The Penicillium chrysogenum Q176 Antimicrobial Protein PAFC Effectively Inhibits the Growth of the Opportunistic Human Pathogen Candida albicans

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five β-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candida albicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.


Strains and Growth Conditions
Fungal strains used in this study are listed in Supplementary Materials, Table S1 and the composition of the media is described in Supplementary Materials, Table S2. For the generation of conidia, P. chrysogenum Q176 was grown on 1 × PcMM for 96 h at 25 • C. Conidia were harvested and washed in spore buffer (0.9% NaCl (w/v), 0.01% Tween (v/v)) before use. Germlings were generated by growing conidia in 1 × PcMM at 25 • C in static culture until the germ tubes were twice the length of the conidia diameter (11 h). For P. chrysogenum submersed shaking cultures 200 mL 1 × PcMM was inoculated with 2 × 10 8 spores and cultivated for up to 96 h at 25 • C and 200 rpm. Synchronized surface cultures were generated on 1 × PcMM, containing 1.5% (w/v) agar at 25 • C as described in Hegedüs et al., 2011 [24]. For the generation of fungal exudates, 5 µL aliquots containing 1 × 10 6 P. chrysogenum conidia were point inoculated on 1 × PcMM or double-concentrated PcMM (2 × PcMM), containing 1.5% (w/v) agar. The surface cultures were grown for up to 144 h at 25 • C in a box lined with wet paper towels. Single colonies of Candida strains grown on potato dextrose broth (PDB) agar plates (Sigma-Aldrich, St. Louis, MO, USA) were used to inoculate 10 mL ten-fold diluted PDB (0.1 × PDB). After overnight cultivation at 30 • C and 160 rpm the cells were washed in 0.1 × PDB before experimental use.

Detection of PafC mRNA
The expression of pafC was investigated in conidia, germlings, and mycelia of submersed and synchronized surface cultures of P. chrysogenum. Total RNA was extracted from cells with TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instruction. Ten µg RNA per lane were loaded onto a 1.2% (v/v) formaldehyde gel, electrophoresed and blotted onto a Hybond N membrane (Amersham Biosciences, Little Chalfont, UK). The pafC transcripts were detected with a digoxigenin-labelled hybridization probe (Roche, Basel, Switzerland) amplified by PCR (Table S3) from pafC cDNA using pafC specific primers (Table S4).

Detection of Native PAFC
Submersed cultures of P. chrysogenum were prepared as described above and the supernatant was used for Western blot analysis at different time points. Fungal exudates, the liquid secreted underneath the fungal colony and the droplets on top of the colony, were harvested as described in Supplementary Materials (experimental procedures; Figure S1) after 120 h of incubation. Samples (20 µL per lane) were loaded onto a 18% (w/v) SDS-polyacrylamide gel, electrophoresed and Western blotting performed as described (Supplementary Materials, experimental procedures). PAFC was detected using rabbit anti-PeAfpC serum (1:2500) [12]. PAF and PAFB in the exudate were detected using IgG purified rabbit anti-PAF serum (1:500) [25] and rabbit anti-PAFB serum (1:1000) [9], respectively (Supplementary Materials, experimental procedures).

Generation of Recombinant PAFC
Recombinant PAFC was prepared using a P. chrysogenum-based expression system [26]. The cloning of expression vector pSK275_pafC ( Figure S2) and transformation into the P. chrysogenum ∆paf mutant strain [26] are described in Supplementary Materials (experimental procedures). The strain with the highest PAFC content in the culture broth was named P. chrysogenum OEpafC and used for large-scale expression. The cell-free supernatant was processed as described previously [26] with minor modification. The pH of the supernatant was adjusted to 4.0 with 1 M citric acid before it was filter sterilized, degassed and loaded onto a BioPro S30 cation exchange column (YMC CO. LTD, Kyoto, Japan). The column was washed with 0.1 M equilibration buffer (0.1 M citrate buffer pH 4, 0.5 M EDTA, 25 mM NaCl) and PAFC was eluted with 0.1 M elution buffer (0.1 M citrate buffer pH 4, 100 mM NaCl). Fractions containing the protein peak were checked for PAFC content and purity on a 18% (w/v) SDS-polyacrylamide gel and by silver staining. The pure PAFC containing fractions were concentrated and dialyzed against ddH 2 O using a Vivaspin®500 device (3 kDa MWCO; GE Healthcare, Chicago, IL, USA). The identity of PAFC was verified by electrospray ionization mass spectroscopy (ESI-MS) (Protein Micro-Analysis Facility; Biocenter, Medical University of Innsbruck) and the purity of the protein confirmed by reverse-phase high-performance liquid chromatography (RP-HPLC). The RP-HPLC run was performed on a Phenomenex Jupiter 10 µM C18 300 Å column using the  To study the effect of serum and ions on the antifungal activity, increasing concentrations of heat-inactivated fetal calf serum (5-15%) and NaCl, CaCl 2 and MgCl 2 (1.25-10 mM) were included in broth microdilution assays together with 1 × IC 90 PAFC and C. albicans in 0.1 × PDB as described above. C. albicans cells exposed to the supplements without PAFC served as controls. The thermal tolerance of PAFC was studied by incubating the protein for 5 min at 95 • C and determining the antifungal activity immediately after cooling to 25 • C. PAFC that had not been exposed to serum, ions or heat served as full-activity control. The optical density (OD 620nm ) was determined after static cultivation for 24 h at 30 • C using a multi-mode microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany) operating in well-scanning mode. All assays included a blank (medium without cells) and an untreated growth control representing 100% growth. All experiments were done using technical triplicates and repeated at least twice.

