Name: small-scale plasma membrane preparation for the analysis of candida albicans cdr1-mgfphis
Uploaded: 2021-06-13T15:00:00.000+00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis at JoVE.com

The authors gratefully acknowledge funding from the New Zealand Marsden Fund (Grant UOO1305), and a block grant from Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (M. Niimi). They wish to thank the University of Otago for providing G. Madani with a PhD Scholarship. The authors also wish to express their gratitude to Professor Stefan Raunser and his colleagues, Dr Amir Apelbaum, and Dr Deivanayagabarathy Vinayagam, for their support and supervision during a 6-month visit of G. Madani at the Max Planck Institute of Molecular Physiology (MPIMP), Dortmund, Germany. The authors also thank the German Academic Exchange Service (DAAD) for providing G. Madani with a research grant (57381332) to visit the MPIMP.

1. Preparation of fresh or frozen stocks of transformation competent AD&#916; and AD&#916;&#916; cells
<ol>
	<li>Inoculate 25 mL of 2x YPCD [i.e., 2x YPD; 2% (w/v) yeast extract, 2% (w/v) peptone, 4% (w/v) dextrose), 0.079 % (w/v) CSM (complete supplement mixture)]35 medium with a single yeast colony and incubate overnight (o/n) for 16 h at 30 &#176;C with shaking at 200 revolutions per minute (rpm).</li>
	<li>Inoculate 225 mL of 2x YPCD medium with the 25 mL&#160;o/n culture and check the cell optical density at 600 nm (OD600); the OD600 is usually ~0.5-1.0. 
	NOTE: At this stage make sure that all materials that are required for the following transformation experiment are readily available.</li>
	<li>Grow the culture at 30 &#176;C for a further ~6-8 h with shaking at 200 rpm until the cell density reaches an OD600 of ~6-8. 
	NOTE: The following steps are performed at room temperature (RT) unless otherwise stated.</li>
	<li>Harvest these logarithmic-phase cells by centrifugation at 3,000 x g for 3 min.</li>
	<li>Resuspend the cells and wash them twice with sterile double-distilled water (ddH2O; i.e., 200 mL and then 20 mL).</li>
	<li>Harvest the cells at 3,000 x g for 3 min.</li>
	<li>Slowly (i.e., add 30 equal aliquots of frozen competent cell (FCC) solution every minute for 30 min) resuspend the cell pellet in X mL of FCC [5% (w/v) glycerol, 10% (v/v) dimethyl sulfoxide (DMSO)] on ice (where X = OD600; e.g., if OD600 = 6 resuspend in 6 mL, or if OD600 = 3 resuspend in 3 mL). 
	NOTE: The correct FCC composition is critical for the transformation success.</li>
	<li>Keep cells on ice for 2 h before transformation or store aliquots at -80 &#176;C until required. 
	NOTE: AD&#916; and AD&#916;&#916; cells are very sensitive to freezing. Thus, cells must be cooled down slowly to -80 &#176;C: place ice-cold microcentrifuge tubes containing 50-600 &#181;L cell aliquots into a plastic storage box (RT). Place the box in a larger polystyrene container (RT) and close the container with a fitting polystyrene lid. Then, put the container into the -80 &#176;C freezer. 
	&#8203;CAUTION: Slow freezing is critical for cell survival.</li>
</ol>
2. Transformation of AD&#916;&#160;and AD&#916;&#916;&#160;with CaCDR1-XLmGFPHis and confirmation of correct transformants by colony PCR and DNA sequencing
NOTE: Plasmid pABC3-CDR1-mGFPHis was created using conventional cloning strategies described in detail in Lamping et al., 201055 and is illustrated in Figure 1B. Wild-type C. albicans CDR1 was isolated as a PacI/NotI fragment from plasmid pABC3-CaCDR1A-GFP14 and cloned into pABC3-XLmGFPHis26.
<ol>
	<li>PCR amplify the entire CDR1 transformation cassette with a high-fidelity DNA polymerase and primer pair PDR5-pro/PDR5-ter35 using 1-10 ng of pABC3-CaCDR1-XLmGFPHis as a DNA template or, alternatively, digest 2 &#181;g of pABC3-CaCDR1-XLmGFPHis to completion with 10 U restriction enzyme AscI at 37 &#176;C (Figure 1A,B). 
	NOTE: The CDR1 transformation cassette (Figure 1A) comprises the PDR5 promoter - CaCDR1-mGFPHis - PGK1 terminator - URA3 selection marker - PDR5 downstream region.</li>
	<li>Perform agarose gel electrophoresis and gel extract the ~8 kb transformation cassette. 
	NOTE: Gel purification of the ~8 kb CaCDR1 transformation cassette removes any possible undigested plasmid DNA, which could lead to incorrect transformants that have the entire plasmid, rather than the linear transformation cassette, integrated at the genomic PDR5 locus.</li>
	<li>Denature the required amount of salmon sperm carrier DNA (2 mg/mL; 10 mM Tris, 1 mM EDTA; pH 7.5) for 10 min in a boiling water bath and keep on ice. Use screw-capped tubes for boiling salmon sperm DNA to avoid opening of the lid.</li>
