Nva-FMDP (F2) and Lys-Nva-FMDP (F3) were synthesized as described previously (Andruszkiewicz et al., 1987, 1990). Lys-Leu-Pro-Val-Met (P5), Lys-Leu-Pro-Val-Met-FMDP (F6), Arg-Lys-Lys-Trp-Phe-Trp (P6), FMDP-Arg-Lys-Lys-Trp-Phe-Trp (F7), Lys-Lys-Val-Val-Phe-Trp-Val-Lys-Phe-Lys (P10), and FMDP-Lys-Lys-Val-Val-Phe-Trp-Val-Lys-Phe-Lys (F11) were synthesized by the solid phase method using the Fmoc/But^t strategy. The crude peptides were purified by HPLC using the Agilent 1290 Infinity system, on Kromasil C8 or Cosmosil C18 column, developed with a linear acetonitrile gradient. Purity of the peptides was determined using analytical HPLC and mass spectrometry with Agilent Technologies 6540 UHD Accurate—Mass Q-TOF LC/MS.

Reference and non-reference C. albicans strains used in this study are listed in Table 1. Strains were grown at 30°C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) and stored on YPD plates containing 2% agar. Susceptibility testing was performed in YPD and other three media: (a) RPMI-1640 w/o sodium bicarbonate, with L-glutamine + 2% glucose + 3.45% MOPS, pH adjusted to 7.0; (b) YNB-AS—Yeast Nitrogen Base w/o amino acids (contains ammonium sulfate 5 g L^-1) + 2% glucose; (c) YNB-SG—Yeast Nitrogen Base w/o amino acids and ammonium sulfate + 2% glucose + 0.2 g L^-1 sodium glutamate. In their solid version, used for the mutant generation and isolation, YNB-AS and YNB-SG contained 2% agar.

The in vitro growth inhibitory activity of antifungals was quantified by determination of minimal inhibitory concentration (MIC) values by the serial twofold dilution method, using the 96-well microtiter plates in three media: buffered RPMI-1640, YNB-AS, and YNB-SG. Conditions of the RPMI-1640-based assay were the same as outlined in the CLSI recommendations (Clinical Laboratory Standards Institute, 2008), except for the end-point readout that was done by spectrophotometric determination of cell density at 531 nm. Turbidity in individual wells was measured with a microplate reader (Victor³; Perkin Elmer). MIC was defined as the lowest drug concentration that gave at least an 80% decrease in turbidity, relative to that of the drug-free growth control.

The 96-well microtiter plates were also used for determination of in vitro growth inhibitory activity in YPD and YNB-based media. Individual wells were inoculated with 5 × 10³ cfu mL^-1 of C. albicans cells from the overnight culture in YPD medium. The inoculated plates were incubated 37°C for 24 h and then turbidity was measured with a microplate reader at 531 nm, as described above for the RPMI-1640-based assay.

Candida albicans cells from the overnight cultures in YPD were harvested, washed with sterile saline and suspended in the RPMI-1640 medium, to the final cell density of 10⁵ cfu mL^-1. The compounds tested were added at appropriate concentrations and cultures were incubated for 6 h at 30°C, with shaking. At time intervals, samples of 0.1 mL were collected and cells were stained for chitin. The culture samples were combined with 0.1 mL aliquots of the Calcofluor White solution [0.3 mg mL^-1 in 25 mM phosphate-buffered saline (PBS), pH 7.0] and incubated for 5 min at 25°C. The cells were then harvested, washed four times with PBS, re-suspended in 0.1 mL of the fresh PBS and transferred to a microscopic slide. The stained cells were examined with the Olympus BX 60 F5 fluorescence microscope and photographs were taken using the Studio Lite software (Pixera Corporation, United States).

