This work was supported by the National Institutes of Health (R03DE029270, T32AI089621B), the National Science Foundation (CBET 1511718), the Department of Education (GAANN-P200A180093), and a University of Maryland Cross-Campus Seed Grant.

Approval was obtained from the University of Maryland, College Park, Institutional Biosafety Committee (IBC) for the work with C. albicans in this protocol (PN 274). The C. albicans strain SC5314 (see Table of Materials) was used in the present study; however, any other strain may also be used.
1. Preparation of the buffer, sterile water, and culture medium
<ol>
	<li>Prepare sterile 0.1 M sodium phosphate buffer (NaPB)26 at pH 7.4, and dilute to 2 mM and 1 mM with sterile water. Preparing 100 mL each of 2 mM NaPB and 1 mM NaPB will be more than sufficient for most applications of this protocol.</li>
	<li>Prepare sterile liquid yeast-peptone-dextrose (YPD, see Table of Materials) medium (10 g/L yeast extract, &#160;&#160;20 g/L dextrose, 20 g/L peptone). Preparing 100 mL of YPD will be more than sufficient for most applications of this protocol.</li>
	<li>Prepare sterile ultrapure water. Preparing 100 mL of water will be more than sufficient for most applications of this protocol. 
	&#8203;NOTE: The buffers, media, and water are sterilized by autoclaving at 121 &#176;C on a liquid cycle for an appropriate time based on the volume in the containers being autoclaved27. For example, for bottles containing 500 mL, the exposure time should be 40 min.</li>
</ol>
2. Inoculation, culturing, and subculturing of C.&#160;albicans
CAUTION: Follow all institutional and governmental regulations for working with C. albicans, which is often classified by academic research institutions as a biosafety level (BSL) 2 organism regardless of drug resistance and by the American Type Culture Collection (ATCC) as a BSL1 or BSL2 organism, depending on the drug resistance of the strain28.
NOTE: Perform the inoculation and culturing portions of this step (steps 2.1-2.2) on the day before beginning the testing for antifungal activity. If possible, perform all the protocol steps with the cells (and the solutions that will be incubated with the cells) in a biosafety cabinet.
<ol>
	<li>Inoculate the desired C. albicans strain&#160;into 10 mL of YPD medium in a culture tube. 
	NOTE: The culture can be inoculated from a freezer stock or from an agar plate, but a consistent source must be used for all data that will be compared.</li>
	<li>Grow the culture overnight (~12-16 h) at 30 &#176;C and 230 rpm on a rotary shaker.</li>
	<li>Subculture the overnight culture of C. albicans, and grow to an OD600 of ~1.0-1.2.
	<ol>
		<li>Measure the OD600 of the overnight culture using a UV spectrophotometer (see Table of Materials).</li>
		<li>Based on the OD600 of the overnight culture, use the overnight culture to inoculate a subculture at an OD600 of 0.1 in 10 mL of YPD.</li>
		<li>Grow the subculture at 30 &#176;C on a rotary shaker at 230 rpm until the OD600 reaches ~1.0-1.2, which is likely to take 4-6 h. Complete step 3 (preparation of peptide solutions)&#160;while the subculture is growing; the subculture will be diluted for use in the assay in step 4.</li>
	</ol>
	</li>
</ol>
3. Preparation of the peptide solutions in a 96-well plate
NOTE: This step may be done ahead of time,&#160;if the peptide stock solutions are stable during storage. Typically, the peptide solutions are stored at &#8722;20 &#176;C until use. They can be thawed in a room temperature water bath before proceeding to the next step.
<ol>
	<li>Determine the highest concentration of each peptide to be tested in the assay. This will vary based on the peptides selected. For the data presented in the representative results section, histatin 5 and engineered analogs22 were tested, and the highest concentration tested was 50 &#181;M. 
	NOTE: The highest concentration to be tested is determined using data reported in the literature or by performing preliminary experiments over a broad range of peptide concentrations. When possible, the highest concentration should be selected to observe a plateau for a complete reduction in viability at the highest concentrations tested and a plateau for no reduction in viability at the lowest concentrations tested, allowing the quantification of the full range of peptide activity.</li>
	<li>Dissolve each peptide in sterile water at twice the desired highest concentration. For the data presented in the representative results section, 150 &#181;L of 100 &#181;M solutions of histatin 5 and engineered analogs were prepared in sterile water. 
	NOTE: If a peptide is not soluble in water, solubilization in another solvent may be necessary prior to this step. Dimethyl sulfoxide (DMSO) is commonly used to solubilize peptides at a high concentration before diluting to the working concentration. Ensure that the final concentration of the DMSO is 1% (v/v) or lower29 and that alternative solvents do not affect the growth of the cells. Also, ensure that the solvent concentration is kept constant in all the wells in steps 3.4-3.5.</li>
	<li>Add 40 &#181;L of the desired peptide stock solutions to the first well (Column 1) of each row in a 96-well, round-bottomed culture plate (see Table of Materials). This plate is Plate 1 (Figure 1). 
