability of the C. albicans CAF2-1 strain decreased on the YPD medium. The decrease in viability of the C. albicans CAF2-1 remained constant for cultures grown in the YPL medium, along with the increased concentration of FLC. These values fluctuated for FLC concentrations of 2-16 µg/mL but fell within the range of a 40-50% reduction in C. albicans growth (Table S1). Subsequently, we monitored the growth of C. albicans CAF2-1 in YPD or YPL medium supplemented with increasing concentrations of FLC (in the range of 0-16 μg/mL). The obtained results expressed by optical density (OD600) indicated a significantly weaker growth of C. albicans on the YPL medium both in the presence and without FLC ( Figure 1B). In turn, the viability analysis (expressed in % of viability) gave detailed information on the effect of FLC on C. albicans' CAF2-1 growth ( Figure 1C). We observed that lactate exerted a moderate impact on C. albicans CAF2-1 in the presence of FLC starting from 0.5 μg/mL (82% of viability), whereas the growth inhibition on glucose started at 1.0 μg/mL of FLC (87.6% of viability). Along with the increasing concentration of the drug, the viability of the C. albicans CAF2-1 strain decreased on the YPD medium. The decrease in viability of the C. albicans CAF2-1 remained constant for cultures grown in the YPL medium, along with the increased concentration of FLC. These values fluctuated for FLC concentrations of 2-16 μg/mL but fell within the range of a 40-50% reduction in C. albicans growth (Table S1). The growth of C. albicans CAF2-1 strain on (A) YPD (glucose) and YPL (lactate) medium; in increasing concentration of FLC (µg/mL) expressed as (B) the optical density (λ = 600 nm) and (C) the % of viability (**, p < 0.01; ***, p < 0.001).
As the concentration of the antifungal agent in the medium increased, it was possible to observe a decrease in the percentage of cell viability in both types of culture. The obtained results allow us to conclusively state that in the presence of FLC, the growth of C. albicans CAF2-1 strain cells in the YPL medium is visibly lower than in the YPD medium.
Based on the results of this experiment, we decided that the following concentrations of FLC would be used in further experiments: 0 (as a control); 0.25; 1; 4 and 16 µg/mL. The protocol of cultures' preparation, cultivation and collection of biomass for point mutation testing is described in Section 4.2.2. Obtained biomass was a source of the genomic DNA isolation from C. albicans CAF2-1 to indicate point mutations resulting from various culture conditions.

FLC and Lactate
Synergistically Impact the Porosity of C. albicans CAF2-1 As we have shown previously, FLC with lactate co-actively reduces ergosterol levels in PM [18]. Hence, we investigated here if the usage of lactate and FLC synergistically contributes to the permeabilization of C. albicans CAF2-1 PM by propidium iodide (PI) staining. As PI is a membrane-impermeable fluorescent DNA stain, the fluorescent cells indicate that FLC and lactate have an impact on the permeability across the PM. In our experiment, we observed progressive permeabilization (59.44%) of PM in C. albicans CAF2-1 strain cultured on YPL with the addition of FLC ( Figure 2). In contrast, PI measurements on YPD with FLC indicated a 3.79% of permeabilization. Control conditions (culture on YPL and YPD) did not show any increased permeability of PM (data not shown). ture conditions.

FLC and Lactate
Synergistically Impact the Porosity of C. albicans CAF2-1 As we have shown previously, FLC with lactate co-actively reduces ergosterol levels in PM [18]. Hence, we investigated here if the usage of lactate and FLC synergistically contributes to the permeabilization of C. albicans CAF2-1 PM by propidium iodide (PI) staining. As PI is a membrane-impermeable fluorescent DNA stain, the fluorescent cells indicate that FLC and lactate have an impact on the permeability across the PM. In our experiment, we observed progressive permeabilization (59.44%) of PM in C. albicans CAF2-1 strain cultured on YPL with the addition of FLC ( Figure 2). In contrast, PI measurements on YPD with FLC indicated a 3.79% of permeabilization. Control conditions (culture on YPL and YPD) did not show any increased permeability of PM (data not shown).