Yeast cells grown exponentially in the YNB-SG medium were harvested by centrifugation (3000 ×g, 5 min), washed with 50 mM potassium phosphate buffer (pH 5.0 or 6.5), and suspended in the same buffer containing 1% glucose, to a final cell density corresponding to A₆₆₀ ≅ 1.0. The cell suspension was incubated at 30°C. After 10 min, an oligopeptide solution was added, to give a final concentration of 100 μM. At that moment and at 5 min intervals thereafter, 2 mL samples of the cell suspension were withdrawn, immediately filtered through the Whatman GF/A filters and the filtrates were used to determine the residual peptide concentration. Then, the 1 mL portions of the filtrates were taken and combined with 1.25 mL aliquots of a solution containing 4% Na₂B₄O₇ × 10 H₂O and 0.8 mg mL^-1 of 2,4,6-trinitrobenzenesulfonate. The reaction was carried out at 37°C for 30 min. The A₄₂₀ values were measured and the peptide concentration was read from the appropriate standard curve. Data were plotted as nanomoles of an oligopeptide, taken up by 1 mg (dry weight) of cells versus time. The initial uptake velocities were determined from the slopes of the linear part of the curves, in the 0- to 10-min region.

Yeast cells from the overnight culture in YNB-SG were harvested by centrifugation and washed with cold 25 mM potassium phosphate buffer (pH 6.5). Cells were then suspended in a minimal amount of the buffer and disrupted with the French press. The resulting suspension was centrifuged (35,000 ×g, 4°C, 45 min), and the supernatant was used as a cell-free extract for the determination of peptide cleavage rates.

The incubation mixtures, consisting of 10 mL of a 200 mM peptide solution in 50 mM potassium phosphate buffer (pH 6.5) and 2 mL of an appropriately diluted crude extract (final protein concentration, 0.1–0.5 mg mL^-1), were incubated at 30°C. At 5 min intervals, 2 mL aliquots were withdrawn and heated at 100°C for 3 min. The resulting suspensions were centrifuged to remove the protein precipitates and the concentration of free amino acids in the supernatant was determined by the Cd-ninhydrin procedure (Doi et al., 1981).

Candida albicans cells, apparently resistant to FMDP-peptides, were isolated from the individual colonies formed on the surface of YNB-SG agar medium containing FMDP-peptide, 10 μg mL^-1 or within the growth inhibitory zones around the paper discs saturated with 10 μL of the FMDP-peptide solution, 1 mg mL^-1. In both versions, the agar surface was inoculated with 10⁵ cells from the overnight culture in YPD medium, washed three times with sterile water, suspended in 0.2 mL of sterile water and the cell suspension was spread on the whole surface with a sterile glass rod. Plates were incubated for 48 h at 30°C and isolated colonies were picked with a platinum loop.

The yeast isolates were grown on YPD agar plates for 18–20 h at 30°C. Small amounts of biomass from single colonies of each tested strain were suspended in 4 mL of YPD broth and incubated with continuous shaking for about 5 h at 30°C, to achieve OD₆₆₀ = 0.6. The yeast cells were then harvested by centrifugation (5000 rpm, 5 min) and mRNA was isolated using the ISOLATE II RNA Mini Kit (Bioline, United Kingdom), according to the manufacturer’s protocol. Immediately after mRNA isolation, a reverse transcription reaction with TranScriba kit (A&A Biotechnology, Poland) was carried out. In the first step, 140 ng of isolated RNA was incubated at 65°C for 5 min with 1 μL of oligo(dT)₁₈ solution (100 μM) and RNase-free water added up to volume of 10 μL. After adding the remaining reagents: 4 μL of 5× Reaction Buffer, 2 μL of dNTPs Mix solution (2.5 mM of each), 4 μL of TranScriba Reverse Transcriptase (20 U μL^-1), the final mixture was incubated for 60 min at 41°C. The reaction was terminated by heating the specimen at 70°C for 5 min.

