Candidalysin is a fungal peptide toxin critical for mucosal infection methods
Aim. Evidence-backed execution summary for Candidalysin is a fungal peptide toxin critical for mucosal infection methods from Candidalysin is a fungal peptide toxin critical for mucosal infection.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Cytokine determination
reagent used in the protocol.
- Use
- Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analyzed using Bioplex Manager 6.1 software to determine analyte concentrations.
Cell damage assay
reagent used in the protocol.
- Use
- Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) activity using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions. Recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve.
Protein database search
reagent used in the protocol.
- Use
- Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz;status: 2015/0...
METHODS
reagent used in the protocol.
- Use
- Experiments were carried out using the TR146 buccal epithelial squamous cell carcinoma line obtained from the European Collection of Authenticated Cell Cultures (ECACC) and grown in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-s...
RNA isolation and real-time PCR analysis
reagent used in the protocol.
- Use
- C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incuba...
Western blotting
reagent used in the protocol.
- Use
- TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge....
Transcription factor DNA binding assay
reagent used in the protocol.
- Use
- DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described,. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Act...
Epithelial adhesion assay
reagent used in the protocol.
- Use
- Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 10 5 ) were added into 1 ml serum-free DMEM, incubated for 60...
Cytokine determination
Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analyzed using Bioplex Manager 6.1 software to determine analyte concentrations.
- Use
- Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analyzed using Bioplex Manager 6.1 software to determine analyte concentrations.
Protein database search
Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz;status: 2015/0...
- Use
- Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz;status: 2015/0...
Generation of C. albicans ECE1 mutant strains
ECE1 deletion was performed as previously described. Deletion cassettes were generated by PCR. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17, was sequentially transformed with the ECE1 -HIS1 and ECE1 -ARG4 deletion cassettes and then transformed with CIp1...
- Use
- ECE1 deletion was performed as previously described. Deletion cassettes were generated by PCR. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17, was sequentially transformed with the ECE1 -HIS1 and ECE1 -ARG4 deletion cassettes and then transformed with CIp1...
Construction of C. albicans ECE1 promoter-GFP strain
ECE1 promoter (primers 5′ ECE1 prom-NarI / 3′ ECE1 prom-XhoI) and terminator (5′ ECE1 term-SacII / 5′ ECE1 term-SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-p ECE1 -GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette....
- Use
- ECE1 promoter (primers 5′ ECE1 prom-NarI / 3′ ECE1 prom-XhoI) and terminator (5′ ECE1 term-SacII / 5′ ECE1 term-SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-p ECE1 -GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette....
RNA isolation and real-time PCR analysis
C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incuba...
- Use
- C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incuba...
Transcription factor DNA binding assay
DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described,. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Act...
- Use
- DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described,. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Act...
Imaging of C. albicans growth and invasion of epithelial cells
TR146 cells (10 5 /ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 10 4 cfu/ml) in DMEM and incubated for 6 h (37°C/5% CO 2 ). Cells were washed with PBS, fixed overnight (4°C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 µ...
- Use
- TR146 cells (10 5 /ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 10 4 cfu/ml) in DMEM and incubated for 6 h (37°C/5% CO 2 ). Cells were washed with PBS, fixed overnight (4°C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 µ...
Scanning Electron Microscopy
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4°C with 2.5%...
- Use
- For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4°C with 2.5%...
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Ece1p encodes a cytolytic peptide toxin
The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicated that this fungal peptide may act similarly to cationic antimicrobial peptides and peptide toxins such as melittin (honey bee), magainin 2 (African clawed frog) and alamethicin ( Trichoderma viride ). Cytolytic peptide toxins have not previously been found in human pathogenic fungi but bacterial cytolytic toxins are known to induce lesions after binding to target cell membranes,. To investigate the importance of lipid composition for Ece1-III 62-93 -mediated cytolysis, we used Förster resonance energy transfer (FRET) and electrical impedance spectroscopy to analyze the interactions of Ece1-III 62-93 with model membranes comprised of lipid bilayers of dioleoylphosphatidylcholine (DOPC) with o...
RNA isolation and real-time PCR analysis
C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65°C) before subjected to 2x freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at -20°C. Precipitated pellets were washed (2 × 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at -80°C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicenter) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used...
Western blotting
TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 µg of total protein was separated on 12% SDS-PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-Ray film (Fuji film). Human α-actin was used as a loading control.
Epithelial adhesion assay
Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 10 5 ) were added into 1 ml serum-free DMEM, incubated for 60 min (37°C/5% CO 2 ) and non-adherent C. albicans cells removed by aspiration. Following washing (3 × 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high power fields of 200 µm × 200 µm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip.
