A new hydrophobin candidate from Cladosporium macrocarpum with super-hydrophobic surface

Abstract Hydrophobins are amphipathic proteins with small molecular weights produced in filamentous fungi. These proteins are highly stable due to the disulfide bonds formed between the protected cysteine residues. They have great potential for usage in many different fields such as surface modifications, tissue engineering, and drug transport systems because hydrophobins are surfactants and soluble in harsh mediums. In this study, it was aimed to determine the hydrophobin proteins responsible for the hydrophobicity of the super-hydrophobic fungi isolates in the culture medium and to carry out the molecular characterization of the hydrophobin producer species. As a result of measuring surface hydrophobicity by determining the water contact angle, five different fungi with the highest hydrophobicity were classified as Cladosporium by classical and molecular (ITS and D1-D2 regions) methods. Also, protein extraction according to the recommended method for obtaining hydrophobins from spores of these Cladosporium species indicated that the isolates have similar protein profiles. Ultimately, the isolate named A5 with the highest water contact angle was identified as Cladosporium macrocarpum, and the 7 kDa band was appointed as a hydrophobin since it was the most abundant protein in protein extraction for this species.


Introduction
Hydrophobins are small, amphipathic, and surface-active proteins produced in filamentous fungi. [1]They have characteristically 8 cysteine residues which form 4 disulfide bridges (C1-C6, C2-C5, C3-C4, C7-C8) each other [2] Although hydrophobins have little similarity in amino acid sequence except having eight cysteine residues they show similar hydrophobicity. [3]Due to their self-assembly and amphipathic properties, they can form rodlet structures at the hydrophilic-hydrophobic interface; for instance, they selfassemble at air-water, water-oil, water-hydrophobic solid interface. [4,5]Based on solubility, rodlet structure they form, hydropathy pattern and also spacing between cysteine residues, they are divided into two classes, class I and II [6][7][8][9] Class I hydrophobins are soluble in TFA and formic acid, and also form rodlet structure [9,10] On the contrary of class I, class II hydrophobins are soluble in some organic solvents (e.g., 60% ethanol and acetonitrile) and 2% SDS solution and can't form amyloid-like structures. [5]ydrophobins are expressed at different stages during the life cycle of filamentous fungi and different parts of the organism. [11]Hydrophobins coating hyphae and fruiting body are surfactants, so they bring about a decrease in the surface tension to help aerial growth at the air-water interface and these fungal sub-structures become water resistant [12] Likewise, spores of filamentous fungi have hydrophobic surfaces because the outer surface of spores is coated with hydrophobins which are amphipathic proteins.Therefore, hydrophobins facilitate spore disposal in the air.Also, fungal sub-structures coated by hydrophobins become water resistant. [3,13]On the other side, hydrophobin layers provide attachment to hydrophobic surfaces; in particular, they are essential for interactions between host and pathogen or symbiotic relationship. [14,15]ecause of the properties mentioned above, hydrophobins have potential usage in various areas especially surface modification.There is a need to discover new fungal species which produce hydrophobins and hydrophobins with unique properties.According to the Interpro database, 1251 hydrophobins from ascomycetes, 2945 hydrophobins from basidiomycetes, and 4196 hydrophobins in total were identified on 5th September 2022 and this number has nearly doubled in the last four years.The rate indicates the importance of the discovery of new hydrophobins.Therefore, the purpose of this study, the purpose was to determine new hydrophobins from Cladosporium isolates that have super-hydrophobic properties in solid culture and are responsible for the hydrophobicity of the isolates and, to characterize these Cladosporium species by using molecular and conventional methods.To our best knowledge, this is the first large-scale study to search for a hydrophobin associated with the surface hydrophobicity of fungi as it can be used to generate super-hydrophobic surfaces.