Analysis of expression of OPT1, OPT2, OPT3, PTR2, PTR22, and reference ACT1 genes was performed by real-time PCR with the Light-Cycler 480 (Roche, Switzerland). All primers (sequences shown in Table 2) were purchased from Sigma. RT-PCR was performed with a 20 μL volume containing the following reagents: 10 μL of RealTime 2× FastStart Essential DNA Green Master Mix (Roche, Switzerland), 1.5 μL of each primer solution (10 μM), total cDNA sample solution (40 ng), and distilled water up to the final volume of 20 μL. Standard curves, for evaluation of RT-PCR efficiency, were prepared in triplicate. Twofold serial dilutions of mixture of the cDNA of all strains tested were used as a template for standard curves in the case of all tested genes. For both genes, the amplification conditions were as follows: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, followed by 15 s primer annealing at 64°C and elongation at 72°C also performed for 15 s. The temperature transition rate was set at 20°C/s. Genes for gene expression analysis were amplified in the same conditions as in the case of preparing standard curves, with 40 ng of cDNA as a template. The experiments were repeated in duplicate for every specimen. Acquisitions of fluorescence signals were carried out at the end of every annealing-extension step for 100 ms.

Quantitative analysis of the results obtained was carried out by using the 2(-Delta Delta C(T)) method (Livak and Schmittgen, 2001). The method allows determination of the relative differences in the expression level of analyzed target genes between the investigated strains and reference strain—also called the calibrator. For our research, the C. albicans SC5314 strain was used as a calibrator. The level of expression of target genes in a particular strain was determined based on comparison of CT (threshold cycle) values of amplification of gene of interest and the reference gene—internal control (formula presented below). As a reference gene for this research, the ACT1 gene, one of the housekeeping genes of C. albicans, expressed in the cells in a constitutive manner was used.

The Livak’s equation value of parameter R = 1 indicates that the level of the target gene expression in the investigated sample (strain) and calibrator are the same. A value greater than 1 indicates a higher level of expression of the tested gene in the cells of the investigated strain in comparison to the cells of the calibrator, whereas a significant increase in the level of the gene expression is considered to have occurred when the value of the parameter R is higher than 2.

The OPT1, OPT2, OPT3, PTR2, and PTR22 genes were amplified, using the sets of primers presented in Table 2. Because of the large size, the OPT1–3 genes were amplified in the form of two fragments. All PCR reactions were performed with a 50 μl volume containing the following reagents: 25 μL of the 2× PCR Mix Plus High GC (A&A Biotechnology, Poland), 17 μL of nuclease-free water, 2.5 μL of each primer solutions (10 μM) and 3 μL of isolated DNA solution. Amplification conditions were as follows: 5 min of initial denaturation at 95°C, 30 s of denaturation at 95°C, 30 s of annealing (52°C for PTR2; 56°C for PTR22; 56°C for C-terminal fragment of OPT1 and 63°C for N-terminal fragment of OPT1; 52°C for C-terminal fragment of OPT2 and 59°C for N-terminal fragment of OPT2; 56°C for C-terminal fragment of OPT3 and 59°C for N-terminal fragment of OPT3), 90 s of primer extension at 72°C repeated for 35 cycles, and 10 min of final extension at 72°C. The PCR products (2110 bp—PTR2; 1731—PTR22; 1365—N-terminal fragment of OPT1 and 1224—C-terminal fragment of OPT1; 1416—N-terminal fragment of OPT2 and 1501—C-terminal fragment of OPT2; 1560—N-terminal fragment of OPT3 and 1310—C-terminal fragment of OPT3) were detected on 1.5% agarose gel stained with ethidium bromide. The products of amplification were then purified according to the A&A Biotechnology protocol enclosed with the Clean up AX kit. Sequencing was carried out by Macrogen (Netherlands). The analysis of sequences was performed using MEGA 6.1 (Molecular Evolutionary Genetics Analysis) software and statistical analysis was carried out with the GraphPad Prism 5.0 program.

Lys-Leu-Pro-Val-Met (P5) is a member of family of the so called “cell-penetrating pentapeptides” (Gomez et al., 2010), Arg-Lys-Lys-Trp-Phe-Trp (P6), known also as PAF26, was reported as one of the hexapeptides easily penetrating fungal cell membranes (Lopez-Garcia et al., 2007) and Lys-Lys-Val-Val-Phe-Trp-Val-Lys-Phe-Lys (P10) is a non-amidated analog of the antimicrobial decapeptide KSL-W (Semlali et al., 2011). Conjugates of these oligopeptides with FMDP (F6, F7, and F11) were prepared, their in vitro growth inhibitory activity against the wild type C. albicans SC5314 and SC5314-derived peptide permease deficient mutants was determined and compared to that of Nva-FMDP (F2) and Lys-Nva-FMDP (F3). The MIC values were determined using the serial dilution microplate method in three different growth media: RPMI-1640 and two minimal media based on Yeast Nitrogen Base (YNB) with different nitrogen sources, ammonium sulfate (YNB-AS) and sodium glutamate (YNB-SG). Results are shown in Tables 3–5.