Agarose Gel Electrophoresis, Purification of the PCR Product and Sanger Sequencing
The obtained genomic DNA was a matrix for the PCR reaction (performed according to the protocol described in Section 4.2.5), in which fragments of the ERG11 gene were amplified. The efficiency of the PCR reaction was checked using the gel electrophoresis technique (described in Section 4.2.6). The illustration below ( Figure 3) shows a pictorial result of this procedure. Expected product sizes were: 785 bp (fragment 5′′  3′) and 826 bp (fragment 3′′  5′). Next, the products were cut from the agarose gel, purified as described in Section 4.2.6 and analysed qualitatively and quantitatively. Prepared samples were sequenced by Eurofins Genomics (Ebersberg, Germany).

Agarose Gel Electrophoresis, Purification of the PCR Product and Sanger Sequencing
The obtained genomic DNA was a matrix for the PCR reaction (performed according to the protocol described in Section 4.2.5), in which fragments of the ERG11 gene were amplified. The efficiency of the PCR reaction was checked using the gel electrophoresis technique (described in Section 4.2.6). The illustration below ( Figure 3) shows a pictorial result of this procedure. Expected product sizes were: 785 bp (fragment 5 → 3 ) and 826 bp (fragment 3 → 5 ). Next, the products were cut from the agarose gel, purified as described in Section 4.2.6 and analysed qualitatively and quantitatively. Prepared samples were sequenced by Eurofins Genomics (Ebersberg, Germany).

Identification of Point Mutations in ERG11 Gene
The obtained nucleotide sequences were exported to FASTA format and aligned using the EMBOSS Needle program (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 17 April 2020) using standard settings. The identified nucleotide substitutions were collected and are presented in Table 1. Among 60 types of point mutations, 11 substitutions turned out to be silent point mutations that did not result in a codon change of the amino acid (underlined). In turn, 12 substitutions (A357C, A383C, A798G, C799A, C799R, T819M, T822G, T822M, G823Y, A824G, T826G, A828T) were located in the hot-

Identification of Point Mutations in ERG11 Gene
The obtained nucleotide sequences were exported to FASTA format and aligned using the EMBOSS Needle program (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 17 April 2020) using standard settings. The identified nucleotide substitutions were collected and are presented in Table 1. Among 60 types of point mutations, 11 substitutions turned out to be silent point mutations that did not result in a codon change of the amino acid (underlined). In turn, 12 substitutions (A357C, A383C, A798G, C799A, C799R,  T819M, T822G, T822M, G823Y, A824G, T826G, A828T) were located in the hot-spots regions (marked in bold). Eight of them occurred on the YPD medium, whereas six were on the YPL medium. Table 1. Nucleotide substitutions of the ERG11 gene identified in cultures of C. albicans CAF2-1 strain under various conditions. Hot-spot nucleotide mutations are marked in bold; previously identified mutations are marked in italics; mutations that did not result in an amino acid substitution are underlined. Since only missense mutations can modify the protein conformation, the nucleotide sequences were translated into amino acid sequences using the EMBOSS Transeq tool (https: //www.ebi.ac.uk/Tools/st/emboss_transeq/ accessed on 19 April 2020) and compared using the EMBOSS Needle program (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 19 April 2020). Among 46 identified amino acid substitutions listed in Table 2, one nonsense mutation (K251Stop) and six missense mutations (K119N, K128T, D225H, D225Y, R265G and W520R) should be distinguished. Interestingly, these mutations have been described in the literature up to now. Among the remaining 38 substitutions, 10 of them are located in the previously mentioned hot-spot regions (E266G, E266K, E266R, R267G, R267S, N273K, D275C, D275G, D275R and L276V) (marked in bold), 7 of them were found on the YPD medium and 4 of them on the YPL medium. In considering the presence of individual substitutions on each of the carbon sources, it was found that 14 of them occur only in strains growing on the YPD medium (D9E, I11T, F31V, L35F, A249D, Q250H, R264E, E266G, E266R, N273K, D275C, D275G, D275R and S506G). Moreover, 14 substitutions were found only in the strains growing on the YPL medium (D9Q, G10D, G10S, G10V, I11L, I11R, N12L, K259I, E260D, K262I, E266K, R267G, R267S and E517D). In turn, 11 substitutions were identified in both the strains growing on the YPD and YPL medium: I11A, I11G, I11M, I11S, I11V, N12I, N12Y, Y13N, R265K, L276V and N490K.