Determination of Colony Forming Units (CFU)
To determine the killing efficacy of PAFC, C. albicans planktonic cells from an overnight culture were diluted to 2 × 10 5 cells·mL −1 in 0.1 × PDB and treated with 1 × IC 90 (2.5 µM), 2 × IC 90 (5 µM) or 4 × IC 90 (10 µM) PAFC for 1 h, 8 h and 24 h at 30 • C under continuous shaking (160 rpm). Untreated cells were used as a growth control. Cells were collected by centrifugation, resuspended in 1 mL 0.1 × PDB and a serial dilution up to 10 −6 was prepared. From each dilution, 100 µL were streaked onto 2% (w/v) PDB agar plates and incubated at 30 • C for 24 h before the colony forming units (CFU) were counted. The CFU of the untreated control was set at 100%. All experiments were done using technical quadruplicates and repeated twice.
To evaluate the activity of PAFC on sessile cells of C. albicans, biofilm formation was induced by distributing 100 µL aliquots of a cell suspension (1 × 10 6 cells·mL −1 in 0.1 × PDB) in the wells of a 96-well, flat-bottom microtiter plate (Thermo Scientific, Waltham, MA, USA) followed by a static incubation for 24 h at 30 • C. The resulting biofilm was checked microscopically for cell attachment and pseudo-hyphae formation before treatment. The biofilm was gently washed with 0.1 × PDB before 100 µL of 1 × IC 90 (2.5 µM) and 10 × IC 90 (25 µM) PAFC was added in respective wells. For the negative control, the cells were incubated with 0.1 × PDB and for the positive control the cells were exposed to 10 µg·mL −1 amphotericin B. After 24 h of incubation, the biofilm was disrupted by vigorous pipetting, and 10 µL of the detached cells were mixed with 40 µL fresh 0.1 × PDB. Serial dilution of this sample up to 10 −6 were made and 100 µL streaked out onto 2% (w/v) PDB agar plates and incubated at 30 • C for 24 h for CFU determination. The CFU of the untreated control was set at 100%. All experiments were conducted using technical triplicates and repeated twice.

Scanning Electron Microscopy (SEM)
C. albicans cells (5 × 10 5 , diluted in 0.1 × PDB) from an overnight liquid culture were incubated with 1 × IC 90 PAFC for 1 h, 12 h, and 24 h at 30 • C under continuous shaking at 160 rpm. Untreated cells were used as a negative control. Samples were washed, resuspended in phosphate buffered saline (PBS) and 8 µL samples spotted onto a silicon disc coated with 0.01% (w/v) poly-l-lysine (Merck Millipore, Billerica, MA, USA). Cells were fixed with 2.5% (v/v) glutaraldehyde and 0.05 M cacodylate buffer (pH 7.2) in PBS overnight at 4 • C. The discs were washed twice with PBS and dehydrated with a graded ethanol series (30%, 50%, 70%, 80%, 100% ethanol (v/v), for 1.5 h each at 4 • C). Samples of untreated cells served as negative control. The samples were dried with a critical point dryer, followed by 12 nm gold coating (Quorum Technologies, Laughton, East Sussex, UK) and observed under a JEOL JSM-7100F/LV scanning electron microscope (JEOL Ltd., Tokyo, Japan).

Fluorophore-Labeling of PAFC
For visualization in uptake studies PAFC was labeled with the green fluorophore BODIPY™ FL EDA (Bd) (Invitrogen, Carlsbad, CA, USA) as described previously [27]. In brief, 0.4 mM PAFC was labeled with 10 mM Bd in the presence of 10 mM EDAC and 5 mM Sulfo-NHS (Invitrogen, Carlsbad, CA, USA) in 100 M MES-buffer (pH 4.5). The reaction mixture was incubated with continuous shaking at 200 rpm overnight at 25 • C in the dark. The labeled AMP (PAFC-Bd) was dialyzed against ddH 2 O to remove excess Bd and concentrated using Amicon®Ultra centrifugal filters (3 kDa MWCO; Merck Millipore, Burlington, MA, USA). The antifungal activity of PAFC-Bd was tested by broth microdilution assay as described above.

Confocal Laser Scanning Microscopy (CLSM)
Candida cells adjusted to 2 × 10 5 cells·mL −1 in 0.1 × PDB were treated with 1 × IC 90 PAFC-Bd (2.5 µM) for 3 h and 6 h at 30 • C under continuous shaking at 160 rpm. Untreated cells were used as negative control. Samples were washed with PBS and stained consecutively with 5 µg mL −1 PI (Sigma-Aldrich, St. Louis, MO, USA) and 5 µg·mL −1 calcofluor white (CFW) (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature in the dark. After washing with PBS, samples were mounted on a glass slide, covered with 2% (w/v) agar slices and observed with an Olympus Fluoview FV 1000 confocal laser microscope with 60× magnification objective (Olympus, Shinjuku, Japan). A 488 nm laser was used for excitation. The excitation and emission wavelengths were 380 nm and 475 nm for CFW, 504 nm and 512 nm for PAFC-Bd and 535 nm and 617 nm for PI, respectively. Sequential scanning was used to avoid crosstalk between the fluorescent dyes.

Fluorescence Activated Cell Sorting (FACS)
Candida cells diluted to 2 × 10 5 cells·mL −1 in 0.1 × PDB were exposed to 1 × IC 90 PAFC for 1 h, 8 h and 24 h at 30 • C under continuous shaking at 160 rpm. Untreated cells were used as negative control and 70% (v/v) EtOH treated cells as positive control for PI staining. The cells were washed in PBS, collected by centrifugation at 11,000× g for 5 min and stained with 5 µg·mL −1 PI for 10 min at room temperature. PI positive cells were counted by a FlowSight imaging flow cytometer (Amins, Merck Millipore, Billerica, MA, USA) with at least 1000 cells were counted per run. For data analysis, the Image Data Exploration and Analysis software (IDEAS; Amins, Millipore, Billerica, MA, USA) was applied. Gates for the data analyses were established according to the unstained control. Experiments were performed using independent triplicates.
Plates were incubated at 37 • C for 24 h before documentation.