All the FMDP-oligopeptides tested demonstrated high activity against the wild type SC5314 cells in YNB-SG medium (MICs in the 0.25–1.0 μg mL^-1 range). In YNB-AS, the MIC values of Nva-FMDP and Lys-Nva-FMDP were still low but these of FMDP-oligopeptides F6, F7, and F11 were 8–16 times higher. All the FMDP-oligopeptides demonstrated much lower growth inhibitory activity in RPMI-1640. This decrease was particularly profound for FMDP-oligopeptides F6, F7, and F11, which apparently lost their anticandidal potency.

Peptide permeases-deficient mutant opt1–opt5Δ ptr2Δ ptr22Δ was not sensitive to all FMDP-oligopeptides in RPMI-1640 and YNB-AS, whereas in YNB-SG a moderate activity of F6 and F7 was observed. The ptr2Δ ptr22Δ mutant, lacking the di-tripeptide permease, was not sensitive to F2 and F3 in all media, while its sensitivity to F6, F7, and F11 was the same as that of the parent strain. Disruption of genes encoding OPT permeases did not affect sensitivity to F2 and F3 but strongly diminished the growth inhibitory activity of F6, F7, and F11. A reduced activity of these FMDP-oligopeptides was also noted against mutants lacking particular OPT permease(s) and a magnitude of this reduction was especially high in the case of the opt2Δ opt3Δ mutant. Interestingly, oligopeptide P10 demonstrated constant moderate growth inhibitory activity, not affected by gene disruption and by growth medium composition. No antifungal activity (MIC >> 128 μg mL^-1) was found for oligopeptides P5 and P6.

The observed lack of anticandidal activity of FMDP-oligopeptides F6, F7, and F11 in RPMI-1640 medium, compared to their good or very good activity in YNB-based minimal media, suggested that any components of the former medium may influence the growth inhibition. Therefore, the activity against the wild type SC 5314 strain was measured in YNB media supplemented with particular components of RPMI-1640, originally not present in YNB broths. Little, if any, effect (maximum twofold MIC enhancement) was found for supplementation with glutathione (reduced) 1 mg L^-1, NaCl 6 g L^-1 or Phenol Red but a strong effect was noted for supplementation with a mixture of amino acids (composition as in RPMI-1640). As shown in Table 6, oligopeptides F6, F7, and F11 lost their growth inhibitory activity in YNB + amino acids, while activity of F2, F3 was almost the same as in YNB-SG. Presence of L-glutamine, 300 mg L^-1 (the same concentration as in RPMI-1640), strongly diminished an activity of F6, F7, and F11, especially in growth medium buffered to pH = 7.0. Another factor that might influence the activity of FMDP-oligopeptides was the pH. The RPMI-1640 medium used in the CLSI assay is buffered to pH = 7.0 with MOPS, while pH of the unbuffered YNB-AS is ∼5.4. Therefore the MIC values of FMDP-oligopeptides in RPMI-1640 buffered to 5.0, 6.0, and 7.0 and in YNB-AS buffered to 4.5, 5.5, and 6.5 (above pH = 6.5 a precipitation of some components was observed) against SC 5314 were determined. Data presented in Figure 1 indicate that the activity of all FMDP-oligopeptides tested was in fact pH-dependent and lower MICs were found at lower pH values. Decrease of growth inhibitory activity at higher pH was slightly more remarkable for peptides F6, F7, and F11, compared to F2 and F3. Antifungal activity of P10 was the same (MIC = 64 μg mL^-1) at different pH values.