	NOTE: Each plate has eight rows that are used for testing peptides. These rows can be used for different peptides or for replicates of the same peptides. Include only a single peptide in each row.</li>
	<li>Add 20 &#181;L of sterile ultrapure water to Column 2 to Column 12 of the rows containing peptide (Figure 1). 
	NOTE: If the peptide stocks were prepared with a solvent other than pure water in step 3.2, the sterile water in this step should be replaced with the solvent present in the peptide stock added to the first column. For example, if the peptide was solubilized in DMSO and the peptide stock contains 1% (v/v) DMSO in water, water containing 1% DMSO must be added to Column 2 to Column 12.</li>
	<li>Serially dilute the peptide stock solutions across the plate to Column 10 (Figure 1). 
	NOTE: Rather than performing the serial dilutions in the plate as described below, the dilutions could also be performed in microcentrifuge tubes and transferred to the 96-well plate.
	<ol>
		<li>Remove 20 &#181;L from Column 1, transfer it to Column 2, and mix by pipetting up and down. Column 2 now contains 40 &#181;L of peptide solution at a 1:2 dilution of the concentration in Column 1.</li>
		<li>Remove 20 &#181;L from Column 2, transfer it to Column 3, and mix by pipetting up and down, so Column 3 now contains 40 &#181;L of peptide solution at a 1:4 dilution of the concentration in Column 1.</li>
		<li>Repeat this process until Column 10 contains 40 &#181;L of peptide solution at a 1:512 dilution of the concentration in Column 1.</li>
		<li>Remove 20 &#181;L of peptide solution from Column 10 and discard it. Each column now contains 20 &#181;L of peptide solution (Columns 1-10) or water (Column 11 and Column 12). 
		&#8203;NOTE: The wells in Column 11 serve as sterility controls, and the wells in Column 12 serve as controls containing no peptide.</li>
	</ol>
	</li>
</ol>
4. Dilution of the C. albicans subculture
NOTE: Begin this step after the subculture has reached an OD600 of ~1.0-1.2 (step 2.3.3).
<ol>
	<li>Transfer the subculture to a 15 mL centrifuge tube, and centrifuge at 3,900 x g for 3 min at room temperature to pellet the cells. Remove the supernatant by pipetting or decanting.</li>
	<li>Resuspend the pellet with 1 mL of 2 mM NaPB (step 1.1), and transfer the suspension to a 1.7 mL centrifuge tube.</li>
	<li>Pellet the cells (as in step 4.1), discard the supernatant, and again resuspend the pellet in 1 mL of 2 mM NaPB.</li>
	<li>Repeat step 4.3 two additional times to leave the washed cells in 1 mL of 2 mM NaPB.</li>
	<li>Determine the cell density of the washed suspension, and calculate the dilution factor needed to obtain a cell density of 5 x 105 cells/mL. This concentration will ultimately lead to adding 1 x 104 cells to each well of the 96-well plate. 
	NOTE: The cell density of the suspension may be determined using a number of methods, including a hemocytometer or automated cell counter. A standard curve of cell density versus OD600 for the strain of interest grown under relevant conditions was used for the present study. This curve was prepared using a hemocytometer (see Table of Materials) to determine the cell density of suspensions with varying OD600 values.</li>
	<li>Dilute the cell suspension to 5 x 105 cells/mL in 2 mM NaPB. Preparing 10 mL of this diluted suspension will be more than sufficient for completing this study.</li>
</ol>
5. Incubation of C. albicans with peptide solutions and preparation of the cells for the quantification of viability
<ol>
	<li>Add 20 &#181;L of the diluted C. albicans suspension (step 4.6) to Columns 1-10 and Column 12 of each row (Figure 1). 
	NOTE: The final peptide concentration in the first column is now half of the peptide stock concentration. For the present study, the final peptide concentration was 50 &#181;M at this point. The final cell suspension in each well contains 2.5 x 105 cells/mL. Columns 1-10 serve as experimental wells to evaluate the antifungal activity at different peptide concentrations, and Column 12 serves as a control for growth with no peptide.</li>
	<li>Add 20 &#181;L of 2 mM NaPB to Column 11. All the wells now have a final concentration of 1 mM NaPB. Column 11 serves as a sterility control.</li>
	<li>Cover Plate 1 (containing both cells and peptide), and incubate it at 30 &#176;C for 30 min. 
	NOTE: An incubation time of 30 min is sufficient for many peptides17,21,22,30, but the incubation time can be increased to 60 min if desired to account for peptides that may require a longer time to exert their antifungal activity25,31,32.</li>
	<li>Prepare a new 96-well culture plate (Plate 2) for use in quantifying the viability (Figure 2). 