	<li>Mix 50 &#181;L of denatured salmon sperm DNA with 14 &#181;L of the transformation cassette (500-2,000 ng) and keep the 64 &#181;L of DNA mixture on ice until further use. 
	NOTE: Use 10 ng of an E. coli-yeast shuttle plasmid (e.g., pYES2) as a positive transformation control (Figure 2B).</li>
	<li>Resuspend fresh or frozen competent cells quickly defrosted for 5 min in a 30 &#176;C water bath and divide them into 50 &#181;L aliquots in 1.5 mL microcentrifuge tubes.</li>
	<li>Harvest cells by centrifugation for 1 min in a microfuge at maximum speed (18,000 x g).</li>
	<li>Remove the supernatant and keep the cell pellet at RT.</li>
	<li>For each transformation, mix 296 &#181;L combinations (RT) of 260 &#181;L of 50% (w/v) polyethylene glycol (PEG 3350) and 36 &#181;L of 1 M lithium acetate (LiAc), by repeat pipetting, with the appropriate 64 &#181;L of ice-cold DNA mixtures.</li>
	<li>Add the appropriate mixture immediately to a 50 &#181;L competent cell pellet aliquot.</li>
	<li>Resuspend the cell pellet in the 360 &#181;L of PEG-LiAc-DNA mixture by thoroughly vortexing for about 30 s.</li>
	<li>Incubate the cell mixture in a 30 &#176;C water bath for 1 h. 
	NOTE: AD&#916; and AD&#916;&#916; cells transform better at 30 &#176;C than at 42 &#176;C35.</li>
	<li>Harvest the cells at 18,000 x g for 10 s. Discard the supernatant and resuspend the cell pellet in 80 &#181;L of ddH2O.</li>
	<li>Spread the cells onto a CSM-URA agar plate35 [i.e., 0.67% (w/v) yeast nitrogen base without amino acids, 0.077% (w/v) CSM minus uracil, 2% (w/v) glucose, and 2% (w/v) agar].</li>
	<li>Incubate the plates for 2-3 days at 30 &#176;C until uracil prototroph transformants are clearly visible. 
	NOTE: Expect ~100 transformants per &#181;g of the linear ~8 kb CaCDR1 transformation cassette and ~4 x 104 transformants per &#181;g pYES2.</li>
	<li>Pick five independent transformants and spread them on a fresh CSM-URA plate to separate the uracil prototroph transformants from remnants of non-transformed host cells.</li>
	<li>Remove any possible petite mutants by growing the transformants on YPG-agar plates [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (v/v) glycerol, and 2% (w/v) agar] (Figure 2D). 
	NOTE: Petite mutants have defective mitochondria and, thus, cannot grow on non-fermentable carbon sources. They are quite common in S. cerevisiae56. S. cerevisiae AD&#916; and AD&#916;&#916; are particularly prone to acquire the petite phenotype.</li>
	<li>Perform yeast colony PCR and confirm at least three independent transformants to be correctly integrated into the genomic PDR5 locus (Figure 1C) by amplifying the entire ~8 kb CaCDR1 transformation cassette with a specific DNA polymerase that is optimized for amplifying PCR products from impure DNA template sources. Use a set of primers that bind just outside the integration site and 1 &#181;L aliquots of cell suspensions (in ddH2O) derived from single colonies as DNA templates. 
	NOTE: This particular DNA polymerase reliably amplifies ~8 kb PCR fragments from intact yeast cells. However, for reliable amplification, 45 PCR cycles are required, and the yeast cells must be resuspended at OD600 1-10 in ddH2O.</li>
	<li>Confirm the correct ~8 kb PCR amplification product by DNA agarose gel electrophoresis of a 1 &#181;L portion of the PCR reaction.</li>
	<li>Remove excess amplification primers from a 10 &#181;L portion of the PCR reaction with an enzyme mixture of a single-strand DNA exonuclease and a phosphatase following the manufacturer&#39;s instructions before sequencing the entire ORF using portions of the treated DNA sample with appropriate primers.</li>
</ol>
3. Small-scale yeast plasma membrane isolation protocol
<ol>
	<li>Growing yeast cells
	<ol>
		<li>Pre-culture a single yeast colony in 10 mL of YPD at 30 &#176;C for ~7-8 h with shaking at 200 rpm.</li>
		<li>Inoculate 40 mL of YPD medium with the 10 mL pre-culture and incubate the cells at 30 &#176;C o/n (~16 h) with shaking at 200 rpm until the cell density reaches an OD600 of 1-3.</li>
	</ol>
	</li>
	<li>Harvesting yeast cells
	<ol>
		<li>Harvest 40 OD units (ODU; e.g., 1 mL at an OD600 of 1 = 1 ODU) of logarithmic-phase cells at 4,200 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend and wash cells twice with ice cold sterile ddH2O (i.e., 40 mL and then 1 mL; harvest cells in between steps by centrifugation at 4,200 x g for 5 min at 4 &#176;C).</li>
		<li>Resuspend the pellet in 1 mL of ice cold sterile ddH2O and transfer the cell suspension into a pre-cooled (on ice) 1.5 mL microcentrifuge tube.</li>
		<li>Harvest cells at 3,300 x g for 3 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant.</li>