Concentration of
It should also be noted that in the group of 38 missense mutations mentioned, 26 of them were conservative mutations resulting in an amino acid change with similar properties. The remaining 12 changes were identified as non-conservative mutations resulting in amino acid substitutions with different properties than the original amino acid.

Identification of Amino Acid Substitutions as a Result of Aligning the Known Amino Acid
Sequences of the Erg11 Protein of Selected Candida Species with the Amino Acid Sequence of the Erg11 Protein of C. albicans SC5314 Using the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 24 April 2020), a local alignment of the amino acid sequence of C. albicans SC5314 strain (GenBank accession no.: AOW29509.1) was performed. The strain SC5314 was chosen for the alignment because it is a parental strain for CAF2-1 used in this study. The sequences were compared with sequences of different Candida species as well as of distinct strains of C. albicans available from biological databases. The results of alignment are shown in Tables S2 and S3 in the supplementary materials. In the case of alignments of known amino acid sequences of the Erg11 protein of selected Candida species, 63 missense mutations were identified, of which 45 were found in both FLC-resistant and FLC-susceptible strains. The substitutions Y132F, N418R, D428E and K143R were identified as mutations occurring only in strains resistant to the used antimycotic. Alignment of the Erg11p amino acid sequences belonging to selected C. albicans strains resulted in the identification of 18 missense mutations. This group included one mutation recorded in the drug-sensitive strain, and four mutations were present in both the sequences of the sensitive and resistant isolates. Thirteen mutations were found in the sequences of the strains resistant to FLC.