	NOTE: The culture plate should be prepared during the incubation of Plate 1.
	<ol>
		<li>Add 100 &#181;L of YPD (step 1.2) to all the wells of the plate.</li>
		<li>Add 100 &#181;L of 2 mM NaPB (Step 1.1) to all the wells of the plate.</li>
	</ol>
	</li>
	<li>Dilute the samples in Plate 1 (containing cells and peptides), and transfer to Plate 2 (containing medium and buffer).
	<ol>
		<li>Retrieve Plate 1 from the incubator following the 30 min of incubation.</li>
		<li>Dilute each well of Plate 1 by adding 280 &#181;L of 1 mM NaPB (step 1.1) for a total volume of 320 &#181;L in each well (Figure 2).</li>
		<li>Mix all the wells containing cells and peptides by pipetting up and down to ensure all the cells are resuspended and no settled cells are visible at the bottom of the wells.</li>
		<li>Transfer 8 &#181;L from each well in Plate 1 to the corresponding well of Plate 2. This volume transfers approximately 250 cells from each well of Plate 1 into Plate 2 (Figure 2).</li>
	</ol>
	</li>
	<li>Cover Plate 2 and incubate on a microtiter plate shaker (see Table of Materials) at 350 rpm for 17 h at 30 &#176;C.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64416/64416fig01.jpg" /> 
Figure 1: Preparation of Plate 1 for the incubation of peptide serial dilutions with cells. Serial dilutions of the peptide are made in water, and C. albicans cells are added to Plate 1. A blue color indicates that peptide is present in the well, and gray indicates a well with water and no peptide. Wells containing cells are indicated with a pattern of black dots. After these steps, the plate is incubated to allow the peptide to exert its antifungal activity. <a href="https://www.jove.com/files/ftp_upload/64416/64416fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64416/64416fig2v2.jpg" /> 
Figure 2: Preparation of Plate 2 for quantifying the reduction in viability due to the peptide. YPD medium and NaPB are added to Plate 2. After diluting the contents of Plate 1, an aliquot from each well is transferred from Plate 1 to Plate 2. Plate 2 is then incubated to allow any viable cells to grow for quantification by measuring the OD600. For Plate 2, wells containing only YPD and NaPB are shown in orange. Wells containing aliquots from Plate 1 mixed with the YPD and NaPB are shown in green. See the Figure 1 legend for the description of the colors on Plate 1. <a href="https://www.jove.com/files/ftp_upload/64416/64416fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Determination of antifungal activity
<ol>
	<li>Obtain OD600 readings for each well in Plate 2.
	<ol>
		<li>Remove Plate 2 from the incubator after 17 h of incubation.</li>
		<li>Mix all the wells in Plate 2 by pipetting up and down to ensure all the cells are resuspended and no settled cells are visible at the bottom of the wells. 
		NOTE: Avoid generating bubbles during this step, as they may interfere with the OD600 readings. If bubbles form, they can often be popped with a dry pipet tip or removed by using two pipet tips to lift them out of the well.</li>
		<li>Use an absorbance plate reader (see Table of Materials) at a wavelength of 600 nm to obtain the OD600 for each well.</li>
	</ol>
	</li>
	<li>Calculate the inhibition of growth (as a percentage), and plot it to determine the effect of the peptides on the growth of C. albicans.
	<ol>
		<li>For each well, calculate the reduction in viability compared to the control (as a percentage) using the following equation17,22,23: 
		<img alt="Equation 1" src="/files/ftp_upload/64416/64416eq1.png" style="margin: auto;" /> 
		where ODwith peptide&#160;is the OD600 of a well containing a given concentration of peptide, ODbackground is the OD600 of Column 11 in the same row (control containing no cells), and 
		ODno peptide&#160;is the OD600 of Column 12 in the same row (control containing cells and no peptide).</li>
		<li>For example, use the following expression to calculate the reduction in viability in Well B5 from the OD600 values for Row B: 
		<img alt="Equation 2" src="/files/ftp_upload/64416/64416eq2.png" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Plot the reduction in viability as a function of the peptide concentration.</li>
</ol>

<ol>
	<li>Gulati, M., Nobile, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilms:+Development,+regulation,+and+molecular+mechanisms.">Candida albicans biofilms: Development, regulation, and molecular mechanisms.</a> Microbes and Infection. 18 (5), 310-321 (2016).</li><li>Arya, N. R., Rafiq, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidiasis.">Candidiasis.</a> StatPearls. , (2021).</li><li>de Oliveira Santos, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+infections+and+therapeutic+strategies:+Mechanisms+of+action+for+traditional+and+alternative+agents.">Candida infections and therapeutic strategies: Mechanisms of action for traditional and alternative agents.</a> Frontiers in Microbiology. 9, 1351 (2018).</li><li>Espinel-Ingroff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+resistance+to+antifungal+agents:+Yeasts+and+filamentous+fungi.">Mechanisms of resistance to antifungal agents: Yeasts and filamentous fungi.</a> Revista Iberoamericana de Micolog&#237;a. 25 (2), 101-106 (2008).