Fungi selection depended on water contact angle
Seventy-two different fungi species supplied from E.T.U.fungi collection was grown on potato dextrose agar (PDA) at 25 C for 8 days.For the first selection, 10 ml distilled water (dH 2 O) was dropped onto all fungus.If a fungus absorbed the water drop the fungus would not be used in subsequent experiments.Next, 10 ml and 6 ml dH 2 O drops were dropped onto the outer surface of fungi which were cut pieces (1 Â 2 cm 2 ) from the solid culture (Figure 1).After that, the water drop shapes were monitored by using a microscope as described by Chau et al. [16] and water contact angles (WCAs) were measured by using Image J-DropSnake plugin which is available online for free at https://imagej.nih.gov/ij/download.html. [17]otein extraction from spores For spore isolation, fungal colonies grown for 8 days on PDA were cut into small pieces by using a lancet.The pieces were transferred into a falcon tube and dH 2 O was added; then the tube was vortexed vigorously for 10 min.The final mixture was filtered with sterile gauze folded eight times and washed with water.
For hydrophobin extraction from the isolated spores, a method recommended for hydrohobin extraction from spores was performed. [18,19]Briefly, the spores were isolated before lyophilization.Two solutions were used to extract hydrohobins.Solution A containing 40% acetonitrile, and 0.1% trifluoroacetic acid (TFA), and solution B containing 60% ethanol were added into the tube containing lyophilized spores and sonicated for 15 min in an ultrasonic water bath.100 ml of solution A and B was added onto 10 mg lyophilized spore.After centrifugation at 13000 rpm for 5 min, the proteins were obtained and Clearband Bradford Reagent (Ecotech Biotechnology, T€ urkiye) occurred for protein quantification.
SDS-PAGE (15%) was followed to separate proteins.The gel was prepared according to Table 1.After the separation of the gel in ClearBand Running Buffer (Ecotech Biotechnology, T€ urkiye) at 80 volts for 2.5 h, the gel was stained with silver nitrate.For silver staining, fixation solution (30% EtOH, 10% acetic acid in dH 2 O), reduction solution (30% EtOH, 40 mM sodium acetate, 10% sodium thiosulfate, and 2.6% glutaraldehyde), 0.2% silver nitrate solution with 40 ml formaldehyde and washing solution (130 mM sodium carbonate with 60 ml formaldehyde) were treated respectively.To determine the molecular weight of proteins, a standard curve for the protein ladder used in SDS-PAGE was drawn.

Conventional characterization
The selected fungus isolates grown on PDA were examined depending on their colony formation and morphological properties by using a stereo microscope, and conidia, spore, and hyphae structures by using a light microscope.

Molecular characterization of ısolates
DNA extraction.Mycelium from fungi was scraped and transferred into a tube.After 1 ml lysis buffer (100 mM Tris, 50 mM EDTA and 3% SDS) addition, the mixture was homogenized for 30 min and then centrifuged at 12000 rpm for 10 min. 2 ml RNase was added and incubated at 37 C for 15 min.Phenol:chloroform:isoamyl alcohol (25:24:1) at a ratio 1:1 was added into the tube, and then, centrifuged.DNA was precipitated with 100% ethanol for 1 h and washed with 70% ethanol three times.Finally, DNA was eluted in Tris-EDTA (10 mM and 1 mM, respectively) buffer.The concentration of the isolated DNA was measured by using a mDrop plate in a spectrophotometer (MultiskanTM Go, Thermo Scientific).DNA concentrations were calculated as follows: (A 260 -A 320 ) Â 50 ng/ml Â (10/0.51).The purity of DNA was determined  -625 ml 10% SDS 100 ml 5 0 ml 10% APS (Ammonium per sulphate) 100 ml 5 0 ml TEMED ((N,N,N',N'-tetramethylethylenediamine) 10 ml 5 ml by using A 260 /A 280 ratio.Also, the genome purity was checked by using agarose gel electrophoresis.

Statistical analysis
All experiments were carried out in three replicates in our study.The standard errors of the angles measured in this study were calculated at a ¼ 95% (p < 0.05) significance level by using IBM SPSS Statistics 22 software (SPSS Inc., Chicago, IL, United States).