Statistical Analysis
Data analyses were conducted with Microsoft Excel (2016, Version 16.16.16) software (Microsoft, Redmond, WA, USA). For the calculation of significant differences between the data obtained from treated samples vs. the untreated controls, a two-sample Student's t-test was applied. p-values of ≤0.05 were considered as significant and p-values of ≤0.005 were considered as highly significant.

In Silico Prediction of the PAFC Structure
The alignment of the pre-mature PAFC pre-pro protein sequence with that of PeAfpC from P. expansum and BP from P. bevicompactum revealed 85.2% and 70.1% identity, respectively ( Figure S3). The mature PAFC is 64 aa long, has a predicted molecular mass of 6630 Da (assuming all cysteines in reduced form) and is 100% identical to the Pc-Arctin of the arctic isolate P. chrysogenum A096 [10]. It has 82.8% and 79.7% identity to the mature form of PeAfpC and BP, respectively ( Figure S3). All three proteins possess two putative levomeric γ-core motifs (CX 3-9 CXGX 1-3 ) [29], which are conserved among the ascomycetous AMPs of the BP cluster [13]. One of them-positioned in the center of PAFC (CDRTGIVECKG)-is highly conserved, and the second, shorter one with lower homology resides near the C-terminus (CGGASCRG) ( Figure 1A and Figure S3).
The in silico prediction of the PAFC tertiary structure gave a model showing high fold similarity to the BP of P. brevicompactum [30]. The Ramachandran plot of the model showed 95.2% of the aa positioned in energetically favored regions and three in the allowed regions (4.8%) ( Figure S4). The PAFC model exhibits five antiparallel β-strands (β1-β5), spanning His26-Cys28 (β1), Gly34-Lys39 (β2), Lys42-Asp48 (β3), Arg55-Val57 (β4) and Gly60-Arg63 (β5), which are connected by four loops (L1-L4). The protein model shows an N-terminal three stranded β-sheet (β-sheet 1) with an N-terminal extended structure and a C-terminal two stranded β-sheet (β-sheet 2). The γ-core motif in the protein center (Cys30-Lys39) encompasses L1 and β2, and the shorter γ-core motif (Cys49-Gly56) contains a part of L3 and β4 ( Figure 1B). As already shown for the BP protein [11] the β-pleated structure of PAFC is stabilized by four intramolecular disulfide bonds that are formed between the eight cysteine residues. The predicted disulfide bond pattern (Cys 3/30, Cys 18/38 Cys 28/54, Cys 49/64) of PAFC follows the abcabdcd pattern of the BP [30]. The disulfide bonds Cys 3/30 and Cys 18/38 connect the β-sheet 1 to the N-terminal extended region, while Cys 28/54 and Cys 49/64 connect β-sheet 1 with β-sheet 2 ( Figure 1C). J. Fungi 2020, 6, x FOR PEER REVIEW 7 of 21 49/64) of PAFC follows the abcabdcd pattern of the BP [30]. The disulfide bonds Cys 3/30 and Cys 18/38 connect the β-sheet 1 to the N-terminal extended region, while Cys 28/54 and Cys 49/64 connect βsheet 1 with β-sheet 2 ( Figure 1C). Analysis of the electrostatic surface distribution according to Coulomb's law in UCSF Chimera software [21] indicated that the putative PAFC structure has a cavity similar to BP [30]. The opening of this "mouth-like" structure is dominated by basic aas (Arg12, Arg13, Arg25, Arg32) that are in highly conserved positions in BP and PAFC, with Arg12, Phe27 and Trp43 form the funnel base ( Figure 1D). The opposite side has two negatively charged patches, a smaller one consisting of Asp24 and Glu37, and a bigger one, consisting of Asp31, Asp48 and Glu45 ( Figure 1D; Supplementary Materials, Video S1). According to the Kyte-Doolittle scale [31], PAFC shows an amphipathic surface with the aas Val9, Ile35/Ile46 and Val57 forming three hydrophobic patches ( Figure 1E; Supplementary Materials, Video S2).  Analysis of the electrostatic surface distribution according to Coulomb's law in UCSF Chimera software [21] indicated that the putative PAFC structure has a cavity similar to BP [30]. The opening of this "mouth-like" structure is dominated by basic aas (Arg12, Arg13, Arg25, Arg32) that are in highly conserved positions in BP and PAFC, with Arg12, Phe27 and Trp43 form the funnel base ( Figure 1D). The opposite side has two negatively charged patches, a smaller one consisting of Asp24 and Glu37, and a bigger one, consisting of Asp31, Asp48 and Glu45 ( Figure 1D; Supplementary Materials, Video S1). According to the Kyte-Doolittle scale [31], PAFC shows an amphipathic surface with the aas Val9, Ile35/Ile46 and Val57 forming three hydrophobic patches ( Figure 1E; Supplementary Materials, Video S2).

