ASOs’ design and synthesis
The ASO to hybridize with EFG1 mRNAwas projected by us in our previously work [15]. To design specific ASOs for C. albicans BRG1 and ROB1, the target regions of each gene were selected based on a search conducted at Candida Genome Database (http://www.candidagenome.org/cgi-bin/compute/blast_clade.pl). Firstly, several gene sequences were aligned to make sure that conserved regions were used for the design. Also, a general BLAST search was performed to ensure that the sequences were not targeting any sequence at the human genome neither a similar region in another C. albicans genes. The sequences were selected considering its high specificity to C. albicans genome, non-binding against Homo sapiens genome and the number of nucleotides to include in the sequence [10]. In order to improve the oligomers hybridization and stability specific nucleotides of each sequence were chemically modified using the 2’ribose modification. The 2’OMe modification was selected since it is one of the most used for antisense applications [17,18]. A gapmer region was also introduced in both ASOs to increase the odds of activating the RNase H activity [19].
The ASOs were synthesized at the Nucleic Acid Center, University of Southern Denmark, using the standard phosphoramidite method on an automated nucleic acid synthesizer (PerSpective Biosystems Expedite 8909 instrument) as described previously by Araújo and colleagues 15.
ASOs’ Cytotoxicity
The ASOs’ cytotoxicity was evaluated against the 3T3 cell line (Fibroblast cells, Embryonic tissue, Mouse from CCL 163, American Type Culture Collection) as described in a previous work 15. The cytotoxicity results were expressed as percentage (%) of viable cells, corresponding the OD490 of cells grown without ASO as 100% of cell viability.
Microorganism and growth conditions
The Candida strain used in this study was the reference C. albicans SC5314, belonging to the Candida collection of the Biofilm group at the Centre of Biological Engineering. Its identity was confirmed using a chromogenic medium, CHROMagarTM Candida, through the distinction of colonies’ colours and by PCR-based sequencing with specific primers (ITS1 and ITS4) [20]. The mutant strains C. albicans efg1ΔΔ (HLC52) and brg1ΔΔ(SN76)were also included in this study in order to validate the ASOs performance.
For all experiments, the yeaststrains were subcultured on Sabouraud dextrose agar (SDA; Merck, Germany) and incubated for 24 h at 37 ᵒC. Cells were then inoculated in Sabouraud dextrose broth (SDB; Merck, Germany) and incubated overnight at 37 °C, 120 rpm. After incubation, the cell suspensions were centrifuged for 10 min at 3000 g at 4 ᵒC and washed twice with phosphate-buffered saline (PBS; pH 7, 0.1 M). Pellets were suspended in 5 mL of Roswell Park Memorial Institute medium (RPMI, pH 7, Sigma, St Louis, USA), and the cellular density was adjusted for each experiment using a Neubauer chamber (Marienfild, Land-Konicshofem, Germany) to 1x106 cells mL-1.
ASOs’ effect on biofilm formation
Candida albicans biofilms were developed in RPMI on 24-wells polystyrene microtiter plates (Orange Scientific, Braine- l’Alleud, Belgium). For the individual and combined ASOs to test, 500 µL of ASOs at 40 nM were prepared also in RPMI (concentration selected based on cytotoxicity assays) and added to 500 µL of C. albicans suspension at 1x106 cells mL-1. The positive control was prepared with 1 mL of the same yeast cell concentration on RPMI. The biofilms were incubated at 37 °C under agitation of 120 rpm. After 24 h of biofilm formation, the supernatants were removed and the biofilms were washed twice with PBS to remove the non-adherent cells, and subsequently characterized [21].
ASOs’ effect on gene expression
Reverse transcription-quantitative PCR (qRT-PCR) was used to determine the effect of ASOs on EFG1, BRG1 and ROB1 expression. For that, C. albicans biofilms were developed as described previously, and then the biofilm cells were removed for RNA extraction. Briefly, the developed biofilms were scraped from the microtiter plate wells in the presence of 1 mL of PBS and sonicated (Ultrasonic Processor, Cole-Parmer, Vernon Hills, Illinois) for 30 s at 30 W to separate the yeast cells from matrix [21]. Cells were collected from the suspension by centrifugation for 5 min at 6000 g and 4 °C, and then washed once with PBS. RNA extraction was performed using the PureLink RNA Mini Kit, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) [15]. To avoid potential DNA contamination, samples were treated with DNase I (Deoxyrybonuclease I, Amplification Grade, Invitrogen) and the RNA concentration was determined by optical density measurement (NanoDrop 1000 Spectrophotometer Thermo Scientific®). The complementary DNA (cDNA) was synthesized using the Xpert cDNA Synthesis Mastermix (Grisp, Porto, Portugal) in accordance with the manufacturer’s instructions, and qRT-PCR (CFX96, Biorad) was performed on a 96-well microtiter plate using Eva Green Supermix (Biorad, Berkeley, USA). Each reaction was performed in triplicate and mean values of relative expression were determined by the 2-ΔΔCq method. The expression of each gene was normalized using the ACT1 Candida reference gene [22].Non-transcriptase reverse (NRT) controls were included in each run. The primers were designed using the Primer 3 web-based (Table S1).