Discussion
The studies indicate that Candida sp., specifically C. albicans, is responsible for most fungal infections [19]. The problem is the growing number of Candida strains resistant to conventional antifungal drugs such as azoles which are the drugs of choice for most Candida infections [20]. Various virulence mechanisms developed by Candida play a role in this increasing drug resistance [11]. Current data show that nearly 7% of all systemic candidiasis indicates a reduced sensitivity to the effects of azole drugs [21]. As a result, the need to develop new therapeutic methods is becoming stronger and stronger. Significant efforts have been made toward the chemical development of antifungal molecules. For instance, 1,3-thiazole derivatives having a lipophilic C4-substituent exhibited lower minimum inhibitory concentration (MIC) values compared to FLC [22]. Synthesised 14-helical β-peptides induced toxicity against C. albicans by forming pores in the cell membrane and disrupting intracellular organelles [23]. Modifications of enfumafungin leading to the new drug ibrexafungerp are also worth mentioning [5].
Interestingly, studies related to C. albicans mainly present the growth of fungal cells in the presence of glucose as the sole carbon source. The extent to which alternative carbon sources affect the innate immune response is poorly studied [24]. The available literature presents only a few reports on the impact of non-glucose carbon sources on the physiological processes of C. albicans. The eventual inhibitory effect of these molecules in the growth of Candida sp. has not been examined systematically and comparatively [17]. C. albicans can colonise glucose-poor niches of the human body, e.g., the intestine or the woman's vagina, where lactate is the carbon source [24,25]. Lactate is commonly present in the human body, produced by red blood cells, the brain and muscles [26]. Interestingly, the discovery that lactate in the pathogen's environment may inhibit their resistance to azole drugs is a promising prospect [24]. Recent studies indicate that the presence of lactate as the sole carbon source in the niche inhabited by the pathogen results in the production of a higher amount of biofilm biomass by C. albicans, in which blastoconidia are the dominant morphological form [25]. Importantly, biofilms composed of a mixture of yeast-form cells, pseudohyphal cells and hyphal cells surrounded by an extracellular matrix are less sensitive to antifungal drugs than planktonic cells. The change in the proportion of biofilm elements due to the presence of lactate reduces its resistance to antifungal agents [27,28]. Moreover, lactate lowers the expression of the ERG11 gene of the ergosterol biosynthesis pathway in the early hours of C. albicans culture and induces delocalisation of the Cdr1 pump from the PM to vacuoles [18]. Overall, the use of lactate as a carbon source affects a large part of the genome and hence is likely to reveal many genetic differences. For instance, it has been found that cells grown in the presence of lactate display a significant reduction in the amount of mannan in the cell wall [24]. In addition, exposure to lactate induces β-glucan masking via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other pathways [29]. The presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle, C. albicans cells adapted to lactate show a reduction of their internalisation by immune cells [30].In this study, we investigated the effect of two different carbon sources, glucose and lactate, on the survival of the C. albicans CAF2-1 strain in the presence of increasing concentrations of FLC. Moreover, we focused on identifying single nucleotide polymorphisms (SNP) and determining the effect of identified point mutations on the Erg11p amino acid sequence in C. albicans cells.
Obtained results show that the C. albicans CAF2-1 strain exhibit a diminished growth rate in the presence of lactate in the YPL medium compared to the control condition (YPD medium). Furthermore, cells grown on a medium with lactate as the carbon source show greater sensitivity to FLC than cultures grown on a medium with glucose. Thus, our findings confirm the information about the decrease in resistance of C. albicans to azoles, such as miconazole and FLC, when the sole carbon source is lactate [24,25]. It may be related to the hypothesis confirmed by Dalle et al. (2010), in which the presence of lactic acid salts in the environment of fungi increases the porosity of its cell wall [31]. Therefore, we aimed to investigate whether lactate and FLC synergistically contribute to the PM permeabilisation of C. albicans CAF2-1. In the case of culture in the YPL with the addition of FLC, we observed the permeabilisation of PM expressed as cell fluorescence. This effect might contribute to the entry of the antimycotic into cells. As noticed, due to the increased permeability across the cell membrane, a higher amount of the therapeutic compounds enters the pathogen's cell and can be easily transported to the target [6]. As a result, lower concentrations of the antimycotic prove to be toxic for the fungus. Indisputably, the discovery of the interaction between lactate and resistance of C. albicans to azole drugs could be significant in combating fungal infections caused by strains of fungi of the genus Candida, which are resistant to conventional antifungal compounds. Importantly, Lactobacillus and as well as lactate or lactic acid, can be applied from the outside during the treatment of candidiasis [32].