Epithelial invasion assay
C. albicans invasion of epithelial cells was determined as described previously. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 10 5 ), for 3 h in a humidified incubator (37°C/5% CO 2 ). Following washing (3x PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti- Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalize...
Imaging of C. albicans growth and invasion of epithelial cells
TR146 cells (10 5 /ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 10 4 cfu/ml) in DMEM and incubated for 6 h (37°C/5% CO 2 ). Cells were washed with PBS, fixed overnight (4°C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 µg/ml) for 45 min at room temperature in the dark with gentle shaking (70 rpm) to stain the fungal cell wall. Epithelial cells were permeabilised with 0.1% Triton X-100 for 15 min at 37°C in the dark, then washed and stained with 10 µg/ml Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 µl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase...
Scanning Electron Microscopy
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4°C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies Ltd). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in...
Zebrafish swimbladder mucosal infection model
Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on 2-tails T-test in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 - beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33°C in E3 + PTU and used as previously described. Briefly, 4 day post-fertilization (dpf) larvae were treated with 20 µg/ml...
Measurement outputs
What raw and processed outputs should exist?
Ece1p is an in vitro substrate for Kex2p, a Golgi-located protease that cleaves proteins after lysine-arginine (KR) motifs. Ece1p contains seven KR-processing sites, suggesting...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To confirm that Ece1-III 62-93 drives epithelial activation and fungal pathogenicity, we generated a C. albicans strain lacking only the Ece1-III 62-93 region ( ece1...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www....
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicate...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Ece1p is an in vitro substrate for Kex2p, a Golgi-located protease that cleaves proteins after lysine-arginine (KR) motifs. Ece1p contains seven KR-processing sites, suggesting...; To confirm that Ece1-III 62-93 drives epithelial activation and fungal pathogenicity, we generated a C. albicans strain lacking only the Ece1-III 62-93 region ( ece1...; Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www....; The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicate....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of...; Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine....; Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King's College London. No statistical method was used t...; TransAM and patch clamp data were analyzed using a paired t-test whilst cytokines, LDH and calcium influx data were analyzed using one-way ANOVA with all compared groups passing...
from paperReporting output
Report representative outputs alongside summary comparisons for Ece1p is an in vitro substrate for Kex2p, a Golgi-located protease that cleaves proteins after lysine-arginine (KR) motifs. Ece1p contains seven KR-processing sites, suggesting..., To confirm that Ece1-III 62-93 drives epithelial activation and fungal pathogenicity, we generated a C. albicans strain lacking only the Ece1-III 62-93 region ( ece1..., Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database ( http://www...., The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicate....
inferred from protocolStructured statistical methods
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of...; Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine....; Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King's College London. No statistical method was used t...; TransAM and patch clamp data were analyzed using a paired t-test whilst cytokines, LDH and calcium influx data were analyzed using one-way ANOVA with all compared groups passing...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicated that this fungal peptide may act similarly to cationic antimicrobial peptides and peptide toxins such as melittin (honey bee), magainin 2 (African clawed frog) and alamethicin ( Trichoderma viride ). Cytolytic peptide toxins have not previously been found in human pathogenic fungi but bacterial cytolytic toxins are known to induce lesions after binding to target cell membranes,. To investigate the importance of lipid composition for Ece1-III 62-93 -mediated cytolysis, we used Förster resonance energy transfer (FRET) and electrical impedance spectroscopy to analyze the interactions of Ece1-III 62-93 with model membranes comprised of lipid bilayers of dioleoylphosphatidylcholine (DOPC) with or without cholesterol. While Ece1-III 62-93 was able to efficiently intercalate into DOPC membranes ( ), Ece1-III 62-93 permeabilization was enhanced in the presence of cholesterol (,). Ece1-III 62-93 -induced lesions were heterogeneous and transient ( ), indicating that the pepti...
C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65°C) before subjected to 2x freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at -20°C. Precipitated pellets were washed (2 × 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at -80°C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicenter) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 - ) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the...
TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 µg of total protein was separated on 12% SDS-PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-Ray film (Fuji film). Human α-actin was used as a loading control.
Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 10 5 ) were added into 1 ml serum-free DMEM, incubated for 60 min (37°C/5% CO 2 ) and non-adherent C. albicans cells removed by aspiration. Following washing (3 × 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high power fields of 200 µm × 200 µm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip.
C. albicans invasion of epithelial cells was determined as described previously. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 10 5 ), for 3 h in a humidified incubator (37°C/5% CO 2 ). Following washing (3x PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti- Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip.