All the FMDP-oligopeptides tested demonstrated no activity in YPD medium (MIC >> 128 μg mL^-1), while P10 was still active, with MIC = 64 μg mL^-1. Addition of Bacto Peptone, 5 g L^-1, completely eliminated growth inhibitory activity of all FMDP oligopeptides in YNB-AS and YNB-SG. Similar effect was observed for peptides F2 and F3 upon supplementation of the YNB-based media with Ala-Ala or Ala-Ala-Ala, 1 g L^-1, while supplementation with Ala-Ala-Ala-Ala 1 g L^-1 resulted in very slight (two- or fourfold) increase of MIC. On the other hand, supplementation of YNB-based media with Ala-Ala or Ala-Ala-Ala, 1 g L^-1 had no effect on activity of F6, F7, and F11, whereas presence of Ala-Ala-Ala-Ala 1 g L^-1 resulted in 16- to 64-fold increase of MICs in YNB-SG.

The antifungal activity of oligopeptide P10 was not affected by any supplementation of YNB medium.

The observed effect of peptides built of proteinogenic amino acids on growth inhibitory activity of FMDP-oligopeptides suggested competition for oligopeptide transporters and possible crucial importance of uptake for growth inhibitory activity of FMDP-oligopeptides, so that the initial rates of uptake of oligopeptides P5, P6, P10, FMDP-oligopeptides and model oligopeptides (Ala)₂, (Ala)₃, and (Ala)₄ to C. albicans wild type and mutant cells were determined.

Data presented in Table 7 show that all the oligopeptides tested, except for P10, were taken up by the wild type SC5314 cells, with initial uptake rates in the 2.22–5.62 nmol min^-1 (mg dry weight)^-1 range. Deletion of the PTR genes (ptr2Δ ptr22Δ) completely eliminated uptake of (Ala)₂, (Ala)₃, and Nva-FMDP and very strongly reduced that of Lys-Nva-FMDP but have very slight, if any effect on the uptake of P5, P6, F6, F7, and F11. The OPT permeases-deficient opt1–opt5Δ mutant was not able to take up F11, demonstrated markedly lower ability to take up (Ala)₄, P5, P6, F6, and F7 and almost unchanged potency for the uptake of Nva-FMDP, (Ala)₂, and (Ala)₃. Analysis of uptake rates determined for the particular OPT mutants leads to the conclusion that permeases Opt2p and Opt3p seem to be responsible for the bulk uptake of P5, P6, F6, and F7, while the tetrapeptide (Ala)₄ is mainly transported by Opt1p. Nevertheless, it is clear that all five permeases of the OPT system participate in transport of oligopeptides (Ala)₄, P5, P6, F6, F7, and F11, although Opt4p and Opt5p do it with minor efficiency.

Data presented in Table 7 indicate, that the decapeptide P10 was not taken up by the wild type cells and the permease-deficient mutants. It is therefore clear that internalization is not a prerequisite for the intrinsic growth inhibitory activity of this oligopeptide. On the other hand, the FMDP-containing derivative of P10, i.e., F11, was taken up by the wild type cells, apparently by permeases Opt2p/Opt3p.

Since the anticandidal activity of P10 was moderate but independent on medium content and that of F11 was higher but strongly influenced by medium composition, it seemed obvious that the molecular bases of growth inhibitory activity of P10 and F11 were different and that a higher activity of F11 resulted from the presence of FMDP in its structure. To confirm whether the anticandidal activity of F11 was a consequence of GlcN6P synthase inhibition by FMDP, a morphology of SC 5314 cells treated with F11 was compared to that of the untreated cells. The cells were stained with Calcofluor White, the fluorescent brightener that binds specifically to chitin in the fungal cell wall. Results are shown in Figure 2. Cells treated with F11 were apparently defective in formation of a primary septum separating the parent and daughter cells during budding (Figure 2B). Since it is well known that the primary septum is built of chitin, this finding confirms that the action of F11 results in inhibition of chitin biosynthesis. The morphological changes, analogous to those observed for F11-treated cells were previously reported for C. albicans cells treated with F2 and F3 (Milewski et al., 1991) and now for cells treated with F6 or F7. Conversely, such changes were not noted now for cells treated with P10. It was also found that supplementation of the YNB-SG medium with N-acetyl-D-glucosamine, 10 mM, resulted in complete elimination of growth inhibitory activity of F11, thus indicating that the growth inhibition by F11 is a consequence of cessation of intracellular glucosamine supply for chitin biosynthesis, most likely by the blockage of GlcN6P synthase activity by FMDP.