Results and discussion
The selection of fungi isolates The surfaces of some filamentous fungi is more hydrophobic than other depending on their living area conditions during evolution.In our study, to determine the hydrophobicity of the surface of filamentous fungi, WCAs were measured, and then, the species of fungi having the most hydrophobic surface were determined.Our fungi collection includes 72 different species belonging to various genus such as Fusarium, Cladosporium, Alternaria, Trichoderma, Penicillium and Aspergillus.For the first selection, 10 ml dH 2 O was dropped onto the surface of fungi, and those which don't absorb the water drop were selected.WCAs were measured as specified in 2.1.WCA measurement indicates the hydrophobicity of a solid surface; the surface having WCA lower than 90 is hydrophilic and a surface with the angle higher than 90 is called hydrophobic as stated earlier [21] Also, super-hydrophobicity requires contact angle in between 140 and 160 .According to the WCA measurements in this study, the hydrophobic fungi surfaces with WCA between 120 and 160 belonged to Cladosporium, Aspergillus and Penicillium genus but there were five different Cladosporium isolates (named A5, A12, A25, A31, D7) with the most hydrophobicity between 140 and 160 (Figure 2; Table 2).Because of the most hydrophobic surfaces, these five isolates were selected for further analysis.It was measured that the higher WCAs were obtained from the fungi isolate A5 with left 153 ± 2.7 and right 150.6 ± 9.6 (Table 2).The second with high WCA, isolate A31, had WCA left 153.6 ± 5 and right 161.2 ± 5.3.Moreover, the effect of the drop volume (6 and 10 ml) on WCAs measurements was evaluated, and no significant difference is found.Chau et al. [16] determined the hydrophobicity of fungi surfaces found by using WCA measurement, and they found that Cladosporium cladosporioides and C. minourae are the most hydrophobic surfaces with 142 ± 1 among their species.The measurement was approximately equal to isolate A25 in this study.Cladosporium genus is generally plant pathogens; so the hydrophobic surface of these fungi may facilitate the attachment to the plant surfaces, and there can be a correlation between the surface hydrophobicity of Cladosporium and its pathogenicity. [14,22]drophobin extraction and comparision among Cladosporium species In this section, it was tried to extract spore proteins including hydrophobins from five Cladosporium species selected the previous section.Protein concentrations were determined as 295.0, 140.3).The band pointed with the red arrow in Figure 3 exists in all extractions.Because species in the same genus can include very similar proteins even if the amino acid sequences differ from each other, the band pointed by the red arrow was appointed as a hydrophobin, and the band molecular weight is calculated as 6,798 kDa ($7 kDa) according to the standard curve of protein ladder or R f value.In addition, the thickest band found in the isolate A5 column and the band found the same line as the other columns on the gel were selected as hydrophobin because it was used a specific method recommended for the extraction of hydrophobins coating spore surface [19,23] For example, HCF-1 isolated from spores of C. fulvum, was 8 kDa similar to our determination. [24][26][27] The difference in the band profile of the isolates is not clear except for isolate A5.Isolate A5 extraction with solution A includes several bands when compared to solution B.
][30] For example, DewA which is a hydrophobin extracted from Aspergillus nidulans was used to coat the silicon surface and the contact angle of this silicon surface was changed from 0 to 67 which indicates decreasing of the silicon surface hydrophilicity. [29]The surface activity of PfaH2 from Paecilomyces farinosus was determined by using a glass slide and Teflon tape, a hydrophilic and a hydrophobic material respectively.PfaH2 altered contact angle values for the different types of surfaces, hydrophobic to hydrophilic and vice versa. [30]Therefore, these researches indicate that the hydrophobin determined in this study has a significant potential to shift the surface hydrophobicity in two directions since there is a correlation between hydrophobins and the surface hydrophobicity of filamentous fungi.

Identification of the selected isolates
According to classical methods, the selected species belong to Cladosporium genus due to green-pigmented vegetative hyphae, conidiophores and conidia, branched conidiophores,  long branched spore chain and producing sphere, oval or lemon-like conidia. [31]The structure of hyphae, conidiophores and spores were obtained by using the light microscope as shown in Figure 4.
In order to molecular characterization of these species, it was studied with ITS and D1-D2 regions.After DNA extraction and purity control of them, PCR was performed by using ITS1, ITS4, NL1, and NL4 primers with the conditions given in 2.3.2.2.The used ITS and NL primers replicate approximately 500-600 bp region [32,33] (Figure 5).The obtained sequences (ITS and D1-D2 regions) after Sanger Sequencing were first uploaded to GeneBank (NCBI) database in order to compare the other ITS and D1-D2 region sequences.As a result of the sequence comparison, the sequences belonging to the isolate A5 and D7 had the same Query Ratio (99%) for two fungal species, C. macrocarpum and C. limoniforme.Similarly, some species (C.cladosporioides and C. herbarum) from Cladosporium genus had very close or the same query ratio in between 98 and 100 percent for A12 and A31.However, isolate A25 had by far the most query ratio for C. ramotenelllum and it was identified without further evaluation.Because of this, sequence comparison was inadequate for species identification.To overcome the problem, classical characterization was detailed.When MycoBank Database (http://www.mycobank.org/)was researched, the genus can be distinguished from many morphological properties such as curved or straight aerial hyphae, the size of conidia and the roughness of the surface of conidia, whether conidiophore structure is tumid or not.Thus, isolates A5 and D7 were identified as C. macrocarpum owing to having the same properties; A12 and A31 were identified as C. cladosporioides.To identify the sequence relations, it was carried out neighborhood analysis by using MEGA 6 (Figures 6 and 7).Based on this analysis, A5 and D7, A12, and A31 are very related species.This analysis  confirmed that the molecular and microscopic characterization of our isolates were true.