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+strategies+of+amphotericin+B+for+invasive+fungal+infections.">Delivery strategies of amphotericin B for invasive fungal infections.</a> Acta Pharmaceutica Sinica B. 11 (8), 2585-2604 (2021).</li><li>Struyfs, C., Cammue, B. P. A., Thevissen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-interacting+antifungal+peptides.">Membrane-interacting antifungal peptides.</a> Frontiers in Cell and Developmental Biology. 9, 649875 (2021).</li><li>Huan, Y., Kong, Q., Mou, H., Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+Classification,+design,+application+and+research+progress+in+multiple+fields.">Antimicrobial peptides: Classification, design, application and research progress in multiple fields.</a> Frontiers in Microbiology. 11, 582779 (2020).</li><li>Sarkar, T., Chetia, M., Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+and+proteins:+From+nature's+reservoir+to+the+laboratory+and+beyond.">Antimicrobial peptides and proteins: From nature's reservoir to the laboratory and beyond.</a> Frontiers in Chemistry. 9, 691532 (2021).</li><li>Mahlapuu, M., Bjorn, C., Ekblom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+as+therapeutic+agents:+Opportunities+and+challenges.">Antimicrobial peptides as therapeutic agents: Opportunities and challenges.</a> Critical Reviews in Biotechnology. 40 (7), 978-992 (2020).</li><li>Lei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptides+and+their+potential+clinical+applications.">The antimicrobial peptides and their potential clinical applications.</a> American Journal of Translational Research. 11 (7), 3919-3931 (2019).</li><li>Mercer, D. K., O'Neil, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+inspiration:+Antifungal+peptides+and+other+immunotherapeutics+from+the+host+immune+response.">Innate inspiration: Antifungal peptides and other immunotherapeutics from the host immune response.</a> Frontiers in Immunology. 11, 2177 (2020).</li><li>Bin Hafeez, A., Jiang, X., Bergen, P. J., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides:+An+update+on+classifications+and+databases.">Antimicrobial peptides: An update on classifications and databases.</a> International Journal of Molecular Sciences. 22 (21), 11691 (2021).</li><li>Xu, T., Levitz, S. M., Diamond, R. D., Oppenheim, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticandidal+activity+of+major+human+salivary+histatins.">Anticandidal activity of major human salivary histatins.</a> Infection and Immunity. 59 (8), 2549-2554 (1991).</li><li>Helmerhorst, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphotericin+B-+and+fluconazole-resistant+Candida+spp.,+Aspergillus+fumigatus,+and+other+newly+emerging+pathogenic+fungi+are+susceptible+to+basic+antifungal+peptides.">Amphotericin B- and fluconazole-resistant Candida spp., Aspergillus fumigatus, and other newly emerging pathogenic fungi are susceptible to basic antifungal peptides.</a> Antimicrobial Agents and Chemotherapy. 43 (3), 702-704 (1999).</li><li>Andra, J., Berninghausen, O., Leippe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecropins,+antibacterial+peptides+from+insects+and+mammals,+are+potently+fungicidal+against+Candida+albicans.">Cecropins, antibacterial peptides from insects and mammals, are potently fungicidal against Candida albicans.</a> Medical Microbiology and Immunology. 189, 169-173 (2001).</li><li>do Nascimento Dias, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+antimicrobial+peptides+ToAP2+and+NDBP-5.7+against+Candida+albicans+planktonic+and+biofilm+cells.">Mechanisms of action of antimicrobial peptides ToAP2 and NDBP-5.7 against Candida albicans planktonic and biofilm cells.</a> Scientific Reports. 10, 10327 (2020).</li><li>Ikonomova, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+histatin+5+modifications+on+antifungal+activity+and+kinetics+of+proteolysis.">Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis.</a> Protein Science. 29, 480-493 (2020).</li><li>Clinical Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> M27-A3. Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard - Third edition. , (2008).</li><li>Lupetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidacidal+activities+of+human+lactoferrin+peptides+derived+from+the+N+terminus.">Candidacidal activities of human lactoferrin peptides derived from the N terminus.</a> Antimicrobial Agents and Chemotherapy. 44 (12), 3257-3263 (2000).</li><li>Han, J., Jyoti, M. A., Song, H. Y., Jang, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+and+action+mechanism+of+histatin+5-halocidin+hybrid+peptides+against+Candida+ssp.">Antifungal activity and action mechanism of histatin 5-halocidin hybrid peptides against Candida ssp.</a> PLoS One. 11 (2), 0150196 (2016).</li><li>den Hertog, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candidacidal+effects+of+two+antimicrobial+peptides:+histatin+5+causes+small+membrane+defects,+but+LL-37+causes+massive+disruption+of+the+cell+membrane.">Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane.</a> Biochemical Journal. 388, 689-695 (2005).</li><li>Ikonomova, S. P., Moghaddam-Taaheri, P., Jabra-Rizk, M. A., Wang, Y., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+improved+variants+of+the+antifungal+peptide+histatin+5+with+reduced+susceptibility+to+Candida+albicans+secreted+aspartic+proteases+and+enhanced+antimicrobial+potency.">Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.