Antibiofilm Activity on Candida albicans and Mechanism of Action on Biomembrane Models of the Antimicrobial Peptide Ctn[15–34]

Ctn[15–34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15–34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15–34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

Fungal biofilms differ from planktonic cells by their higher virulence, better adherence to mammalian tissue, and greater resistance to antifungals, especially azoles [30]. Although all Candida species are potentially prone to form biofilms, a well-recognized fact in clinical settings, the formation of biofilms depends on several factors, involving both the microorganisms themselves and the host [25]. Surfaces can play a determinant role on the formation of Candida biofilms [25,30]. Here, the biofilms of wild-type and fluconazole-resistant C. albicans were grown on polystyrene surfaces and differences in the amount of cells in the initial inoculum necessary to establish the biofilm were evidenced ( Figure S1). As observed, despite the cell number-and time-dependence for formation, both strains formed biofilms (Figures 1 and 2).

Microorganisms
Two C. albicans strains were used: a wild-type standard strain (ATCC 90028) and a fluconazole (FCZ)-resistant clinical isolate (LABMIC 0125), provided by the Santa Casa de Misericordia Hospital in Sobral (Sobral, Brazil). The identification of the clinical isolate was performed using a CHROMagar-Candida (CHROMagar, Paris, France) and VITEK 2 automated identification system (BioMérieux, Lyon, France) with an YST card, and by PCR-AGE using the transcribed internal spacer (ITS) region. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the peptides were determined by the broth microdilution method, using 96-well plates, according to the procedures of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 standard [36]. Candida cells were exposed to peptide concentration ranging from 0.03 to 71.9 µg mL −1 (0.02 to 40 µM), in culture medium, in a microplate, incubated at 35 • C and fungal growth and/or inhibition was observed after 24 h. The MIC was defined as the lowest concentration where no visual growth (no turbidity) was observed, corresponding to 90% inhibition of fungal growth. The MFC was determined after the transference of 100 µL Candida suspension in which no turbidity was observed to capped test tubes containing SDA and incubated at 35 • C, for 48 h. The MFC was determined in accordance with fungal growth in the culture medium. Each experiment was performed in duplicate.

C. albicans Culture Preparation for Antibiofilm Assays
Stock cells were inoculated on 4% glucose SAD plates and incubated for 24 h at 35 • C. One colony was grown overnight in SDB and incubated at 35 • C with shaking at 180 rpm. Then, 100 µL of the overnight culture were diluted in 5 mL of SDB and incubated for 3 h with shaking at 35 • C. The cell concentration was measured by optical density at 660 nm.

Biofilm Development and XTT/Menadione Assay
For biofilm formation, RPMI 1640/MOPS supplemented with 2% glucose was used as culture medium. In this assay, wild-type C. albicans (ATCC 90028) and the clinical isolate LABMIC 0125 inocula were prepared as described above, but at cell suspensions of 10 4 , 10 5 , 10 6 , and 10 7 CFU mL −1 . Then, 100 µL of these cell suspensions were individually transferred to wells in a 96-well round bottom polypropylene plate. The plate was incubated for 12, 24, 48, 72, and 96 h, at 37 • C, with shaking at 180 rpm. At the end of the incubation period, the biofilm was washed three times with PBS to remove planktonic cells. Then, 100 µL of 200 µg of XTT/mL with 25 µM menadione was added to the wells of each plate and incubated for 2 h, at 37 • C, protected from light. The supernatants were transferred to a flat bottom plate and the optical density was measured at 490 nm [37].

Atomic Force Microscopy Imaging
C. albicans biofilms were prepared as described for confocal microscopy assays (Section 4.6). After biofilm treatment with and without peptide, the cellular mass was carefully washed with Milli-Q water and air-dried at 25 • C. Images of untreated and treated yeast cells were conducted using a JPK NanoWizard IV atomic force microscope (Berlin, Germany) mounted on a Zeiss Axiovert 200 inverted microscope (Jena, Germany). Measurements were carried out in intermittent contact mode (air) using ACL silicon cantilevers (AppNano, Huntingdon, UK). Height and error images were recorded with similar AFM parameters (setpoint, scan rate, and gain). Scan rate was set between 0.3 and 0.6 Hz and setpoint close to 0.3 V. Images were analyzed with the JPK image processing software v. 6.1.149.