		<li>Resuspend the cell pellet in 0.5 mL homogenizing buffer [HB; 50 mM Tris, 0.5 mM EDTA, 20% (v/v) glycerol; pH 7.5] freshly supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). 
		NOTE: Always add PMSF fresh because PMSF is inactivated quickly upon exposure to water. 
		CAUTION: PMSF is a serine protease inhibitor, which is extremely corrosive and destructive to tissues. It may cause irreversible eye damage.</li>
		<li>Store the cell suspension at -80 &#176;C or use&#160;immediately.</li>
	</ol>
	</li>
	<li>Isolation of plasma membranes
	<ol>
		<li>If frozen, defrost the cells on ice for ~1 h.</li>
		<li>Add ice-cold 0.5 mm diameter silica beads to the 0.5 mL cell suspension to reach a total volume of 1 mL.</li>
		<li>Break cells with 6 cycles of vortexing at maximum shaking intensity for 1 min interspersed with 3 min cooling periods on ice.</li>
		<li>Make a thin hole at the bottom of the tube with a heated scalpel blade.</li>
		<li>Collect the broken cell homogenate through the bottom of the tube fitted into another ice-cold 1.5 mL microcentrifuge tube with a 10 s low-speed (~200 rpm) spin. 
		NOTE: This ensures that the silica beads remain in the original tube.</li>
		<li>Centrifuge the cell homogenate at 5,156 x g for 5 min at 4 &#176;C to remove cell debris, unbroken cells, and nuclei.</li>
		<li>Transfer 450 &#181;L of supernatant into an ice cold 1.5 mL microcentrifuge tube and add an additional 1 mL of ice-cold HB supplemented with fresh PMSF (1 mM). 
		&#8203;NOTE: This dilution step is critical for the high-quality plasma membrane protein recovery.</li>
		<li>Harvest plasma membranes at 17,968 x g for 1 h at 4 &#176;C and resuspend the plasma membrane pellet, by repeat pipetting, in 100 &#181;L of HB freshly supplemented with 1 mM PMSF. Loosen the cell pellet for proper plasma membrane homogenization by stirring the cell pellet with the 100 &#181;L pipette tip before releasing the 100 &#181;L of HB and up and down pipetting.</li>
		<li>Measure the protein concentration of the plasma membrane preparation with a protein assay kit that is compatible with buffers containing reducing agent and detergent.</li>
		<li>Store the plasma membranes at -80 &#176;C or keep on ice for immediate use.</li>
	</ol>
	</li>
</ol>
4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
<ol>
	<li>Assemble the apparatus for preparing polyacrylamide gels.</li>
	<li>For two separating gels (7% polyacrylamide), mix 2.1 mL of 40% acrylamide/bis-acrylamide, 3 mL of 4x separating buffer (1.5 M Tris, 0.4% sodium dodecyl sulfate [SDS] (w/v); pH 8.8), and 6.9 mL of ddH2O. Add 8 &#181;L of tetramethylethylenediamine (TEMED) and 60 &#181;L of 10% ammonium persulphate (APS) to initiate polymerization of acrylamide. 
	CAUTION: Acrylamide/bis-acrylamide is very toxic. It causes skin irritation, peripheral neuropathy and is a carcinogen. TEMED is harmful if swallowed or inhaled. APS is harmful if swallowed. It causes serious eye and skin irritations.</li>
	<li>Pour ~4-5 mL of this mixture into the assembled gel apparatus, up to ~2 cm from the top.</li>
	<li>Carefully layer ~1-2 mL of 0.1% SDS on top to create a planar meniscus.</li>
	<li>Allow the polyacrylamide to set for ~60 min at RT.</li>
	<li>Prepare a stacking gel mixture for two gels by mixing 0.5 mL of 40% acrylamide/bis-acrylamide, 1 mL of 4x stacking buffer (0.5 M Tris, 0.4% SDS (w/v); pH 6.8), and 6.9 mL of ddH2O. Add 2 &#181;L of TEMED and 30 &#181;L of 10% APS to initiate polymerization of acrylamide.</li>
	<li>Remove the 0.1% SDS layer from the polymerized separating gel and rinse with ddH2O to remove traces of SDS.</li>
	<li>Pour the stacking gel mix onto the separating gel.</li>
	<li>Place a comb into the stacking gel and remove any air bubbles from around the comb.</li>
	<li>Allow the stacking gel to set for ~60 min at RT.</li>
	<li>Remove the comb and rinse the gel slots with water. Put the gel into the gel tank and fill the gel tank to the top with 1x running buffer (24.8 mM Tris, 190 mM glycine, 0.1% SDS). 
	NOTE: Prepare 1x running buffer from a 10x buffer stock (248 mM Tris, 1.9 M glycine, 1% SDS (w/v) in ddH2O) stored at RT.</li>
	<li>Mix 5-10 &#181;L plasma membrane samples (i.e., 10-20&#160;&#181;g protein) with equal volumes of 2x protein loading dye [120 mM Tris-HCl (pH 6.8), 20% glycerol, 0.02% bromophenol blue, 4% SDS, 200 mM dithiothreitol (DTT)] and immediately load into individual gel slots submerged in running buffer. 
	CAUTION: DTT is harmful if swallowed. It causes serious eye and skin irritations. 