</a> The FEBS Journal. 285 (1), 146-159 (2018).</li><li>Moghaddam-Taaheri, P., Leissa, J. A., Eppler, H. B., Jewell, C. M., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histatin+5+variant+reduces+Candida+albicans+biofilm+viability+and+inhibits+biofilm+formation.">Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation.</a> Fungal Genetics and Biology. 149, 103529 (2021).</li><li>Gong, Z., Doolin, M. T., Adhikari, S., Stroka, K. M., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+charge+and+hydrophobicity+in+translocation+of+cell-penetrating+peptides+into+Candida+albicans+cells.">Role of charge and hydrophobicity in translocation of cell-penetrating peptides into Candida albicans cells.</a> AIChE Journal. 65 (12), 16768 (2019).</li><li>Gong, Z., Karlsson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+cell-penetrating+peptides+into+Candida+fungal+pathogens.">Translocation of cell-penetrating peptides into Candida fungal pathogens.</a> Protein Science. 26 (9), 1714-1725 (2017).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. Fourth edition. 3, (2012).</li><li>Consolidated Sterilizer Systems. Laboratory and Research Autoclaves Available from: <a target="_blank" href="https://consteril.com/wp-content/uploads/2020/12/CSS-Product-Brochure.pdf">https://consteril.com/wp-content/uploads/2020/12/CSS-Product-Brochure.pdf</a> (2022)</li><li>Rodriguez-Tudela, J. L., Cuenca-Estrella, M., Diaz-Guerra, T. M., Mellado, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+antifungal+susceptibility+variables+for+a+semiautomated+methodology.">Standardization of antifungal susceptibility variables for a semiautomated methodology.</a> Journal of Clinical Microbiology. 39 (7), 2513-2517 (2001).</li><li>Mbuayama, K. R., Taute, H., Strmstedt, A. A., Bester, M. J., Gaspar, A. R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+and+mode+of+action+of+synthetic+peptides+derived+from+the+tick+OsDef2+defensin.">Antifungal activity and mode of action of synthetic peptides derived from the tick OsDef2 defensin.</a> Journal of Peptide Science. 28 (5), 3383 (2022).</li><li>Rossignol, T., Kelly, B., Dobson, C., d'Enfert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis-mediated+vacuolar+accumulation+of+the+human+ApoE+apolipoprotein-derived+ApoEdpL-W+antimicrobial+peptide+contributes+to+its+antifungal+activity+in+Candida+albicans.">Endocytosis-mediated vacuolar accumulation of the human ApoE apolipoprotein-derived ApoEdpL-W antimicrobial peptide contributes to its antifungal activity in Candida albicans.</a> Antimicrobial Agents and Chemotherapy. 55 (10), 4670-4681 (2011).</li><li>Helmerhorst, E. J., Reijnders, I. M., van't Hof, W., Veerman, E. C., Nieuw Amerongen, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+comparison+of+the+hemolytic+and+fungicidal+activities+of+cationic+antimicrobial+peptides.">A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.</a> FEBS Letters. 449 (2-3), 105-110 (1999).</li><li>Kerenga, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt-tolerant+antifungal+and+antibacterial+activities+of+the+corn+defensin+ZmD32.">Salt-tolerant antifungal and antibacterial activities of the corn defensin ZmD32.</a> Frontiers in Microbiology. 10, 795 (2019).</li><li>Lee, I. H., Cho, Y., Lehrer, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH+and+salinity+on+the+antimicrobial+properties+of+clavanins.">Effects of pH and salinity on the antimicrobial properties of clavanins.</a> Infection and Immunity. 65 (7), 2898-2903 (1997).</li><li>Li, X. S., Reddy, M. S., Baev, D., Edgerton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+Ssa1/2p+is+the+cell+envelope+binding+protein+for+human+salivary+histatin+5.">Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary histatin 5.</a> Journal of Biological Chemistry. 278 (31), 28553-28561 (2003).</li><li>Rothstein, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticandida+activity+is+retained+in+P-113,+a+12-amino-acid+fragment+of+histatin+5.">Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5.</a> Antimicrobial Agents and Chemotherapy. 45 (5), 1367-1373 (2001).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+Volume+transfers+with+serological+pipettes+and+micropipettors.">Aseptic laboratory techniques: Volume transfers with serological pipettes and micropipettors.</a> Journal of Visualized Experiments. (63), e2754 (2012).</li><li>Mansoury, M., Hamed, M., Karmustaji, R., Al Hannan, F., Safrany, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+edge+effect:+A+global+problem.+The+trouble+with+culturing+cells+in+96-well+plates.">The edge effect: A global problem. The trouble with culturing cells in 96-well plates.</a> Biochemistry and Biophysics Report. 26, 100987 (2021).</li><li>Goughenour, K. D., Balada-Llasat, J. M., Rappleye, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microplate-based+growth+assay+for+determination+of+antifungal+susceptibility+of+Histoplasma+capsulatum+yeasts.">Quantitative microplate-based growth assay for determination of antifungal susceptibility of Histoplasma capsulatum yeasts.</a> Journal of Clinical Microbiology. 53 (10), 3286-3295 (2015).</li></ol>

The authors declare that they have no competing financial interests.