Fungal Protoplast Preparation
Protoplasts were prepared by removing the cell wall of C. albicans, which consist in a microfibrillar network composed by glucans and chitin embedded in an amorphous material composed mainly of mannoproteins that together provide rigidity to the cell wall [38,39]. Protoplasts were isolated with a yeast lytic enzyme (Zymolyase-20T, Grisp Research Solutions, Porto, Portugal), as described elsewhere [40]. Briefly, C. albicans (ATCC 90028) cultured in SDA was inoculated into yeast peptone glucose (YPG) broth and incubated overnight at 37 • C. After this period, cells were harvested by centrifugation at 3000× g, for 10 min, at room temperature. The pellet was washed with sterile Milli-Q water, resuspended, and incubated in pre-incubation medium (50 mM EDTA, pH 9.0, with 35 mM β-mercaptoethanol), for 30 min, at 37 • C, with shaking at 180 rpm. Cells was centrifuged and washed once with washing solution I (1.2 M sorbitol, 50 mM EDTA, pH 7.5). A second wash was carried out with washing solution II (1.2 M sorbitol, Tris 50 mM, pH 7.5). Cell concentration was determined by optical density at 660 nm. A suspension of 5 × 10 6 CFU mL −1 was washed with enzyme buffer (1.2 M sorbitol, 50 mM Tris, 0.1 mM calcium acetate, 0.5 mM magnesium acetate). After washing, cells were resuspended in enzyme solution (enzyme buffer and 3 mg mL −1 Zymolyase-20T) and incubated for 2 h at 37 • C, with shaking at 180 rpm. Protoplasts were imaged with an optical microscope, by Gram staining, and maintained in HEPES buffer, at 4 • C, for up to 24 h after isolation.

C. albicans Strains and Culture Conditions
C. albicans strains were maintained and grown as previously described [32]. From stock cultures kept at −80 • C until use, 10 µL of each strain were plated in SDA and incubated overnight, at 37 • C. An isolated colony was inoculated into 5 mL of SDB and allowed to grow overnight, at 37 • C, with shaking at 180 rpm. On the day of the measurement, 100 µL of the cell suspension were diluted in 5 mL of SDB and left at 37 • C until reaching log-phase, with a final cell counting of 10 × CFU mL −1 . Then, cells were washed 3 × with SDB and centrifuged at 4000× g, at 10 • C, for 25 min. Finally, cells were suspended in HEPES or SDB, and reserved until use. Excitation spectra and the ratio of intensities were obtained at the excitation wavelengths of 455 and 525 nm (R = I 455 /I 525 ), with emission at 670 nm. Excitation and emission slits were set to 5 and 10 nm, respectively. The variation of the ratio with the peptide concentrations was analyzed by a single binding site model using the expression where R is divided by R 0 , the normalized ratio of intensities obtained without treatment with peptide, R min is the minimum asymptotic value of R and K d is the apparent dissociation constant [43]. Fitting of the equation to the experimental data was performed by non-linear regression with GraphPad Prism 6.

Dynamic Light Scattering and Zeta Potential Measurements
Dynamic light scattering (DLS) and zeta potential measurements were carried out on a Malvern Zetasizer Nano ZS device (Malvern, UK) and data was processed using the Malvern's DTS software [44]. DLS was performed to assess possible changes induced by Ctn [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] in the size of LUVs. LUVs were diluted in 200 µM HEPES buffer, filtered through a 0.45 µM filter, and treated with 5, 10, or 50 µM peptide. An untreated control was also used. All measurements were conducted at 25 • C, using 1 cm optical path disposable cells, with 15 measurements (100 runs each) for each sample, after 15 min of equilibration time. Viscosity and refractive index were set at 0.8872 cP and 1.330, respectively. The results presented correspond to the average of three independent samples. Zeta potential measurements were performed with LUVs, and with C. albicans cells and protoplasts. LUVs were diluted in HEPES buffer at a lipid concentration of 200 µM and filtered through a 0.45 µM pore size filter. Vesicles were incubated for 1 h with 5, 10, or 50 µM of peptide prior to measurements. C. albicans and protoplasts were tested at 5 × 10 5 CFU mL −1 , in HEPES buffer. Applied current was set to 40 mV, with 60 s waiting between measurements.