Expression of PAFC in P. chrysogenum
Northern blot experiments indicated that the expression of the PAFC encoding gene (pafC) peaked after 72 h of cultivation under standard submersed conditions ( Figure 2A). Expression of pafC was also detected in synchronized P. chrysogenum surface cultures and correlated with the onset of conidiation ( Figure S5), with gene expression reaching its maximum after 36 h of incubation ( Figure 2B). No pafC expression was detected in conidia or 11-h-old germlings. These results imply that PAFC is produced under submersed and surface growth conditions in P. chrysogenum. To demonstrate PAFC in the fungal culture broth, we performed a Western blot analysis ( Figure 2C) probed with a rabbit polyclonal antibody generated against the PAFC-related PeAfpC [12]. This antibody recognized the purified recombinant PAFC ( Figure S6) and did not bind to the purified recombinant PAF or PAFB ( Figure 3B, upper panel). A faint band with the molecular weight corresponding to mature native PAFC was detected in the crude fermentation broth of submersed cultures after 72 h and 96 h of cultivation ( Figure 2C). However, the anti-PeAfpC antibody also bound to secreted proteins with molecular weight > 6.6 kDa in the crude supernatant. This may be due to (I) the polyclonal character of the anti-PeAfpC antibody [12] and (II) the low PAFC amount in the culture broth, which necessitated a long development time to obtain a detectable signal intensity for the PAFC specific band.
J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 21 visualization and analysis software [21] depicting the structure of the β-strands (blue) and location of the two γ-cores (red), the N-and C-terminus is indicated. (C) Disulfide bonding of PAFC is shown with yellow sticks. (D) Surface representation of PAFC in two orientations colored according to electrostatic potential (blue: electropositive, red: electronegative). The model depicted on the right side visualizes the "mouth-like" cavity indicated by a black arrow in the left model. The basic aas surrounding the opening and the aa forming the funnel base are indicated. (E) Distribution of hydrophobic and hydrophilic patches on the surface of PAFC in two orientations colored according to the Kyte-Doolittle scale (blue: hydrophilic, orange: hydrophobic). The aas forming the hydrophobic patches are indicated in the model on the right.

Expression of PAFC in P. chrysogenum
Northern blot experiments indicated that the expression of the PAFC encoding gene (pafC) peaked after 72 h of cultivation under standard submersed conditions ( Figure 2A). Expression of pafC was also detected in synchronized P. chrysogenum surface cultures and correlated with the onset of conidiation ( Figure S5), with gene expression reaching its maximum after 36 h of incubation ( Figure  2B). No pafC expression was detected in conidia or 11-h-old germlings. These results imply that PAFC is produced under submersed and surface growth conditions in P. chrysogenum. To demonstrate PAFC in the fungal culture broth, we performed a Western blot analysis ( Figure 2C) probed with a rabbit polyclonal antibody generated against the PAFC-related PeAfpC [12]. This antibody recognized the purified recombinant PAFC ( Figure S6) and did not bind to the purified recombinant PAF or PAFB ( Figure 3B, upper panel). A faint band with the molecular weight corresponding to mature native PAFC was detected in the crude fermentation broth of submersed cultures after 72 h and 96 h of cultivation ( Figure 2C). However, the anti-PeAfpC antibody also bound to secreted proteins with molecular weight > 6.6 kDa in the crude supernatant. This may be due to (Ι) the polyclonal character of the anti-PeAfpC antibody [12] and (ΙΙ) the low PAFC amount in the culture broth, which necessitated a long development time to obtain a detectable signal intensity for the PAFC specific band.

Expression of PAFC, PAF and PAFB in Fungal Exudates
Seibold et al. (2011) [11] found the BP in exudates of P. brevicompactum surface cultures. This prompted us to check the droplets formed on the colony surface of P. chrysogenum grown on solid medium. These droplets emerged after 120 h of cultivation on colonies that had been point inoculated on solid P. chrysogenum minimal medium (1 × PcMM). On this medium the droplets formed a ring around the center of the P. chrysogenum colony ( Figure 3A). These droplets originated from the fungal exudate that the colony secreted basally. The amount of liquid underneath the colony and the size of droplets could be augmented by cultivating P. chrysogenum on agar containing double-concentrated nutrients (2 × PcMM) ( Figure 3A). Here the droplets accumulated on top of the colony. When the exudate that had formed underneath the colony after 96 h was harvested, no further droplets emerged on top of the colony with further incubation (Figure S1), indicating the same origin of the exudate and the droplets. After an extended incubation of 144 h the exudate had completely disappeared ( Figure S1). PAFC was detected by Western blot analysis in both, the exudate formed underneath the colony and the droplets ( Figure 3B). We also tested the samples for the presence of the other two P. chrysogenum AMPs and could detect PAF and PAFB by using polyclonal anti-PAF and anti-PAFB antibodies [9,25] ( Figure 3B).

Expression of PAFC, PAF and PAFB in Fungal Exudates
Seibold et al. (2011) [11] found the BP in exudates of P. brevicompactum surface cultures. This prompted us to check the droplets formed on the colony surface of P. chrysogenum grown on solid medium. These droplets emerged after 120 h of cultivation on colonies that had been point inoculated on solid P. chrysogenum minimal medium (1 × PcMM). On this medium the droplets formed a ring around the center of the P. chrysogenum colony ( Figure 3A). These droplets originated from the fungal exudate that the colony secreted basally. The amount of liquid underneath the colony and the size of droplets could be augmented by cultivating P. chrysogenum on agar containing double-concentrated nutrients (2 × PcMM) ( Figure 3A). Here the droplets accumulated on top of the colony. When the exudate that had formed underneath the colony after 96 h was harvested, no further droplets emerged on top of the colony with further incubation (Figure S1), indicating the same origin of the exudate and the droplets. After an extended incubation of 144 h the exudate had completely disappeared ( Figure S1). PAFC was detected by Western blot analysis in both, the exudate formed underneath the colony and the droplets ( Figure 3B). We also tested the samples for the presence of the other two P. chrysogenum AMPs and could detect PAF and PAFB by using polyclonal anti-PAF and anti-PAFB antibodies [9,25] (Figure 3B).   Figure 2C indicates that the amount of native PAFC in the supernatant of P. chrysogenum was insufficient for purification and further functional investigations. Therefore, we expressed recombinant PAFC using a P. chrysogenum-based expression system [26] by cloning pafC into the pSK275 vector between the strong paf promoter and the paf terminator sequence ( Figure S2). The P. chrysogenum ∆paf strain transformed with linearized pSK275_pafC served as cell factory for PAFC expression [32,33]. The strain secreting the highest PAFC quantity (P. chrysogenum OEpafC ) was selected for protein production and PAFC was purified from 96 h cell culture supernatant by one-step cation-exchange chromatography ( Figure S7A). A protein yield of up to 105 ± 15 mg L −1 fermentation broth was obtained. RP-HPLC gave a single elution peak suggesting high purity of the sample ( Figure S7B). The identity of PAFC was verified by ESI-MS. The detected mass of 6622 Da matched the predicted mass of oxidised PAFC which contains four disulfide bonds and lacks the pre-pro sequence ( Figure S7C).