Biofilm biomass analysis
The ASOs effect on biofilm formation was determined by crystal violet (CV) methodology [21]. For that, firstly the biofilms were fixed with 500 µL of methanol, for 15 min. After the methanol removal, the biofilms were dried at room temperature, and then 500 µL of CV (1% v/v) were added to each well. The stain was aspirated after 5 min and its excess was removed by washing the biofilms twice with sterile ultra-pure water. Finally, 500 µL of acetic acid (33 % v/v) were added to each well to release and dissolve the CV stain. The absorbance of the CV solutions was then measured, at 570 nm, and the results presented as absorbance per unit are (Abs CV cm-2).
Simultaneously, the number of cultivable cells on C. albicans biofilm was also estimated using the colony forming units (CFUs) counting methodology [23]. Briefly, the developed biofilms were scraped from the microtiter plate wells in the presence of 1 mL of PBS and disrupted was previously described to separate the yeast cells from the matrix (Silva et al. 2009). The cells suspensions were then collected by centrifugation for 5 min at 6000 g and 4 °C and serial decimal dilutions (in PBS) were plated onto SDA. Agar plates were incubated for 24 h at 37 °C and the total enumerated and presented per unit area (Log CFU cm-2) [23].
Biofilm structure analysis
In order to study the oligomers’ effect in the biofilm thickness the confocal laser scanning microscopy (CLSM) was used. For that, the 24 h biofilms were stained with 1% (v/v) of Calcofluor white (Sigma-Aldrich, St Louis, MO, USA) for 10 min at room temperature in the dark and then observed with a CLSM (Olympus BX61, Model FluoView 1000, Portugal). The excitation line 405 and the emission filters BA 430-470 (blue channel) were used, and images were acquired with the program FV10-ASW 4.2 (Olympus).
Biofilm matrix analysis
The combined effects of ASOs on biofilm matrix composition was evaluated as described by Silva et al [23]. For that, the biofilms were scraped from the wells and then disrupted as described before. The suspensions were vortexed for 2 min and centrifuged at 5,000 g for 5 min. The pellets were dried at 37 °C until a constant weight was obtained. The matrix-containing supernatants were filtered through a 0.2 mm nitrocellulose filter and then the protein and total carbohydrate contents were estimated as described next.
Total protein quantification
The protein content was measured using the BCA Kit (Bicinchoninic Acid, Sigma-Aldrich, St Louis, Missouri) and bovine serum albumin (BSA) as standard [21]. Briefly, 0.2 mL of BCA solution was added to 25 mL of matrix sample and incubated for 30 min at 37 °C. Then, the absorbance was determined in a microplate reader at 562 nm. The protein concentration was extrapolated from a calibration curve (abs ¼ 0.009 x [protein] þ 0.1685) performed with standard concentrations of BSA. The results were normalized with the dry weight of biofilm cells, previously determined, and presented as mg of protein per g of biofilm (mg gbiofilm-1).
Total carbohydrate quantification
The total carbohydrate content was estimated using the phenol-sulfuric method [24] and glucose as standard. Briefly, 0.5 mL of phenol (50 g L -1) and 2.5 mL of sulfuric acid (95–97%) were added to 0.5 mL of matrix sample. The solution was vortexed for 30 s and incubated for 15 min at room temperature. The absorbance was determined in a microtiter plate reader at 490 nm. The concentration of carbohydrate was extrapolated from a calibration curve (Abs ¼ 0.2955 x [carbohydrate] þ 0.114) performed with standard glucose concentrations. The results were normalized with the dry weight of biofilm cells and presented as mg of carbohydrate per g of biofilm (mg gbiofilm-1).
Statistical analysis
All experiments were performed in triplicate and in a minimum of three independent assays. Data are expressed as the mean ± standard deviation (SD) of a least three independent experiments. Results were compared using t test analysis with a confidence level of 95%.All tests were performed with GraphPad Prism 6® (GraphPad Software, CA, USA).