This protocol describes an efficient approach for obtaining quantitative data on the antifungal activity of AMPs against the fungal pathogen C. albicans. One common alternative approach to testing peptides and other antifungal agents is the broth microdilution described in the Clinical Laboratory Standards Institute&#39;s (CLSI) standard M2718, but this standard focuses on obtaining qualitative visual results instead of quantitative results. Another alternative approach is to use a method similar to the one described in this protocol to prepare peptides and incubate them with cells and then plate the contents of the wells on agar to count the number of CFUs and quantify the effect of the peptides. As noted in the representative results section, this use of OD600 for measuring cell growth in this protocol substantially reduces the time burden compared to plating and counting CFUs. Eliminating the need to plate the contents of wells on agar also reduces the amount of plastic waste generated by the peptide-testing protocol.
Several aspects of this protocol are important for its success. Since many cationic antimicrobial peptides perform poorly in physiological salt concentrations in assays performed in vitro8,32,33,34, a 1 mM NaPB buffer was selected for this assay. Additionally, the peptides and C. albicans cells are incubated for a short time (30 min) in buffer rather than a long time in growth medium. This reduces the potential growth and division of the cells, which would affect the subsequent quantification of the viable cells remaining following the incubation with the peptides. Following the incubation of the peptides with the cells, the wells are diluted to reduce the concentration of the peptide, reducing the potential for antifungal activity continuing beyond the desired 30 min incubation35. The potential for continued activity of the peptides is further reduced by transferring the cell-peptide solution to a plate containing YPD medium; many peptides lose their activity in the YPD medium (unpublished data), possibly because of the presence of mono- and divalent cations, which are known to reduce the activity of some antimicrobial peptides6,7,34,36. However, a lack of activity in the YPD medium is not necessary for the success of the protocol. Finally, the C. albicans cells are grown at 30 &#176;C for the relevant steps in the protocol. By growing the cells at this temperature, the cells should be primarily in the yeast morphology, which is important for obtaining accurate estimations of the number of cells in step 2 and step 4 and for the reliable quantification of the reduction in viability in step 6.
Users may encounter challenges during this protocol, including contamination with undesired microbial organisms or the evaporation of liquid from the wells on the edges of the 96-well plate. The protocol includes wells for sterility controls to identify if contamination has occurred; contamination invalidates the assay results. If users observe contamination, care must be taken to ensure that all the buffers and media are properly sterilized and that proper sterile technique is used when pipetting37. Working in a biosafety cabinet will also reduce the potential for contamination. The evaporation of liquid from the edge wells of 96-well plates is a known challenge with microtiter plates38 and could affect the results of this protocol. While the short incubation time of the cells with the peptide in step 5.3 is not likely to lead to issues with edge effects, the longer incubation of the remaining viable cells in step 5.6 could lead to the observance of evaporation. If evaporation is observed, using different 96-well plates or filling the space around the wells with water may reduce evaporation38. Users could also consider not using Row A and Row H and Column 1 and Column 12 of the plate to obtain data, instead filling them with water, medium, or buffer.
Modifications to many aspects of the protocol are possible. For example, a different buffer (or medium) could be used when incubating the cells and peptides together (steps 4-5.3), or another fungal culture medium could be used for cell growth following incubation with the peptides (steps 5.4-5.6). The use of alternative buffers and media could allow for benchmarking with other methods published in the literature or with the CLSI standard18 and will be important for testing peptides under conditions that mimic their intended in vivo use. Another modification that some users may find helpful is to dilute the peptides down the rows of the plate rather than across the columns. Changing the orientation would reduce the number of concentrations that could be tested for a given peptide, but it would allow more peptides to be tested in a single plate, which could be useful for experiments requiring the testing of a large number of different peptides.