Growth inhibitory activity of FMDP-oligopeptides against C. albicans SC 5314 was also determined by the disc diffusion method. As shown in Figure 3, growth inhibitory zones were formed around discs saturated with F2, F3, F6, F7, and F11 oligopeptide solutions (10 μL of 1 mg mL^-1 stocks). The zones formed around discs saturated with F6, F7, and F11 were clear, but numerous colonies were observed inside the zone formed around the F2 disc (Figure 3A) and a few colonies appeared inside the F3-induced zone (Figure 3B). Cells grown in these colonies were collected, re-grown in YNB-AS and tested for sensitivity to FMDP-oligopeptides. Surprisingly enough, only 6 out of 200 isolates demonstrated remarkably lower than the SC 5314 strain sensitivity to F2 and F3. The six resistant isolates (three from the colonies grown inside the F2-induced zone and three from the F3-induced zone) were cross-resistant to F2 and F3 but demonstrated unchanged sensitivity to F6, F7, and F11 (Table 8).

All six isolates demonstrated remarkably lower initial uptake rates of dipeptides F2 and (Ala)₂ then the wild type SC5314 cells (Figure 4A) and lower rates of uptake of tripeptides (Ala)₃ and F3 (Figure 4B), while the rates of uptake of (Ala)₄ and F6 were nearly the same or even somewhat higher (Figure 4C). It is therefore clear that the diminished sensitivity of all six isolates to F2 and F3 was due to the reduced uptake, most likely by permease(s) of the PTR system.

Resistance of some of the six isolates was not stable. When all isolates were passaged 10 times in rich YPD medium and then tested for sensitivity to F2 and F3, two isolates originating from colonies from the F3-induced zone were found to fully restore the original sensitivity and one isolate from the F2-induced zone exhibited diminished sensitivity but was not entirely resistant (Table 9). Determination of oligopeptide uptake rates confirmed recovery of original di- and tripeptide uptake potency for revertants LNF1′ and LNF2′ and its partial restoration for the revertant NF3′. It seems therefore that resistance of isolates NF1, NF2, and LNF3 might be due to the stable impairment of the di- tri-peptide transport system.

No substantial difference was found for velocities of proteolytic cleavage of FMDP-oligopeptides by peptidases present in cytoplasmic extracts obtained from NF1, NF2, NF3, LNF1, LNF2, LNF3, and SC 5314 cells. The velocities, determined by the Cd-ninhydrin method, were in the 6.2–25 nmol min^-1 (mg protein)^-1 range, generally two to three times higher for F2 and F3 than for F6, F7, and F11 but those for a particular FMDP-oligopeptide were almost the same in all seven extracts studied.

All six isolates and the SC5314 cells were examined for the level of expression of the OPT1, OPT2, OPT3, PTR2, and PTR22 genes and possible mutations in their nucleotide sequences. No substantial difference between NF1-3 and LNF1-3 isolates and the wild type SC 5314 strain in expression profiles of these genes were found, since the R parameters of Livak’s equations were in the 0.87–1.7 range (Supplementary Figure S1). On the other hand, sequencing of the OPT1, OPT2, OPT3, PTR2, and PTR22 genes amplified from the genomic DNA of isolates, revealed some single mutations in the coding sequences of OPT1 and PTR2. These mutations should result in the following substitutions in the amino acid sequence: NF1: M217L in Opt1p and T422H in Ptr2p; NF2: A320S in Ptr2p; LNF3: D119V and A320S in Ptr2p. All the substituted residues, except for the D119 of Ptr2p, are located in the putative transmembrane spanning regions of either Opt1p or Ptr2p. No mutations were found in OPT1–3, PTR2, and PTR22 genes derived from NF3, LNF1, and LNF2. i.e., isolates that lost their initial apparent resistance upon several passages in oligopeptides-containing rich medium.