	NOTE: Make a 10 mL stock of 2x protein loading dye, aliquot and store at -20 &#176;C. Do not heat the mixed samples but immediately load them into individual gel slots so that the GFP tag is not denatured, and the in-gel fluorescence signals can be detected.</li>
	<li>Load protein molecular weight markers (10-245 kDa range) into a separate slot to enable the size estimation of individual protein fragments.</li>
	<li>Perform gel electrophoresis at 200 V until the blue loading dye reaches the bottom of the gel (usually 45-55 min).</li>
	<li>Examine the gel for in-gel GFP-fluorescence with a gel imaging system (excitation and emission wavelengths are 475-485 nm and 520 nm, respectively).</li>
	<li>Following in-gel fluorescence imaging, fix proteins by gentle agitation of the gel in ~10-20 mL Protein Gel Fixing Solution (40% ethanol, 10% acetic acid) for 15 min at RT.</li>
	<li>Rinse the gel twice for 10 min with 10 mL of ddH2O and visualize protein bands by placing the gel in 10 mL colloidal Coomassie stain solution with gentle shaking for ~1 h at RT.</li>
	<li>For improved visualization of protein bands, de-stain the gel once or twice in ~20 mL of ddH2O for ~1 h before recording images with the gel imaging system.</li>
</ol>
5. Determination of Cdr1 ATPase Activities&#160;57
<ol>
	<li>Dilute plasma membrane samples &#62;2.2 mg/mL to 1-2 mg/mL in HB.</li>
	<li>Equilibrate the ATPase assay cocktail (75 mM MES-Tris, 75 mM potassium nitrate, 0.3 mM ammonium molybdate, 7.5 mM sodium azide; pH 7.5) and Mg-ATP (28.8 mM ATP disodium salt, 28.8 mM MgCl2; pH 7.0) to 30 &#176;C in a 30 &#176;C incubator. 
	CAUTION: Sodium azide is highly toxic. 
	NOTE: Ensure all buffer stocks and bottles are phosphate free; i.e., wash glassware with 1% (vol/vol) HCl and rinse it several times with ddH2O. Also, keep it separate from other glassware that likely contains traces of phosphate commonly present in detergents used to wash glassware.</li>
	<li>Add 90 &#181;L of assay cocktail with or without Cdr1-ATPase inhibitor (20 &#181;M of oligomycin) into individual wells of a 96-well microtiter plate. 
	NOTE: The assay is performed in triplicate.</li>
	<li>Add 5 &#181;L of the isolated plasma membranes (~5-10 &#181;g protein) or phosphate standards (0-100 nmoles Pi) into the appropriate wells of the microtiter plate. 
	NOTE: Keep the first and last columns for two separate sets of phosphate standards.</li>
	<li>Start the assay by adding 25 &#181;L of prewarmed 28.8 mM Mg-ATP (6 mM final concentration) with a multi-channel pipette into individual wells and incubate at 30 &#176;C for 30 min.</li>
	<li>Stop the reaction by adding 130 &#181;L of development reagent (1.6% sodium L-ascorbate, 1% SDS, 12% ammonium molybdate in 6 M sulfuric acid). 
	CAUTION: Concentrated sulfuric acid reacts violently with water. It is corrosive and may cause skin and lung damage.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Read the absorbance of the microtiter plate wells at 750 nm with a microtiter plate reader. 
	&#8203;NOTE: Sticking to the 10 min incubation time for blue dye development (i.e., a reduced phosphor-molybdenum complex) is critical for assay accuracy because the blue dye development continues with time.</li>
	<li>Obtain the Cdr1-specific ATPase activity (i.e., the oligomycin-sensitive ATPase activity) by subtracting the ATPase activity in the presence of oligomycin from the total ATPase activity in the absence of oligomycin.</li>
</ol>
6. Small scale detergent screen
<ol>
	<li>Combine the plasma membrane preparations (i.e., 2.5 mg of plasma membrane protein) of cells overexpressing Cdr1-mGFPHis with GTED-20 buffer [10 mM Tris, 0.5 mM EDTA, 20 % (w/v) glycerol; pH 7.5; freshly supplemented with 1 mM PMSF] containing 5 mg of the test detergent, to reach a total volume of 0.5 mL supplemented with 1% (w/v) detergent.</li>
	<li>Rotate the mixture at 4-8 &#176;C for 2 h, with a rotation device.</li>
	<li>Centrifuge the mixture at 141,000 x g at 4 &#176;C for 1 h.</li>
	<li>Transfer the supernatant containing solubilized material to a fresh microcentrifuge tube.</li>
	<li>Add 0.5 mL of GTED-20 buffer supplemented with 2% (w/v) SDS to the insoluble pellet fraction and incubate at 30 &#176;C o/n in a shaking incubator to extract all detergent-insoluble membrane protein.</li>
	<li>Analyze and compare the supernatant and solubilized pellet fractions by SDS-PAGE.</li>
	<li>Photograph gels containing GFP-tagged proteins for in-gel GFP-fluorescence before Coomassie staining and quantify expression levels with the imaging system.</li>
	<li>Use the soluble membrane protein fraction for downstream applications such as fluorescence size exclusion chromatography (FSEC)58 to identify suitable detergent(s).</li>
</ol>