Anti-Candida Activity of PAFC in Broth Microdilution Assays
The recombinant PAFC was tested for anti-Candida activity in broth microdilution assays and the inhibitory concentration that reduces growth ≥90% (IC 90 ) was determined (Table 1). PAFC inhibited the growth of the Candida spp. tested (Table S1) at low µM concentrations, and the fluconazole sensitive C. albicans fluS and a fluconazole resistant clinical isolate C. albicans fluR exhibited the same PAFC sensitivity (IC 90 2.5 µM) ( Table 1 and Table S1). For comparison, we also tested the susceptibility of Candida spp. for the commonly used antifungal drugs fluconazole, amphotericin B and nystatin ( Table 1). The C. albicans fluR strain still proliferated in the presence of the highest fluconazole concentration tested (125 µg mL −1 ) which confirmed its resistance against this common drug under the experimental conditions applied in this study.

Impact of PAFC on C. albicans Biofilm
We investigated the antifungal effect of PAFC on sessile cells of the most prevalent pathogen in this group using C. albicans fluS and C. albicans fluR . A 24-h-old biofilm was treated with 1 × IC 90 (2.5 µM) and 10 × IC 90 (25 µM) PAFC because biofilms are generally less accessible to antifungal compounds [34] and the established biofilm contains a higher cell number than the experiments performed with planktonic cells.
The survival of the sessile cells evaluated by CFU determination using a plating assay revealed that PAFC inhibited biofilm formation in a concentration dependent manner. Table 2 shows that the number of viable C. albicans fluS cells decreased significantly after the 24-h treatment with 10 × IC 90 PAFC compared to the untreated control. The C. albicans fluR seemed to be more sensitive because a lower PAFC concentration (1 × IC 90 ) significantly increased cell death. This was further aggravated when PAFC was applied at 10 × IC 90 . In both strains, however, a PAFC concentration as high as 10 × IC 90 could not completely impede biofilm growth as shown with 10 µg mL −1 amphotericin B (no growth).

Effect of PAFC on the C. albicans Cell Morphology
The effect of PAFC on the morphology of C. albicans fluS and C. albicans fluR cells was evaluated using SEM. Untreated control cells exhibited a typical ovoid shape with budding sites and a smooth cell surface (Figure 4). In contrast, a 1 h treatment with 1 × IC 90 PAFC (2.5 µM) showed cells with a wrinkly surface and amorphous material on and between cells, which may indicate loss of osmotic pressure and leakage of cell contents, respectively. With longer incubation time (12 h), C. albicans cells showed severe signs of cell leakage and lost their ovoid shape. After 24 h morphologically intact C. albicans fluS cells were observed again, suggesting that some cells survived the treatment and resumed growth ( Figure 4A). In contrast, C. albicans fluR cells still appeared severely damaged after 24 h of PAFC exposure ( Figure 4B).

Effect of PAFC on the C. albicans Cell Morphology
The effect of PAFC on the morphology of C. albicans fluS and C. albicans fluR cells was evaluated using SEM. Untreated control cells exhibited a typical ovoid shape with budding sites and a smooth cell surface (Figure 4). In contrast, a 1 h treatment with 1 × IC90 PAFC (2.5 μM) showed cells with a wrinkly surface and amorphous material on and between cells, which may indicate loss of osmotic pressure and leakage of cell contents, respectively. With longer incubation time (12 h), C. albicans cells showed severe signs of cell leakage and lost their ovoid shape. After 24 h morphologically intact C. albicans fluS cells were observed again, suggesting that some cells survived the treatment and resumed growth ( Figure 4A). In contrast, C. albicans fluR cells still appeared severely damaged after 24 h of PAFC exposure ( Figure 4B).

Cellular Localization of PAFC and Cell Death Induction
To investigate whether PAFC interacts only with the outer cell layers (cell wall or plasma membrane) or enters the cytosol of the C. albicans cells we used CLSM to detect PAFC labeled with the green fluorophore BODIPY (PAFC-Bd). The fluorophore-labelling of PAFC had no adverse