The proven effect of lactate on decreasing the survival rate of C. albicans in the presence of FLC inspired us to analyse the point mutations of the ERG11 gene as a result of culturing the fungus under various conditions. As a result of missense mutations causing amino acid substitutions in the protein, we observed the reduction of the degree of affinity of the active site of the Erg11 enzyme towards azoles. As a consequence, the susceptibility of the strain to a particular antifungal drug decreased. In the case of the ERG11 gene sequence, we recorded the highest number of mutations in three hot-spot regions. Importantly, hot-spot regions are located close to the active site of the enzyme [33]. The analysis of the obtained nucleotide sequences revealed 60 types of single-point mutations. Among them, A383C, C658T, A1440G and T1470C nucleotide substitutions are already described in the literature [34]. Identification of SNP resulted in finding 45 amino acid substitutions. Up to now, six of these mutations, K119N, K128T, D225H, D225Y, R265G and W520R, are described in the literature [21,35,36]. However, none of these mutations is associated with the phenomenon of azole resistance of C. albicans. Hence, all identified mutations, which have not been described in the literature so far, were analysed. As amino acid substitutions may be associated with C. albicans' resistance to FLC, changes present only in amino acid sequences isolated from cultures showing a high viability % (≥80%) in the presence of increasing antifungal agent concentration were considered. As a result, the following hypothetical mutations may be associated with the reduced susceptibility of C. albicans to FLC: G10D, G10V, I11M, I11R, Y13N, F31V, L35F, A249D, Q250H, E266G, R267G, N273K, D275C, D275G, D275R. Only four indicated mutations were identified in the ERG11 gene sequences from fungal cells grown on the YPL medium, while nine were located in those from the YPD cultures. Substitutions found in Erg11 sequences isolated from FLC-resistant cultures and substitutions located in FLC-sensitive and FLC-resistant cultures were considered possibly unrelated to the resistance mechanism of C. albicans. The number of this type of mutation was 23.
Next, we aligned the amino acid sequences of the Erg11 protein derived from isolates of different Candida species known from the available literature with the Erg11p sequence of the C. albicans SC5314 strain (Table S2). Six of the analysed sequences came from isolates sensitive to FLC activity, while the remaining nine belonged to isolates showing resistance to the antimycotic. We identified 64 missense mutations, of which only four (Y132F, N418R, D428E, K143R) were recorded in the sequences of resistant isolates. The Y132F mutation has been described before as related to the resistance of the genus Candida to azole drugs. The mutations were described in the isolates of C. albicans, C. orthopsilosis, C. parapsilosis and C. tropicalis [7,12,35,[37][38][39]. In turn, the N418R substitution was reported and related to the resistance in C. auris [40]. So far, it has not been recorded in the C. albicans. The D428E substitution was identified in resistant (MIC ≥ 256 µg/mL) C. tropicalis isolate IHEM21234 [41]. In turn, K143R substitution found in C. albicans 10C1B1M1 had a ≥4-fold increase in FLC MIC [7]. Furthermore, the K143 substitutions were also identified in other azole-resistant clinical isolates, Candida spp., which demonstrated the importance of this amino acid for the proper binding of azoles to Erg11p [42,43].
Unfortunately, with the sequences of the Erg11 protein belonging to different isolates of the C. albicans species, it was not possible to link the amino acid substitutions that sensitise pathogen cells to FLC (Table S3) with the results obtained by aligning the sequence of the Erg11p from C. albicans CAF2-1 cultured on the YPL (Table 2). We found two-point mutations in the E266 position (E266G and E266R) described before by Flowers et al. (2015). However, in their study, the E266D substitution occurred only in combination with other amino acid substitutions. It did not contribute any additional effect on azole susceptibility beyond what authors observed with the single amino acid substitution [7]. Moreover, we detected two kinds of mutations at position D225 (D225H and D225Y). These mutations have been described earlier. However, D225 is located in the F helix of Erg11p, far away from the I helix or the active centre. Thus, substitutions of D225 cannot influence the affinity between FLC and the target [44]. Similarly, the amino acid substitutions K128T and K119N reported previously were also determined not to confer resistance to FLC [7,12,36,45]. The contribution to the resistance of other mutations (R265G and W520R) is uncertain [35,44].
Nonetheless, our results suggest that the carbon source may play a role in inducing the formation of amino acid substitutions in the Erg11p. We observed a twice higher number of amino acid mutations in the YPD medium compared to the YPL medium. This result may suggest that lactate causes a lower ability of C. albicans to mutate. However, a lower number of lactate-induced mutations may appear due to the lower growth rate of Candida on the YPL compared to the YPD medium. Before, we proposed synergistic effects of lactate and FLC, where C. albicans was four-fold more sensitive to FLC in the presence of lactate than glucose [18]. In addition to mutations in the ERG11 gene, a factor influencing the altered strain sensitivity of C. albicans to FLC may naturally be a higher or lower level of MDR proteins (Cdr1, Cdr2 and Mdr1) activity [44]. Interestingly, Derkacz et al. (2022) noticed that point mutations in the ERG11 gene not only result in lower susceptibility to FLC. They also contribute to higher levels of ergosterol after FLC treatment or may result in chitin and β-glucan unmasking on the cell surface [46], which proves the complexity of the effects of single-point mutations in the ERG11 gene. Moreover, single nucleotide changes in the ERG11 gene may not affect the sensitivity of C. albicans strains, and only multiple substitutions may indicate a possible relation with the increase in resistance to azole drugs [45]. We should be aware that mutations can lead to the loss of function of genes. For instance, the loss of function of ERG11 genes leads to the exchange of ergosterol for alternate sterols such as lanosterol, eburicol and 4,14-dimethyl-zymosterol in PM [47]. This mechanism may result in the acquisition of resistance of C. albicans to polyenes, which interact with ergosterol.
The research carried out in this study allowed us to confirm the information on the increased sensitivity of fungal cultures grown in the presence of FLC on a medium with lactic acid salts as a sole carbon source. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity. We have identified 15 amino acid substitutions in C. albicans cultures that may be part of the fungal resistance mechanism to the commonly used azole compound. These findings confirm the thesis that mutations in the ERG11 gene are prevalent among Candida species, especially in clinical azole-resistant isolates, and most mutations result in appreciable changes in FLC susceptibilities [7].