Obtaining replicate data for each peptide tested in this protocol is important. Including technical replicates on a given day of testing allows for understanding the variability of the protocol. Additionally, performing biological replicates is important to understand the variability from random variation in the growth of cells. The authors&#39; lab typically obtains at least six replicate data points for a given peptide. Testing is performed on a minimum of 2 different days, and a minimum of two technical replicates are included on each day. Commonly, three replicates are performed on each of 2 different days or two replicates on each of 3 different days. Figure 3 shows that this protocol produces consistent data with small standard deviations when properly performed.
The simple nature of this protocol allows it to be adapted for use in many experiments not directly described in the protocol. For example, the protocol could be easily adapted for testing other yeasts, including other Candida species. However, it is not well suited for filamentous species or dimorphic species growing in their filamentous morphology. The protocol could also be adapted for use with non-peptide antifungal agents, such as small-molecule drugs, or mixtures of different peptides or peptide fragments. For example, similar protocols have been used previously to test small-molecule drugs, including amphotericin B, fluconazole, and caspofungin, against Histoplasma yeasts39. The authors have used the protocol to compare the antifungal activity of non-degraded peptides with pools of peptide fragments formed by proteolysis of the peptide17,22. Another extension of this protocol would be to explore the kinetics of the antifungal activity of AMPs. This protocol uses an incubation time of 30 min for the peptides to exert their antifungal activity, but shorter or longer incubation times could be used to better determine the time required for the peptides to reduce the viability of cells.

Candida albicans is a member of the human microbiota that colonizes numerous locations, including the oral cavity, skin, gastrointestinal tract, and vagina1. For patients that are immunocompromised due to diseases such as human immunodeficiency virus (HIV) and immunosuppressive treatments, the colonization of C. albicans may lead to local or systemic candidiasis2,3. The use of currently available small-molecule antifungal therapeutics, such as amphotericin B, azoles, or echinocandins, can be complicated by solubility and toxicity issues and by the resistance of infections to the therapeutics4,5. Due to the limitations of current antifungal agents, researchers are continually searching for new antifungal molecules with activity against C. albicans.
Antimicrobial peptides (AMPs) are a potential alternative to the current small-molecule antifungal agents6,7,8 and are proposed to be less susceptible to the development of resistance compared to small-molecule drugs9. AMPs are a diverse set of peptides, but they are often cationic, with a broad spectrum of activity10,11,12. AMPs with activity against C. albicans include well-known peptides from the histatin and cecropin families13,14,15, along with more recently described peptides like ToAP2, NDBP-5.7, and the histatin 5 variant K11R-K17R16,17. Due to their potential for treating Candida infections, identifying and designing new AMPs that target C. albicans is an important goal for many research groups.
As part of the process to develop effective AMPs (and other antifungal agents) that target C. albicans, in vitro testing is commonly used to identify promising peptides. Methods to test antifungal activity against C. albicans typically involve incubating cells with serial dilutions of the AMPs (in buffer or medium) in 96-well plates. Several methods are available to assess the antifungal activity following incubation. A technique described by the Clinical Laboratory Standards Institute uses a purely visual assessment of the turbidity of the wells to determine the minimum concentration (MIC) for the complete inhibition of growth (at least 50% inhibition for selected antifungal agents, like azoles and echinocandins) and provides no quantification of growth at sub-MIC concentrations18. Another commonly used approach involves quantifying the viability following incubation with AMPs by plating the contents of the wells on agar plates, incubating the plates, and then counting the number of colony-forming units (CFUs) on the plate. This method has been used for evaluating a number of peptides, including histatin 5-based peptides, LL-37, and human lactoferrin19,20,21. This technique requires a relatively large volume of agar and a high number of plates and involves the tedious counting of CFUs on the plates. To obtain more quantitative data while generating less plastic waste and avoiding counting CFUs, the contents of the wells can be used to inoculate fresh medium in another 96-well plate. After incubating the newly inoculated plate, the growth can be quantified by measuring the optical density at 600 nm (OD600) on an absorbance plate reader. This method has been used to determine the antifungal activity of histatin 5 and its degradation fragments and cell-penetrating peptides17,22,23,24,25.
This protocol describes how to test the antifungal activity of peptides and uses the OD600 method to quantify the reduction in viability of C. albicans due to peptides.