Cellular Localization of PAFC and Cell Death Induction
To investigate whether PAFC interacts only with the outer cell layers (cell wall or plasma membrane) or enters the cytosol of the C. albicans cells we used CLSM to detect PAFC labeled with the green fluorophore BODIPY (PAFC-Bd). The fluorophore-labelling of PAFC had no adverse impact on its antifungal activity. The application of 2.5 µM PAFC-Bd in a broth microdilution assay resulted in C. albicans growth inhibition of 98.6%, which corresponded to the IC 90 of PAFC. Co-staining with the membrane impermeant fluorescent cell death dye PI visualized cells that were killed by the interaction with PAFC-Bd. Cells were incubated for 3 h and 6 h to ensure that the PAFC-Bd specific fluorescence signal was intense enough for visualization. After 3 h PAFC-Bd attached to the outer layers of C. albicans fluS and this signal co-localized with the cell wall specific blue dye CFW ( Figure 5A). Intracellular fluorescent patches (subcellular structures) indicated that PAFC-Bd was taken up by the cells without cell death induction (no PI signal). After 6 h the positively stained cells showed the PAFC-Bd signal dispersed in the whole cell (cytoplasm) that coincided with a strong intracellular PI signal, whereas cells exhibiting a PAFC-Bd signal that still localized in subcellular structures remained PI-negative ( Figure 5A). A similar uptake and intracellular staining pattern with PAFC-Bd was observed with C. albicans fluR cells, although interaction of PAFC-Bd with cells could be detected only at 6 h of incubation ( Figure 5B). Localization of PAFC-Bd with the nuclei was excluded when co-staining with the nuclei-specific dye Hoechst 33342 and imaged by fluorescence microscopy (Supplementary Materials, experimental procedures; Figure S8). Taken together, these qualitative microscopy-based data revealed that PAFC first enters sensitive cells before the plasma membrane is compromised and cell death is induced. This suggests that an intracellular PAFC target may exist.
J. Fungi 2020, 6, x FOR PEER REVIEW 12 of 21 impact on its antifungal activity. The application of 2.5 μM PAFC-Bd in a broth microdilution assay resulted in C. albicans growth inhibition of 98.6%, which corresponded to the IC90 of PAFC. Costaining with the membrane impermeant fluorescent cell death dye PI visualized cells that were killed by the interaction with PAFC-Bd. Cells were incubated for 3 h and 6 h to ensure that the PAFC-Bd specific fluorescence signal was intense enough for visualization. After 3 h PAFC-Bd attached to the outer layers of C. albicans fluS and this signal co-localized with the cell wall specific blue dye CFW ( Figure 5A). Intracellular fluorescent patches (subcellular structures) indicated that PAFC-Bd was taken up by the cells without cell death induction (no PI signal). After 6 h the positively stained cells showed the PAFC-Bd signal dispersed in the whole cell (cytoplasm) that coincided with a strong intracellular PI signal, whereas cells exhibiting a PAFC-Bd signal that still localized in subcellular structures remained PI-negative ( Figure 5A). A similar uptake and intracellular staining pattern with PAFC-Bd was observed with C. albicans fluR cells, although interaction of PAFC-Bd with cells could be detected only at 6 h of incubation ( Figure 5B). Localization of PAFC-Bd with the nuclei was excluded when co-staining with the nuclei-specific dye Hoechst 33342 and imaged by fluorescence microscopy (Supplementary Materials, experimental procedures; Figure S8). Taken together, these qualitative microscopy-based data revealed that PAFC first enters sensitive cells before the plasma membrane is compromised and cell death is induced. This suggests that an intracellular PAFC target may exist.

Candidacidal Efficacy of PAFC
The time course of cell death caused by PAFC was detected using the cell death specific dye PI with FACS analysis ( Table 3). Exposure of C. albicans fluS to 1 × IC 90 (2.5 µM) PAFC resulted in a time dependent increase in the proportion of PI positive cells after 1 h and 8 h of incubation. Interestingly, after a 24 h incubation the number of cells with a PI-positive phenotype dropped below 1%, suggesting that surviving cells had resumed growth. The treatment of C. albicans fluR with PAFC similarly induced an increase in PI-positive cells between 1 h and 8 h of exposure, though in a slightly delayed manner. In strong contrast to the C. albicans fluS , however, the percentage of C. albicans fluR cells with a PI-positive phenotype further increased with time and reached more than 70% after 24 h, indicating that a high proportion of cells suffered from plasma membrane permeabilization due to PAFC treatment. These data were confirmed by using CFU determinations after the exposure of C. albicans cells to increasing PAFC concentrations (2.5-10 µM) corresponding to 1 × IC 90 , 2 × IC 90 and 4 × IC 90 in a time course (1 h, 8 h, 24 h) ( Figure 6). In C. albicans fluS , only 21% ± 2.0% survived the 8-h treatment with 1 × IC 90 , but after 24 h of exposure the CFU increased again to 49.5% ± 2.5%, indicating that surviving cells had resumed growth ( Figure 6A). In contrast, 1 × IC 90 PAFC treatment of C. albicans fluR gave higher CFU numbers (31.0% ± 0.7%) after 8 h, but very few cells survived the 24-h treatment (1.1% ± 0.001%) ( Figure 6B). This time-dependent trend was strain specific and apparent at all concentrations tested. These results comfirm the data generated by FACS and suggest that while PAFC killed C. albicans fluS cells quickly, the survivors of the treatment proliferated again. In contrast, PAFC had a delayed action on C. albicans fluR but was ultimately more effective as a fungicide.

Intracellular ROS Induction by PAFC
Studies on the mode of action of fungal AMPs have shown that their activity is often closely linked with the induction of iROS [13,35,36]. The formation of iROS in Candida cells in response to PAFC was therefore investigated by fluorescence microscopy (Supplementary Materials, experimental procedures). C. albicans cells exposed to 1 × IC 90 PAFC (2.5 µM) for 8 h were loaded with the non-fluorescent dye dichlorodihydrofluorescein diacetate (H 2 DCFDA). In the presence of iROS, this compound is converted intracellularly to the fluorescent dichlorofluorescein (DCF). As depicted in Figure 7, both C. albicans strains suffered from iROS induction by PAFC, just like the positive control cells, which had been exposed to 10 µg mL −1 of the ROS inducing polyene drug nystatin. Untreated control cells did not show any DCF specific signal (Figure 7).     . Excess of fluorescent dye was removed by washing in PBS and the cells were mounted on glass slides for evaluation of iROS induction by fluorescence microscopy. Cells without treatment and exposed to nystatin (10 µg mL −1 ) were used as negative and iROS-positive controls, respectively. The merged images show the DCF signal (green) superimposed with the Candida cells visualized with brightfield microscopy. One representative experiment out of three independent experiments is shown. Scale bar, 5 µm.