The authors have nothing to disclose.

Despite recent progress in the structural analysis of membrane proteins, no 3D structure for Cdr1, or any other PDR transporter, is currently available. So, gaining knowledge of the Cdr1 structure and its biochemical features is important, as this will not only provide insight into rational design of novel drugs to overcome efflux-mediated drug resistance, but also into the mechanism of function of an important subfamily of ABC proteins.
One of the main requirements for the structural characte...

The extraction of integral membrane proteins from their native lipid environment can dramatically affect their structure and function1,2,3,4. The complex lipid composition of biological membranes5 ensures that critically important protein-lipid interactions can occur6. Lipids maintain the structural integrity of membrane proteins, thus enabling them to function correctly in their membrane compartment destination(s)7,8. Therefore, a critical first step in the membrane protein purification is the extraction of the protein from its native environment without affecting its structure and/or function.
There are many obstacles to characterizing the structure of membrane proteins, most of which are related to their hydrophobic nature, and the difficulties of expressing properly folded and functional membrane proteins in the quantities required for X-ray crystallography or cryo-electron microscopy (cryo-EM)9,10,11,12. There are three types of membrane protein expression systems: homologous9, heterologous13,14,15, and in vitro expression systems16,17. The often-low expression levels, or the prohibitive costs, of many expression systems leave only a few hosts as the preferred option to produce membrane proteins. They include the bacterial host, Escherichia coli, the yeasts S. cerevisiae and Pichia pastoris, and higher eukaryotes such as Sf9 insect cells or mammalian cell lines18. All membrane protein expression technologies have advantages and disadvantages; however, S. cerevisiae is perhaps the best studied eukaryotic model organism suitable for membrane protein production. It is highly versatile with applications in genetic engineering, drug discovery, synthetic biology, and the expression of eukaryotic membrane proteins14,19,20,21.
In this study, a patented S. cerevisiae membrane protein expression technology21 was used, with S. cerevisiae AD&#916;14 and AD&#916;&#916;22 as the preferred hosts (Figure 1A), to overexpress and study the major C. albicans multidrug efflux pump Cdr1. Both the S. cerevisiae strains are derivatives of AD1-8u-&#160;23 that have either the ura3 (AD&#916;) or both the ura3 and his1 (AD&#916;&#916;) genes deleted to eliminate any false positive uracil or histidine prototroph transformants arising through the unwanted integration at the URA3 or the HIS1 genomic loci. The deletion of the 7 major multidrug efflux pumps23, indicated in Figure 1A, makes AD&#916;&#916; exquisitely sensitive to most xenobiotics. The gain-of-function mutant transcription factor Pdr1-3 causes the constitutive overexpression of heterologous membrane proteins such as Cdr1 (red octagons in Figure 1A) after integration of the heterologous-ORF-containing transformation cassette (Figure 1A) at the genomic PDR5 locus (blue rectangle in Figure 1A) via two homologous recombination events. Proper plasma membrane localization of C-terminally mGFPHis tagged proteins can be confirmed by confocal microscopy (Figure 1A), and the His tag can be used for nickel-affinity purification of the tagged protein. Cloning some fungal ABC transporters (e.g., Candida krusei ABC1) into pABC3-derived plasmids was, however, not possible because they could not be propagated&#160;in Escherichia coli due to cell toxicity. This prompted the development of the one-step cloning of membrane proteins14,24 tagged at either their N- or C-terminus with various affinity, epitope, or reporter tags directly into S. cerevisiae AD&#916;&#916; (Figure 1C). S. cerevisiae AD&#916;&#916; strains overexpressing various CDR1 mutants can also be created efficiently this way by using up to five individual PCR fragments that overlap by 25 bp (Figure 1C). Employing this protocol, many ORFs of interest can be cloned, expressed, and characterized at low cost and at high efficiency within a very short time span. The transformation efficiency reduces only ~2-fold with each additional PCR fragment.
If desired, expression levels can also be readily manipulated by primer design to predictably tune expression levels down to anywhere between 0.1%-50% of the usually high, constitutive expression levels25. The optimized, multifunctional, pABC314 derivative cloning vector, pABC3-XLmGFPHis26 (Figure 1B) contains a HRV-3C protease cleavage site (X; LEVLFQ|GP), a protease that performs better at 4 &#176;C than the frequently used tobacco etch virus (TEV) protease27. L is a five amino acid (GSGGS) linker, mGFP is a monomeric mutant (A206K)28,29 version of the yeast enhanced green-fluorescence protein variant yEGFP330, and His is a three amino acid linker (GGS) followed by the six-histidine (HHHHHH) nickel-affinity protein purification tag.
This expression technology has been successfully used in drug discovery and the study of membrane proteins. The first structure for a fungal azole drug target, S. cerevisiae Erg1131, was solved using this technology. It also enabled the detailed characterization of C. albicans Cdr132,33,34 and the creation of a cysteine-deficient Cdr1 molecule35 suitable for cysteine-crosslinking studies to verify any future high-resolution structure. Many other ABC transporters from major human fungal pathogens&#160;(i.e., C. albicans, Candida glabrata, Candida auris, Candida krusei, Candida utilis, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, and the Fusarium solani species complex) have also been studied in detail using this expression platform24,36,37,38,39. This has enabled the generation of a panel of S. cerevisiae strains overexpressing efflux pumps that has been used in high-throughput screens to discover the novel fluorescent efflux pump substrate Nile red40 and specific41 and broad-spectrum14,33,42,43,44&#160;efflux pump inhibitors. The use of this system also enabled the discovery of clorgyline as the first of its kind broad-spectrum fungal multidrug efflux pump inhibitor42.
Complete solubilization of membrane proteins and the creation of a homogeneous membrane protein-micelle preparation devoid of endogenous lipids, requires high detergent concentrations45. But unfortunately, this also often inactivates the membrane protein5,8,45,46. The properties of detergent monomers and their aggregation in solution are affected by the physical properties of the hydrophobic tail, the length and branching of the alkyl chain, the presence of an aromatic nucleus or fluoroalkyl side chain, or the number of polyoxyethylene units. Thus, detergent screening is an important first step to determine the most suitable detergent for membrane protein solubilization and purification.
C. albicans is a major human fungal pathogen of immunocompromised individuals that can cause serious, life threatening invasive infections47, and it can become resistant to azole antifungal drugs48,49. One of the main mechanisms of C. albicans multidrug resistance is the overexpression of Cdr150, which is a type II ATP-binding cassette (ABC) transporter51 of the ABCG subfamily located in the plasma membrane. Full-size fungal ABCG transporters (consisting of two nucleotide binding domains [NBDs] and two transmembrane domains [TMDs]) are more commonly known as pleiotropic drug resistance (PDR) transporters and are characterized by their unique inverted domain topology [NBD-TMD]2. PDR transporters are only found in plants52,53 and fungi54. Despite their importance, there are no structures for PDR transporters, although structures for human half-size ABCG transporters have recently been solved which helped create the first tentative model for Cdr133. Our recent experimental evidence suggests, however, that this model is flawed possibly because fungal PDR transporters have characteristic asymmetric NBDs resulting quite possibly in a unique transport mechanism. A high-resolution structure of Cdr1 is, therefore, required for both the rational design of novel efflux pump inhibitors that may help overcome efflux-mediated drug resistance, and to provide insights into the mechanism of action of this important ABC transporter family.
The objective of this study was to develop reliable protocols for the expression, solubilization, and purification of Cdr1 in the genetically modified S. cerevisiae expression host, with the ultimate aim of obtaining a high-resolution structure for Cdr1. As part of this process, a protease-cleavable mGFPHis double tag (Figure 1B) was designed with a 16-residue linker separating the tag from the C-terminus of Cdr1, which improved binding of the attached 6x His affinity tag to the nickel-affinity resin and enabled the monitoring of Cdr1 expression levels in living cells and during the entire purification process. A reproducible protocol for small-scale yeast plasma membrane protein preparations containing about 10% C. albicans Cdr1 (as estimated by Coomassie staining after SDS-PAGE) was also developed, which could be used for the biochemical characterization of Cdr1.