Conclusion
In this study, it was aimed at the selection of fungi with the super-hydrophobic surface because the hydrophobicity comes from hydrophobins coating the outer surface of filamentous fungi.This study pointed out that the higher the hydrophobicity of the surface of filamentous fungi the higher the hydrophobic of that the hydrophobin so that they can use for obtaining the super-hydrophobic surfaces.Also, WCA measurement is an easy and valuable method to select filamentous fungi with the most hydrophobic surface.However, there is a need for further characterization of the appointed hydrophobin such as the determination of amino acid sequence, surface properties of the hydrophobin in vitro, and also its toxicity should be determined if it will be used in medical applications.The phylogeny was inferred using the Neighborhood analysis method for ITS sequences. [34]The most suitable tree with the sum of the branch length ¼ 0.74476742 is shown.The percentage of duplicate trees where related taxa were assembled in the bootstrap test (1000 replicates) is shown next to the branches. [35]he tree is scaled by the branch lengths in the same unit as the evolutionary distances used in the inference of the phylogenetic tree.Evolutionary distances were calculated using the Maximum Composite Likelihood method [36] and are in units of the number of base substitutions per site.The analysis included 5 nucleotide sequences.Evolutionary analyzes were performed in MEGA6. [37]gure 7.The phylogeny was inferred using the Neighborhood analysis method for the sequences of D1-D2 region. [33]Optimal tree with branch length ¼ 0.02655032 is shown.The percentage of duplicate trees where related taxa were assembled in the bootstrap test (1000 replicates) is shown next to the branches. [34]volutionary distances were calculated using the Maximum Composite Likelihood method [36] and are in units of the number of base substitutions per site.The analysis included 5 nucleotide sequences.Evolutionary analyzes were performed in MEGA6. [37]

Figure 1 .
Figure 1. 6 ml water drop isolate A5 grown on PDA (A), and the drop shape onto a piece (1 Â 2 cm 2 ) of the isolate monitorized by using stereo microscope horizontally (B) 8 ribosomal RNA gene, and internal transcribed spacer 2, and partial sequence of large subunit ribosomal RNA gene, and D1-D2 region includes a partial sequence of large subunit ribosomal RNA gene.For PCR reactions, 50 ml PCR reaction including 50 ng of genomic DNA, 37.5 ml of dH 2 O, 5 ml of 10X reaction buffer, 0.8 ml of 200 mM primers (synthesized by OLIGOMER), dNTP mix (QIAGEN, Germany) containing 10 mM of each dATP, dCTP, dGTP, and dTTP and 2.5 U of Taq DNA polymerase (QIAGEN, Germany) was prepared.As a negative control, dH 2 O instead of gDNA was used in the same composition.The following PCR conditions were used: one cycle at 95 C for 2 min, 35 cycles at 95 C for 45 s, 55 C for 45 s, 72 C for 1 min and a final extension at 72 C for 10 min.The PCR reaction was occurred in a thermal cycler (Sensoquest-Labcycler, Germany).After the PCR reactions were completed, a EcoSpin PCR purification kit (Ecotech Biotechnology, T€ urkiye) was used to purify the PCR products by following the manufacturer's instructions.Sequence analysis.ITS and D1-D2 regions were sequenced by Sanger Sequencing (Oligomer Biotechnology, T€ urkiye).

Figure 2 .
Figure 2. Water contact angle values of five Cladosporium species.WCAs were obtained by using DropeSnake plugin of Image J program.

Figure 3 .
Figure 3.The SDS-PAGE gel image including the extracted proteins by using solution A and B.

Figure 4 .
Figure 4. Conidiophore structures under light microscope for five isolates having the most hydrophobic surfaces in our culture collection.

Figure 5 .
Figure 5. ITS region agarose gel after PCR reaction was performed.

Figure 6 .
Figure 6.The phylogeny was inferred using the Neighborhood analysis method for ITS sequences.[34]The most suitable tree with the sum of the branch length ¼ 0.74476742 is shown.The percentage of duplicate trees where related taxa were assembled in the bootstrap test (1000 replicates) is shown next to the branches.[35]The tree is scaled by the branch lengths in the same unit as the evolutionary distances used in the inference of the phylogenetic tree.Evolutionary distances were calculated using the Maximum Composite Likelihood method[36] and are in units of the number of base substitutions per site.The analysis included 5 nucleotide sequences.Evolutionary analyzes were performed in MEGA6.[37]

Table 2 .
The water contact angle values belonged to the isolates with the most hydrophobic surface.