Testing of Serum-, Ion-, Thermotolerance and Hemolytic Activity of PAFC
The tolerance of PAFC to serum compounds, high ion concentrations and extreme temperature as well as the lack of adverse effects in the host are important prerequisites for considering this AMP for a potential medical application in the future. The presence of 5-15% inactivated fetal calf serum in the medium reduced PAFC activity against C. albicans fluS in a dose-dependent manner (Figure 8). Similarly, the supplementation of the growth medium with 1.25-10 mM MgCl 2 , NaCl and CaCl 2 counteracted the PAFC activity in a concentration dependent manner, with PAFC being most sensitive to MgCl 2 ( Figure S9). The heating of PAFC to 95 • C for 5 min and cooling to 25 • C had no adverse effect on its antifungal efficacy with retention of an IC 90 of 2.5 µM by C. albicans fluS . PAFC reduced the fungal growth after 24 h of incubation to 7.5 ± 2.7% and to 5.8 ± 4.2% before and after its thermal treatment, respectively, which underlined its high thermotolerance. Finally, PAFC showed no hemolytic activity when tested on sheep erythrocytes in an agar diffusion assay using Columbia blood agar plates ( Figure S10).
H2DCFDA (5 ng μL −1 ). Excess of fluorescent dye was removed by washing in PBS and the cells were mounted on glass slides for evaluation of iROS induction by fluorescence microscopy. Cells without treatment and exposed to nystatin (10 μg mL −1 ) were used as negative and iROS-positive controls, respectively. The merged images show the DCF signal (green) superimposed with the Candida cells visualized with brightfield microscopy. One representative experiment out of three independent experiments is shown. Scale bar, 5 μm.

Testing of Serum-, Ion-, Thermotolerance and Hemolytic Activity of PAFC
The tolerance of PAFC to serum compounds, high ion concentrations and extreme temperature as well as the lack of adverse effects in the host are important prerequisites for considering this AMP for a potential medical application in the future. The presence of 5-15% inactivated fetal calf serum in the medium reduced PAFC activity against C. albicans fluS in a dose-dependent manner (Figure 8). Similarly, the supplementation of the growth medium with 1.25-10 mM MgCl2, NaCl and CaCl2 counteracted the PAFC activity in a concentration dependent manner, with PAFC being most sensitive to MgCl2 ( Figure S9). The heating of PAFC to 95 °C for 5 min and cooling to 25 °C had no adverse effect on its antifungal efficacy with retention of an IC90 of 2.5 μM by C. albicans fluS . PAFC reduced the fungal growth after 24 h of incubation to 7.5 ± 2.7% and to 5.8 ± 4.2% before and after its thermal treatment, respectively, which underlined its high thermotolerance. Finally, PAFC showed no hemolytic activity when tested on sheep erythrocytes in an agar diffusion assay using Columbia blood agar plates ( Figure S10).

Discussion
We have predicted the structure, studied the expression and characterized the antifungal mode of action of PAFC, which phylogenetically belongs to the BP group and represents-apart from PAF and PAFB [8,9,13,37]-the third and previously uncharacterized AMP from the industrial strain P. chrysogenum Q176. The mean ± SD (technical triplicate of one representative experiment out of two biological replicates) is shown. A two-sample Student's t-test was applied to calculate the significant difference between the serum-treated samples compared to the untreated growth control (* p ≤ 0.05 and ** p ≤ 0.005).