<table><tbody><tr><td>2-(N-Morpholino)ethane-sulphonic acid (MES)</td><td>Sigma-Aldrich</td><td>M3671</td><td></td></tr><tr><td>2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base; ultra-pure)</td><td>Merck</td><td>77-86-1</td><td></td></tr><tr><td>2,2-Didecylpropane-1,3-bis-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>NG310S</td><td>LMNG</td></tr><tr><td>2,2-Dihexylpropane-1,3-bis-&amp;beta;-D-glucopyranoside</td><td>Anatrace</td><td>NG311S</td><td>OGNG (MNG-OG)</td></tr><tr><td>2,2-Dioctylpropane-1,3-bis-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>NG322S</td><td>DMNG</td></tr><tr><td>4-Trans-(4-trans-propylcyclohexyl)-cyclohexyl &amp;alpha;-D-maltopyranoside</td><td>Glycon Biochemicals GmbH</td><td>D99019-C</td><td>PCC-&amp;alpha;-M</td></tr><tr><td>40% Acrylamide/Bis-acrylamide (37.5:1)</td><td>Bio-Rad</td><td>1610148</td><td></td></tr><tr><td>Acetic acid (glacial)</td><td>Merck</td><td>64-19-7</td><td></td></tr><tr><td>Agar</td><td>Formedium</td><td>&amp;nbsp;009002-18-0</td><td></td></tr><tr><td>Ammonium molybdate</td><td>Sigma-Aldrich</td><td>13106-76-8</td><td></td></tr><tr><td>Ammonium persulphate (APS)</td><td>Bio-Rad</td><td>1610700</td><td></td></tr><tr><td>ATP disodium salt</td><td>sigma-Aldrich</td><td>A-6419</td><td></td></tr><tr><td>Bromophenol blue</td><td>SERVA Electrophoresis GmbH</td><td>34725-61-6</td><td></td></tr><tr><td>CHAPS</td><td>Anatrace</td><td>C316S</td><td></td></tr><tr><td>CHAPSO</td><td>Anatrace</td><td>C317S</td><td></td></tr><tr><td>CSM</td><td>Formedium</td><td>DCS0019</td><td></td></tr><tr><td>CSM minus uracil</td><td>Formedium</td><td>DCS0161</td><td></td></tr><tr><td>Cyclohexyl-1-butyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>C324S</td><td>CYMAL-4</td></tr><tr><td>Cyclohexyl-1-heptyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>C327S</td><td>CYMAL-7</td></tr><tr><td>Cyclohexyl-methyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>C321S</td><td>CYMAL-1</td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>11024-24-1</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Roche Diagnostics</td><td>10197785103</td><td></td></tr><tr><td>DMSO</td><td>Merck</td><td>67-68-5</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>459836</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt (EDTA; Titriplex III)</td><td>Merck</td><td>6381-92-6</td><td></td></tr><tr><td>ExoSAP-IT PCR Product Cleanup Reagent</td><td>Applied Biosystems</td><td>78205</td><td>A blend of exonuclease and phosphatase</td></tr><tr><td>Glucose</td><td>Formedium</td><td>50-99-7</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>56-81-5</td><td></td></tr><tr><td>Glycine</td><td>Merck</td><td>G8898</td><td></td></tr><tr><td>HEPES</td><td>Formedium</td><td>7365-45-9</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>1003172510</td><td></td></tr><tr><td>KOD Fx Neo</td><td>TOYOBO Co</td><td>KFX-201</td><td>Use for reliable colony PCR</td></tr><tr><td>lithium acetate (LiAc)</td><td>Sigma-Aldrich</td><td>546-89-4</td><td></td></tr><tr><td>Magnesium chloride hexa-hydrate</td><td>sigma-Aldrich</td><td>M2393</td><td></td></tr><tr><td>MES</td><td>Formedium</td><td>145224-94-8</td><td></td></tr><tr><td>n-Decanoyl-N-hydroxyethyl-glucamide</td><td>Anatrace</td><td>H110S</td><td>HEGA-10</td></tr><tr><td>n-Decanoyl-N-methyl-glucamide</td><td>Anatrace</td><td>M320S</td><td>MEGA-10</td></tr><tr><td>n-Decyl-phosphocholine</td><td>Anatrace</td><td>F304S</td><td>Fos-choline-10</td></tr><tr><td>n-Decyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>D322S</td><td>DM</td></tr><tr><td>n-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate</td><td>Anatrace</td><td>AZ312S</td><td>Anzergent 3-12</td></tr><tr><td>n-Dodecyl-N,N-dimethylamine-N-oxide</td><td>Anatrace</td><td>D360S</td><td>LDAO</td></tr><tr><td>n-Dodecyl-&amp;alpha;-D-maltopyranoside</td><td>Anatrace</td><td>D310HA</td><td>&amp;alpha;-DDM</td></tr><tr><td>n-Dodecyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>D310S</td><td>&amp;beta;-DDM</td></tr><tr><td>n-Nonyl-&amp;beta;-D-glucopyranoside</td><td>Anatrace</td><td>N324S</td><td>NG</td></tr><tr><td>n-Nonyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>N330S</td><td>NM</td></tr><tr><td>n-Octadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate</td><td>Anatrace</td><td>AZ318S</td><td>Anzergent 