Talaromyces marneffei yeast cells were sub‐cultured on Sabouraud Dextrose Agar (SDA) at 37 °C for 5–7 days until single cells were created. The cells were homogenised in 5 ml PBS (pH 7.2) containing Tween 20 (5%) (Sigma, Singapore). Cell pellets were collected by centrifugation at 8000
Lyticase enzyme
Manufactured by Merck Group
Sourced in United States
Lyticase is an enzyme derived from Arthrobacter luteus that catalyzes the hydrolysis of beta-1,3-glucosidic linkages in yeast cell walls. It is commonly used in the isolation and analysis of yeast genomic DNA and protoplast formation.
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Lab products found in correlation
Tween 20, by Merck Group (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
80i fluorescent microscope, by Nikon (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Merck Group (1 mentions)
Ergosterol standard, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Tetrahydrofuran, by Merck Group (1 mentions)
N heptane, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Peptone, by HiMedia (1 mentions)
Yeast extract, by HiMedia (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Mgcl2, by Merck Group (1 mentions)
Sorbitol, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Lmp agarose, by Merck Group (1 mentions)
Fungi yeast genomic dna isolation kit, by Norgen Biotek (1 mentions)
6 protocols using lyticase enzyme
1
Talaromyces marneffei DNA Extraction Protocol
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Talaromyces marneffei yeast cells were sub‐cultured on Sabouraud Dextrose Agar (SDA) at 37 °C for 5–7 days until single cells were created. The cells were homogenised in 5 ml PBS (pH 7.2) containing Tween 20 (5%) (Sigma, Singapore). Cell pellets were collected by centrifugation at 8000g for 5 min, and after thoroughly washed two times with PBS, cell pellets were resuspended in 5 ml PBS. Fungal DNA extraction was performed as described previously with minor modifications.18 Briefly, fungal cell wall was removed to form spheroplasts by treating 200 μl of resuspended cultured fungal isolates or plasma specimens with 600 μl sorbitol buffer (1 mol l−1 sorbitol, 100 mmol l−1 EDTA and 14 mmol l−1 β mercaptoethanol) supplemented with 200 U lyticase enzyme (Sigma). The mixture was incubated at 30 °C for 30 min. Spheroplast DNA was extracted using QIAamp DNA blood mini kit (QIAGEN, Hilden, Germany) and was eluted in 30 μl of elution buffer.
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Talaromyces marneffei yeast cells were sub‐cultured on Sabouraud Dextrose Agar (SDA) at 37 °C for 5–7 days until single cells were created. The cells were homogenised in 5 ml PBS (pH 7.2) containing Tween 20 (5%) (Sigma, Singapore). Cell pellets were collected by centrifugation at 8000
2
Golgi Apparatus Labeling in Yeast Cells
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For Golgi apparatus labeling, the pmr1/pmr1 GDT1-GFP/gdt1 cells were grown to log-phase in SD-URA, harvested, washed twice with PBS, and suspended in 200 μl PBS supplemented with 4 μl beta-mercaptoethanol and 15 U lyticase enzyme (Sigma). Cells was incubated at 37 °C for 40 min to partially digest the cell wall, and were washed once with PBS and suspended in 200 μl PBS before they were mixed with 2 μl Golgi-Tracker Red dye (33.3 mg ml− 1) (Beyotime Institute of Biochemistry, China) and incubated at 4 °C for 30 min. Cells were then washed twice and incubated in 1 ml SD-URA at 30 °C for 30 min, before they were visualized by the Nikon 80i fluorescent microscope.
3
Microbial Cell Culture and Characterization
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Media chemicals (yeast extract, peptone, glucose, and agar) for culture of the microbial cells were procured from Hi-Media (Mumbai, India) and Fisher Scientific (Hampton, NH, USA). Analytical grade sodium borohydrate, silver nitrate, sodium citrate, EDTA, sorbitol, Tris-Cl, and MgSO4 obtained from Qualigens (Mumbai, India) were used for preparing buffers. All solvents including n-heptane, methanol, ethanol, and water were of high performance liquid chromatography grade, high purity, and procured from Merck Millipore (Burlington, MA, USA). Lyticase enzyme, pyrogallol salts, ascorbic acid (AA), solvents such as dimethyl sulfoxide and tetrahydrofuran, ergosterol standards, and fluorescent probes 1,6-diphenyl-1,3, 5-hexatriene (DPH) and 2,7-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA).