Discussion
We have predicted the structure, studied the expression and characterized the antifungal mode of action of PAFC, which phylogenetically belongs to the BP group and represents-apart from PAF and PAFB [8,9,13,37]-the third and previously uncharacterized AMP from the industrial strain P. chrysogenum Q176.
Similar to the crystal structure of the P. brevicompactum BP, in silico analysis of PAFC suggested a β-fold structure containing five antiparallel β-strands. Notably, for PeAfpC, a β-sheet structure composed of only three β-strands was predicted [12]. This discrepancy could result from the different in silico approach and prediction software applied to model the proteins. The disulfide bonds in cysteine-rich AMPs mediate high tolerance to harsh environmental conditions [38]. For Pc-Arctin, a high thermal stability but low ion tolerance and low proteolytic stability has been reported [10]. In line with this observation, PAFC showed a high sensitivity to serum and ions but thermal tolerance under the experimental conditions applied. The positively charged funnel-like opening predicted for PAFC could be involved in protein function, such as ligand binding and/or interaction with negatively charged membrane compounds of the fungal target cells, as suggested for this motif in BP [30,39,40]. A detailed analysis by nuclear magnetic resonance to resolve the PAFC solution structure is in progress.
The presence of the pafC transcripts in the mycelium of shaking submersed and surface cultures grown on solid medium, but not in dormant conidia or germinated conidia, indicated that PAFC might play a role after colony establishment in fungal growth and/or differentiation of P. chrysogenum. This assumption was supported by the finding that pafC transcription started in the mycelia of surface cultures at the onset of sporulation.
It is generally hypothesized that AMPs from filamentous fungi-apart from their antifungal activity-fullfill additional functions in the host during adaptation to environmental conditions [41,42]. In P. chrysogenum roles of PAF and PAFB were reported in sensing/signaling during growth, differentiation and/or asexual sporulation [8,24,41] but detailed knowledge at the molecular level of the role of fungal AMPs in adaptive responses is still lacking. However, we have shown for the first time that the three P. chrysogenum AMPs PAF, PAFB and PAFC are abundantly secreted together into the exudate of old sporulating surface colonies grown on rich medium. Generally, fungal exudates contain secondary metabolites, enzymes and other by-products that are assumed to support cell survival under unfavorable environmental conditions. These liquid reservoirs can be reabsorbed when needed [43]. Indeed, these droplets and the exudate below the colony disappeared with longer incubation. This observation could indicate that exudate production in P. chrysogenum is related to nutrient access and the age of the mycelium.
The amount of endogenous PAFC was insufficient to provide the purified protein needed for characterization of its antifungal activity. Instead, we used a well-established P. chrysogenum-based expression system to produce recombinant PAFC. We obtained protein yields better than those achieved previously with other recombinant AMPs using this system [9,12,13,26,27,[44][45][46][47][48].
The BP cluster AMPs were poorly characterized with regard to their potential medical applicability. Our study shows the in vitro growth inhibitory efficacy of a BP cluster protein against opportunistic pathogenic fungi of the genus Candida. The anti-Candida effective concentration range of PAFC resembled that of PAF and PAFB, the latter showing the highest efficacy against the NAC species C. glabrata, C. krusei and C. parapsilosis (IC 90 0.6 µM) with PAF being most effective against C. parapsilosis (IC 90 2.5 µM) [7,9]. The growth inhibitory activity of Pc-Arctin from P. chrysogenum A096 was tested on the common environmental mold Paecilomyces variotii, the plant pathogen Alternaria longipes, and the biofungicide Trichoderma viride [10]. For the P. brevicompactum BP, growth inhibitory activity against Saccharomyces cerevisiae was reported [11], whereas no antifungal activity was detected for PeAfpC [12].
To dissect in more detail the antifungal mode of action of PAFC, we used two strains of the most prevalent human pathogenic yeast, the fluconazole-sensitive C. albicans fluS and the fluconazole-resistant clinical isolate C. albicans fluR [16], whereby the latter strain still proliferated in the presence of the highest fluconazole concentration applied in this study. The molecular basis for the fluconazole resistance of C. albicans fluR is not known and subject of current investigations.
Both C. albicans strains exhibited the same IC 90 value (2.5 µM). However, the broth microdilution assays show effects on growth but not on the killing potential of an antifungal compound. Therefore, we applied plating assays for CFU determination plus SEM, CLSM and FACS to visualize and quantitate the fungicidal efficacy of PAFC. Our data indicated that PAFC acted in a fungicidal way on sessile and planktonic cells of both C. albicans strains under the test conditions applied, though the efficacy was higher against C. albicans fluR than against C. albicans fluS . After 24 h of incubation PAFC killed more planktonic and sessile cells of C. albicans fluR than of C. albicans fluS . Interestingly, cells of the latter strain resumed growth after 24 h of PAFC exposure, which suggests an adaptive response to PAFC treatment. In contrast, C. albicans fluR might lack the ability to respond adequately to the antifungal PAFC action [49]. It is known that drug resistance can be acquired at the cost of cellular fitness, leading to a disadvantage for the organism under unfavorable conditions in the absence of the specific drug [50]. Further investigations are needed to clarify, if azole-resistant C. albicans strains are generally more susceptible to AMPs than sensitive ones. In this respect, the comparison of several well characterized drug-resistant clinical isolates with standard strains for PAFC susceptibility or the screening of drug-sensitive parental and resistant daughter strains isolated from the same patient could be supportive.
The cell damage induced by PAFC resembled that reported for the candidacidal-acting Neosartorya fischeri antifungal protein 2 (NFAP2) [16]. However, our qualitative analysis by CLSM revealed that cell death in C. albicans requires PAFC uptake and cytoplasmic localization before plasma membrane permeabilization occurs pointing towards an intracellular target. This mode of action strongly resembles that of P. chrysogenum PAF and PAFB [9,25,27], the N. fischeri antifungal protein NFAP [48] and synthetic antifungal peptides, e.g., PAF26 [51]. Notably, PAFC activity was closely linked to the accumulation of iROS, which is generally recognized as a trigger for apoptotic cell death in C. albicans [52]. Indeed, oxidative stress seems to be a common phenotypic marker of AMP-mediated antifungal activity as reported for PAF [13,53], several plant defensins [54,55] and synthetic antifungal peptides [13,56].

Conclusions
The emerging drug resistance of human pathogenic fungi and the limited number of antifungal drugs requires the urgent identification of new antifungal compounds with novel mechanism of action [2]. We have shown that PAFC is a promising new antifungal biomolecule. It can be produced in high yields and quality in P. chrysogenum, a generally recognized as safe organism [57]. The recombinant PAFC effectively inhibited the growth of the opportunistic human pathogen C. albicans and exhibited candidacidal activity with high efficacy against a fluconazole-resistant C. albicans clinical isolate. The high thermotolerance and the lack of hemolytic activity further supports its potential applicability in clinical therapy. Similar, to many other cysteine-rich, cationic AMPs from filamentous fungi [8,10,27,53,58] PAFC also exhibited serum and cation sensitivity, which may hamper an intravenous application to combat severe systemic fungal infections. Instead, PAFC might help to develop new drugs for topical prevention or cure of fungal nail, skin and mucosal infections while repeated application of PAFC may be required to maintain its candidacidal efficacy. Furthermore, rational design could be a promising option to improve the antifungal efficacy of PAFC and overcome features that limit its potential for medical application.
Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/6/3/141/s1, Table S1. Fungal and bacterial strains used in this study; Table S2. Composition of media and solutions used in this study; Table S3. PCR conditions applied in this study; Table S4. Oligonucleotides used in this study; Figure S1. Exudate formation of P. chrysogenum surface colonies on 2 × PcMM agar; Figure S2. Cloning of the expression vector pSK275_pafC; Figure S3. Amino acid alignment of PAFC and orthologous AMPs; Figure S4. Ramachandran plot of the PAFC model; Figure S5. Expression of pafC, paf and pafB in synchronized surface cultures of P. chrysogenum over a time course of 12-48 h of incubation; Figure S6. Western blot analysis to prove the binding of the PeAfpC antibody to PAFC; Figure S7. Purification of recombinant PAFC; Figure S8. Fluorescence microscopy for the localization of nuclei and PAFC-Bd; Figure S9. Ion tolerance of PAFC; Figure S10. Hemolytic activity of PAFC tested with agar diffusion assay; Video S1. Rotation of the PAFC model colored according to the Coulomb scale (blue: electropositive, red: electronegative); Video S2. Rotation of the PAFC model colored according to the Kyte-Doolittle scale (blue: hydrophilic, orange: hydrophobic).