Chemicals
Chemicals and reagents used in this study were obtained from the following sources: The C. albicans CAF2-1 was pre-grown in YPD medium (28 • C; with shaking: 120 rpm) overnight to determine growth phases. Next, cells were washed with fresh YPD or YPL medium. 100 µL of YPD or YPL medium was added to the sterile 96-well plate (Sarstedt, Nümbrecht, Germany) and inoculated with C. albicans to the final A 600 = 0.05 and incubated for 24 h at 28 • C with shaking (120 rpm). The OD 600 measurements were performed every hour using the Spark multimode microplate reader (Tecan, Männedorf, Switzerland) in three independent repetitions.
In order to determine the viability of C. albicans CAF2-1, a serial dilution of fluconazole (µg/mL) was prepared in YPD or YPL medium using sterile 96-well plates. Wells containing medium were inoculated with C. albicans CAF2-1 strain (final OD 600 = 0.01). The volume of each well was 100 µL. Plates were incubated for 24 h at 28 • C without shaking. Next, the optical density (λ = 600 nm) was measured using Asys UVM 340 (Biogenet, Józefów, Poland). Negative controls were wells with YPD or YPL medium. The experiment was carried out in three independent replications.

Propidium Iodide (PI) Staining of C. albicans Cells
In order to investigate the permeabilisation of C. albicans PM, propidium iodide (PI) staining was used as described before [49], with modifications. C. albicans cells after 24 h of culture on the YPL or the YPD with or without the addition of FLC (4 µg/mL) were washed three times with PBS (5 min, 4500 rpm) and stained with PI (0.006 mM) for 5 min, RT. Then, cells were harvested, washed three times with PBS and concentrated.
The samples (n = 100 cells per each case) were visualised using a Zeiss Axio Imager A2 equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp (Oberkochen, Germany). The percentage of permeabilised cells was evaluated by counting PI-positive cells out of one hundred cells in three independent repetitions for each experiment.

Cultures of C. albicans and Preparation of Material for DNA Isolation
For experiments, 20 mL of YPD or YPL medium (with or without FLU) was inoculated with pre-culture at starting A 600 = 0.1 and grown for 8, 14 or 24 h (28 • C, 120 rpm). The addition of FLC to the culture was 0.25, 1, 4, or 16 µg/mL. At the end of the experiments, the cultures were transferred to 50 mL falcons or 1.5 mL Eppendorf tubes (the final OD 600 = 1.5). Then, the cultures were centrifuged (10 min, 13 400 rpm), the supernatant was discarded, and the biomass was stored at −20 • C.

DNA Isolation
Genomic DNA from C. albicans cultures was isolated using the GeneMATRIX Bacterial & Yeast Genomic DNA Purification Kit (EURx, Gdańsk, Poland) according to the manufacturer's instructions. Purified DNA was eluted with Mili-Q water (ultrapure molecular water) at room temperature.
The amount of isolated DNA and its purity were determined by spectrophotometric analysis using the DS-11 Series Spectrophotometer / Fluorometer (DeNovix, Wilmington, DE, USA).