4
Apoptotic Marker Analysis in C. auris
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For studying apoptotic markers, protoplasts of C. auris MRL6057 were prepared as explained previously [21] (link). Briefly, cells grown for 8 h were then exposed for 4 h to ¼ MIC, ½ MIC and MIC of test compounds. For the purpose of washing and resuspending yeast cells different protoplast buffers (PB) were prepared. After exposure with test compounds, cells were washed and incubated in PB-1 (1 M sorbitol, (Sigma Aldrich Co., USA), 0.05 M tris base (Merck, Germany), 0.01 M MgCl2 (Sigma Aldrich Co., USA), 0.03 M DTT (Merck, Germany), pH 7.4) for 10 min at room temperature. Post-incubation, cells were harvested at 1500 rpm for 5 min and pellet was mixed and incubated in PB-2 (1 M sorbitol, 0.05 M tris base, 0.01 M MgCl2, 0.001 M DTT, pH 7.4) supplemented with lyticase enzyme (1 μg/mL; Sigma Aldrich Co., USA) at room temperature for 1 h. Next, cell suspensions were centrifuged, and pellets were resuspended and incubated in PB-3 (1 M sorbitol, 0.05 M tris base, 0.01 M MgCl2, pH 7.4) at room temperature for 20 min. Post-incubation cell suspension was centrifuged at 1500 rpm for 5 min and pellets with protoplasts were washed and mixed in fresh PBS and stored at 4 °C until further use.
5
Preparation of Yeast Agarose-Plugs for DNA Analysis
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Preparation of S. cerevisiae agarose-plugs was carried out as previously described (33 (link)). Briefly, S. cerevisiae cells were harvested and washed twice in 50 mM EDTA (pH 8.0), then resuspended in digestion solution (0.9 M sorbitol, 0.125 M EDTA,100 mM dithiothreitol (DTT)) containing 2 mg/ml lyticase enzyme (Sigma-Aldrich). Samples were mixed with an equal volume of 1.5% low melting point (LMP) agarose (Sigma-Aldrich) dissolved in 0.9 M sorbitol/0.125 M EDTA. Aliquots were allowed to harden in sample molds at 4°C for 5 min, and then placed into 0.9 M sorbitol/0.125 M EDTA at 37°C for 6 h. Each plug contained ∼3 × 108 cells. The plugs containing yeast spheroplasts were digested with 0.5 mg/ml Proteinase K (Thermo Fisher Scientific) in lysing solution (0.5 M EDTA, 10 mM Tris–HCl, 1% SDS, pH 8.0) at 55°C for 2 days, then washed with TE (10 mM Tris–HCl, 2 mM EDTA, pH 8.0) and treated by 0.75 μM phenyl-methyl-sulfonyl-fluoride (PMSF, Sigma-Aldrich) at 37°C for 10 min in order to inactivate residual proteinase activity. Finally, the plugs were washed with TE and stored in the same buffer at 4°C.
6
Fungal DNA Extraction Protocol
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The DNA from pure fungal cultures was extracted using a Fungi/Yeast Genomic DNA Isolation Kit (Norgen Biotek, Ontario, Canada) following the procedure of Kumar and Mugunthan (2018) (link). In brief, 5 ml of collection solution was poured on the culture plate and the fungal mycelium was centrifugated at 14,000 rpm for 1 min and the pellet, containing the fungal mycelium, was recovered and then resuspended in 250 μl of resuspension solution A. One hundred fifty microliters of proteinase K and 200 units of lyticase enzyme (Sigma Aldrich, St. Louis, Mo, United States) were added to the mixture and incubated for 45 min at 37°C. Then, to this mixture, 500 μl of lysis buffer and glass beads were added, and it was vortexed for 10 min at maximum speed followed by 2 h of incubation at 90°C with intermittent vortexing.
The tubes, containing the mixture, were placed in an ultrasonicator water bath (Bionics) and sonicated for 20 min. The mixture was centrifugation for 2 min at 14,000 rpm and the supernatant was transferred to a fresh microcentrifuge tube with an equal volume of ethanol and mixed. The lysate was placed in the spin column and centrifuged at 10,000 rpm for 1 min followed by two washes with 500 μl of wash buffer. The DNA was recovered in 100 μl of elution buffer by spinning at 10,000 rpm for 2 min and was stored at -20°C prior to the subsequent analyses.
The tubes, containing the mixture, were placed in an ultrasonicator water bath (Bionics) and sonicated for 20 min. The mixture was centrifugation for 2 min at 14,000 rpm and the supernatant was transferred to a fresh microcentrifuge tube with an equal volume of ethanol and mixed. The lysate was placed in the spin column and centrifuged at 10,000 rpm for 1 min followed by two washes with 500 μl of wash buffer. The DNA was recovered in 100 μl of elution buffer by spinning at 10,000 rpm for 2 min and was stored at -20°C prior to the subsequent analyses.
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