3-18</td></tr><tr><td>n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate</td><td>Anatrace</td><td>AZ308S</td><td>Anzergent 3-8</td></tr><tr><td>n-Octyl-phosphocholine</td><td>Anatrace</td><td>F300S</td><td>Fos-choline-8</td></tr><tr><td>n-Octyl-&amp;beta;-D-glucopyranoside</td><td>Anatrace</td><td>O311S</td><td>OG</td></tr><tr><td>n-Tetradecyl-phosphocholine</td><td>Anatrace</td><td>F312S</td><td>Fos-choline-14</td></tr><tr><td>n-Tetradecyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>T315S</td><td>TDM</td></tr><tr><td>n-Tridecyl-phosphocholine</td><td>Anatrace</td><td>F310S</td><td>Fos-choline-13</td></tr><tr><td>n-Tridecyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>T323S</td><td>-</td></tr><tr><td>n-Undecyl-&amp;beta;-D-maltopyranoside</td><td>Anatrace</td><td>U300S</td><td>UM (UDM)</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetramethyl-ethylenediamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Octylphenoxypolyethoxyethanol</td><td>Sigma-Aldrich</td><td>9002-93-1</td><td>TRITON X-100</td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>75351</td><td></td></tr><tr><td>Peptone</td><td>Formedium</td><td>3049-73-7</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Roche Diagnostics</td><td>329-98-6</td><td></td></tr><tr><td>Phusion&amp;nbsp;Hot Start Flex DNA Polymerase</td><td>New England Biolabs</td><td>M0535S</td><td>High-fidelity DNA polymerase</td></tr><tr><td>polyethylene glycol (PEG 3350)</td><td>Sigma-Aldrich</td><td>25322-68-3</td><td></td></tr><tr><td>polyoxyethylenesorbitan monooleate</td><td>Sigma-Aldrich</td><td>9005-65-6</td><td>TWEEN 80</td></tr><tr><td>Potassium nitrate</td><td>Sigma-Aldrich</td><td>P8394</td><td></td></tr><tr><td>Protein Assay Kit</td><td>Bio-Rad</td><td>5000122</td><td>RC DC Protein Assay Kit II</td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>Bio-Rad</td><td>1610803</td><td></td></tr><tr><td>Prism Ultra Protein Ladder (10-245 kDa)</td><td>Abcam</td><td>AB116028</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>71289</td><td></td></tr><tr><td>Sodium dodecyl sulphate</td><td>Sigma-Aldrich</td><td>151-21-3</td><td>SDS</td></tr><tr><td>Sodium L-ascorbate BioXtra</td><td>Sigma-Aldrich</td><td>11140</td><td></td></tr><tr><td>Sucrose Monododecanoate</td><td>Anatrace</td><td>S350S</td><td>DDS</td></tr><tr><td>Sulphuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td></td></tr><tr><td>Yeast extract</td><td>Formedium</td><td>008013-01-2</td><td></td></tr><tr><td>Yeast nitrogen base without amino acids</td><td>Formedium</td><td>CYN0402</td><td></td></tr><tr><td>&lt;strong&gt;&amp;nbsp;Equipment (type)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge&amp;nbsp; (Eppendorf 5804)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Centrifuge (Beckman Ultra)</td><td>Beckman</td><td></td><td></td></tr><tr><td>Centrifuge (Sorvall RC6)</td><td>Sorvall</td><td></td><td></td></tr><tr><td>FSEC apparatus (NGC Chromatography Medium Pressure system equipped with a fluorescence detector, an autosampler, a fractionator)</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Gel imaging (GelDoc EZ Imager)</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Microplate reader (Synergy 2 Multi-Detection)</td><td>BioTek Instruments</td><td></td><td></td></tr><tr><td>PCR thermal cycler (C1000 Touch)</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Power supply (PowerPac)</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>SDS PAGE (Mini-PROTEAN Tetra)</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Shaking incubator (Multitron)</td><td>Infors HT, Bottmingen</td><td></td><td></td></tr><tr><td>Superose 6 Increase 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>GE17-5172-01</td><td></td></tr><tr><td>UV/Visible spectrophotometer (Ultraspec 6300 pro)</td><td>Amersham BioSciences UK Ltd</td><td></td><td></td></tr></tbody></table>