TR146 cells (10 5 /ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 10 4 cfu/ml) in DMEM and incubated for 6 h (37°C/5% CO 2 ). Cells were washed with PBS, fixed overnight (4°C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 µg/ml) for 45 min at room temperature in the dark with gentle shaking (70 rpm) to stain the fungal cell wall. Epithelial cells were permeabilised with 0.1% Triton X-100 for 15 min at 37°C in the dark, then washed and stained with 10 µg/ml Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 µl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase images were taken per picture.
For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4°C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies Ltd). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in high vacuum mode.
Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on 2-tails T-test in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 - beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33°C in E3 + PTU and used as previously described. Briefly, 4 day post-fertilization (dpf) larvae were treated with 20 µg/ml dexamethasone dissolved in 0.1% DMSO 1 h prior to infection and thereafter. For tissue damage and neutrophil recruitment, individual AB or mpo:GFP fish (respectively) were injected into the swimbladder with 4 nl of PBS with/without 25-40 C. albicans yeast cells of ece1 Δ/Δ- dTomato,...
Machine-readable layer
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"name": "Candidalysin is a fungal peptide toxin critical for mucosal infection methods",
"description": "Evidence-backed execution summary for Candidalysin is a fungal peptide toxin critical for mucosal infection methods from Candidalysin is a fungal peptide toxin critical for mucosal infection.",
"totalTime": "PT2075M",
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"name": "Ece1p encodes a cytolytic peptide toxin",
"text": "The amphipathic properties of Ece1-III 62-93 (SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR) coupled with the α-helical structure of the N-terminal hydrophobic region ( ) indicated that this fungal peptide may act similarly to cationic antimicrobial peptides and peptide toxins such as melittin (honey bee), magainin 2 (African clawed frog) and alamethicin ( Trichoderma viride ). Cytolytic peptide toxins have not previously been found in human pathogenic fungi but bacterial cytolytic toxins are known to induce lesions after binding to target cell membranes,. To investigate the importance of lipid composition for Ece1-III 62-93 -mediated cytolysis, we used Förster resonance energy transfer (FRET) and electrical impedance spectroscopy to analyze the interactions of Ece1-III 62-93 with model membranes comprised of lipid bilayers of dioleoylphosphatidylcholine (DOPC) with o..."
},
{
"@type": "HowToStep",
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"name": "RNA isolation and real-time PCR analysis",
"text": "C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 µl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65°C) before subjected to 2x freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at -20°C. Precipitated pellets were washed (2 × 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at -80°C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicenter) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used..."
},
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"position": 3,
"name": "Western blotting",
"text": "TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 µg of total protein was separated on 12% SDS-PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-Ray film (Fuji film). Human α-actin was used as a loading control."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Epithelial adhesion assay",
"text": "Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 10 5 ) were added into 1 ml serum-free DMEM, incubated for 60 min (37°C/5% CO 2 ) and non-adherent C. albicans cells removed by aspiration. Following washing (3 × 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high power fields of 200 µm × 200 µm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Epithelial invasion assay",
"text": "C. albicans invasion of epithelial cells was determined as described previously. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 10 5 ), for 3 h in a humidified incubator (37°C/5% CO 2 ). Following washing (3x PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti- Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalize..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Imaging of C. albicans growth and invasion of epithelial cells",
"text": "TR146 cells (10 5 /ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 10 4 cfu/ml) in DMEM and incubated for 6 h (37°C/5% CO 2 ). Cells were washed with PBS, fixed overnight (4°C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 µg/ml) for 45 min at room temperature in the dark with gentle shaking (70 rpm) to stain the fungal cell wall. Epithelial cells were permeabilised with 0.1% Triton X-100 for 15 min at 37°C in the dark, then washed and stained with 10 µg/ml Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 µl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Scanning Electron Microscopy",
"text": "For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4°C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies Ltd). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Zebrafish swimbladder mucosal infection model",
"text": "Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on 2-tails T-test in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 - beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33°C in E3 + PTU and used as previously described. Briefly, 4 day post-fertilization (dpf) larvae were treated with 20 µg/ml..."
}
],
"tool": [
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"name": "Cytokine determination"
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"name": "Protein database search"
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"@type": "HowToTool",
"name": "Generation of C. albicans ECE1 mutant strains"
},
{
"@type": "HowToTool",
"name": "Construction of C. albicans ECE1 promoter-GFP strain"
},
{
"@type": "HowToTool",
"name": "RNA isolation and real-time PCR analysis"
},
{
"@type": "HowToTool",
"name": "Transcription factor DNA binding assay"
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{
"@type": "HowToTool",
"name": "Imaging of C. albicans growth and invasion of epithelial cells"
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