Polymerase Chain Reaction (PCR)
The amplification of the ERG11 gene sequence was performed using PCR. Due to its length (1587 bp), the sequence was amplified in two fragments (A and B) using the primers shown in Table 3. The starters were designed using the SnapGene program and the OligoAnalyzer tool and were synthesised by Genomed (Warsaw, Poland). The primers were designed to include a sequence of 20 bp downstream and 12 bp upstream (at positions 20 bp ERG11 or +1599 bp ERG11, respectively) to ensure the amplification of the entire ERG11 gene sequence. Reactions were carried out in the following reaction mixture: 12.5 µL iProof ™ HF Master Mix (BioRad, Hercules, CA, USA), 0.5 µL of each of the 5 µM primers, 2 µL of isolated genomic DNA and 9.5 µL of sterile water (total volume 25 µL). Fragment "A" of the ERG11 gene sequence was amplified using a program called ZL-ERG11A and fragment "B" using the ZL-ERG11B program. A C100 Touch Thermal Cycler (BioRad, Hercules, CA, USA) was used to perform the PCR reaction. The reaction programs are shown in Table 4. Table 4. Description of the ZL-ERG11A and ZL-ERG11B programs used for the amplification of "A" and "B" fragments of the ERG11 gene.
Step In order to identify the amplified DNA fragments, 1% agarose gel electrophoresis (Bi Tools, Madrid, Spain) with the addition of the SimplySafe nucleic acid intercalator (5 µg/mL) (EURx, Gdańsk, Poland) was used. GeneRuler ™ 1 kb DNA Ladder (Thermo Fisher Scientific ™, Waltham, MA, USA) was used as the marker. Purification of PCR products from unattached nucleotides and primers was performed using the GeneMA-TRIX Agarose-Out DNA Purification Kit (EurX, Gdańsk, Poland) according to the manufacturer's instructions. The samples were suspended in molecular water (Mili-Q). The purified DNA fragments were stored at −20 • C. The samples were then analysed qualitatively and quantitatively with the DS-11 Series Spectrophotometer/Fluorometer (De-Novix, Wilmington, DE, USA), and then samples were subjected to Sanger sequencing. The templates for the reaction were previously purified PCR products that were diluted according to the manufacturer's instructions to a final concentration of 5 ng/µL with molecular water (Mili-Q). The sequencing reaction was performed by Eurofins Genomics (Ebersberg, Germany).

The Identification of ERG11 Point Mutations among Amino Acid Sequences of C. albicans Strains Available in Databases
The obtained nucleotide sequences were exported to FASTA format using the Jalview application. These were then translated into amino acid sequences using the EMBOSS Transeq tool (https://www.ebi.ac.uk/Tools/st/emboss_transeq/ accessed on 19 April 2020).
Using the BLAST (basic local alignment search tool; https://blast.ncbi.nlm.nih.gov/ Blast.cgi accessed on 24 April 2020), a local alignment of the amino acid sequence of strain SC5314 (GenBank accession no.: AOW29509.1) was performed. The strain C. albicans SC5314 is the parental strain of CAF2-1 used in this study. The sequences were compared with sequences of different Candida species as well as of distinct strains of C. albicans available from biological databases. Nucleotide and amino acid sequences were aligned using the EMBOSS Needle program (https://www.ebi.ac.uk/Tools/psa/emboss_needle/ accessed on 19 April 2020) using standard settings. The identified nucleotide and amino acid substitutions were collected and presented in the form of a table.

Statistical Analyses
For data analysis, statistical significance was determined using a Student's t-test (binomial, unpaired). Data represent the means ± standard errors from at least three biological replicates.

Conclusions
The aim of the study was to determine the effect of point mutations of the Erg11p amino acid sequence in C. albicans depending on the phases of cell growth, the presence of lactate as a carbon source and the influence of FLC. As a result, C. albicans CAF2-1 grown on lactate as the sole carbon source showed increased sensitivity to FLC compared to growth on glucose. The synergistic treatment with lactate or glucose and FLC contributed to the development of point mutations in the C. albicans genome. The analysis of the ERG11 gene derived from the CAF2-1 strain cultivated in various conditions revealed 56 nucleotide substitutions, which resulted in 38 amino acid substitutions. Ten substitutions were located in the hot-spot regions. Interestingly, almost twice a higher number of mutations in the hot-spot regions were found in the cultures grown on lactate, compared to glucose. This result may be related to the increased sensitivity of cells grown on the YPL medium to the antimycotic effect. However, there is no evidence that either mutation is responsible for the increased susceptibility of C. albicans CAF2-1 to FLC. Thus, our study is a good starting point for further research, such as site-directed mutagenesis, to investigate the impact of mutations on C. albicans' resistance/susceptibility to FLC.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens11111289/s1. Table S1: The percentage reduction in the growth of C. albicans CAF2-1 after FLC treatment in the presence of YPD and YPL; Table S2: Identification of amino acid substitutions in hot spots as a result of aligning the amino acid sequences of the Erg11p of Candida species with the sequence of the Erg11p of C. albicans SC5314 strain. "."-no mutation [50][51][52][53][54][55][56][57]; Table S3: Identification of amino acid substitutions in hot spots as a result of aligning the amino acid sequences of the Erg11p C. albicans strains with the amino acid sequence of the Erg11p of C. albicans SC5314 strain. "."-no mutation [58,59].