small-scale plasma membrane preparation for the analysis of candida albicans cdr1-mgfphis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

A high frequency of transformation of S. cerevisiae AD&#916;&#916; (~4 x 104 transformants/&#181;g) was achieved with pYES2 (Figure 2B). As expected, the no DNA (i.e., ddH2O only) control gave no transformants, and 1 &#181;g of the linear CDR1-mGFPHis transformation cassette (Figure 1A) gave ~50 transformants (Figure 2C)&#160;with the optimized AD&#916;&#916; transformation protocol. ...

Watch this Scientific Journal Video about Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis at JoVE.com

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

small-scale-plasma-membrane-preparation-for-analysis-candida-albicans

Research

JoVE Journal

Biology

2.7K Views.  University of Otago. Het kleinschalige plasmamembraanisolatieprotocol en mGFPHis double tag bieden een economische, snelle, betrouwbare en eenvoudige methode om membraaneiwitten te karakteriseren in het eukaryote modelorganisme Saccharomyces cerevisiae. Het grootste voordeel van deze technologie is het gemak en de snelheid waarmee de expressieniveaus van membraaneiwitten, de activiteiten van de ATP en het reinigingsmiddel dat het meest geschikt is voor hun zuivering kunnen worden bepaald. Deze technologie is zeer...

Video: Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Immunology and Infection

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

Medicine

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&amp;RUN Method and Data Analysis Workflow

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&amp;RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&amp;RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&amp;RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&amp;RUN data analysis.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&amp;RUN) in the human fungal pathogen Candida albicans.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

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