<table><tbody><tr><td>96-well plates (round bottom)</td><td>VWR</td><td>10062-902</td><td></td></tr><tr><td>Absorbance microplate reader</td><td>N/A</td><td>N/A</td><td>Any available microplate reader is sufficient</td></tr><tr><td>&lt;em&gt;C. albicans &lt;/em&gt;strain SC5314</td><td>ATCC&amp;nbsp;</td><td>MYA-2876</td><td>Other &lt;em&gt;C. albicans&lt;/em&gt; may also be used</td></tr><tr><td>Hemocytometer</td><td>N/A</td><td>N/A</td><td>Can be used to make a standard curve relating cell number to OD&lt;sub&gt;600&lt;/sub&gt;</td></tr><tr><td>Microplate shaker</td><td>VWR</td><td>2620-926</td><td></td></tr><tr><td>Peptide(s)</td><td>N/A</td><td>N/A</td><td>Peptides can be commercially synthesized by any reliable vendor; a purity of &amp;ge;95% and trifluoroacetic acid salt removal to hydrochloride salt are recommended</td></tr><tr><td>Reagent reservoirs for multichannel pipettors</td><td>VWR</td><td>18900-320</td><td>Simplifies pipetting into multiwell plates with multichannel pipettor</td></tr><tr><td>Sodium phosphate, dibasic</td><td>Fisher Scientific</td><td>BP332-500</td><td>For making NaPB</td></tr><tr><td>Sodium phosphate, monobasic</td><td>Fisher Scientific</td><td>BP329-500</td><td>For making NaPB</td></tr><tr><td>UV spectrophotometer</td><td>N/A</td><td>N/A</td><td>Any available UV spectrophotometer is sufficient</td></tr><tr><td>YPD medium powder</td><td>BD Life Sciences</td><td>242820</td><td>May also be made from yeast extract, peptone, and dextrose</td></tr></tbody></table>

quantifying the antifungal activity of peptides against candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Using OD600 measurements to quantify the reduction in growth due to antifungal peptides saves substantial time compared to plating samples and counting CFUs. The method described in this protocol requires completing the steps on three different days. On the first day, approximately 1 h is needed to prepare the buffers and media (not including the sterilization time) and inoculate the starting culture of C. albicans for overnight incubation. On the second day, the steps require 5-6 h (including subculturing time) to prepare a 96-well plate for the 17 h incubation. On the third day, the steps can be completed in less than 1 h. The portion of the protocol that involves collecting the quantitative data on viability requires approximately 30 min of work (plus 17 h of incubation). In contrast, plating the samples on agar and counting the CFUs for a single replicate of the contents of each 96-well plate would require over 3 h of work (plus 24 h of incubation), resulting in over 2.5 h less time needed to complete the quantification of viability using this protocol. These time savings would be amplified if multiple 96-well plates were needed in an experiment.
The described protocol was used to assay the antifungal activity of histatin 5 and several variants of histatin 5 containing modifications at the lysine residues. After incubating the peptides with the C. albicans cells in step 5, the antifungal activity was quantified using both the CFU counting method and the described protocol incorporating OD600 measurements (Figure 3). Importantly, the two methods produced similar results for all peptides, though each peptide had one concentration with a statistically significant difference in the reduction in viability determined by the two methods.&#160;In each of these cases, the OD600 data showed a lower reduction in viability than the CFU data, indicating that a single method must be used when the data will be compared.&#160;Importantly, data using the OD600 readings are highly reproducible, as indicated by the generally small standard deviations of the replicates in Figure 3 and published results using this protocol17,22,23,24,25.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64416/64416fig03.jpg" /> 
Figure 3: Comparison of the traditional (CFU) and described (OD) methods for quantifying the reduction in C. albicans cell growth. The peptide (A) histatin 5 (Hst-5) and the variants of histatin 5&#160;(B) K5R, (C) K11R, (D) K13R, and (E) K17R&#160;(with lysine to arginine modification at the indicated residues) were incubated with C. albicans cells at concentrations of 50 &#181;M to 0.20 &#181;M, as described in the protocol. After step 5.5.3, the samples were removed for plating on agar (one replicate for each experiment) or transferred to a new plate (two replicates for each experiment) to continue the protocol. The data represent the mean of three independent experiments (N = 3 data points for CFU data, N = 6 data points for OD data), and the error bars indicate the standard deviation of the data. A two-way analysis of variance (ANOVA) with Tukey&#39;s multiple comparisons test (&#945; = 0.05) was performed to identify statistical differences between the two methods. Peptide concentrations with a statistically significant difference are noted by an asterisk (*) on the plots. <a href="https://www.jove.com/files/ftp_upload/64416/64416fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantifying the Antifungal Activity of Peptides Against Candida albicans at JoVE.com

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

quantifying-antifungal-activity-peptides-against-candida

Nanyang Technological University

Research

JoVE Journal

Biology

2.1K Views.  University of Maryland. Peptiden worden bestudeerd als nieuwe antischimmelmoleculen. Dit protocol kan worden gebruikt om de antischimmelwerking tegen de menselijke schimmelpathogeen Candida albicans te bestuderen. Deze techniek biedt kwantitatieve gegevens over antischimmelactiviteit en vermindert de experimentele tijd en het plastic afval in vergelijking met de andere methoden die het maken op agar vereisen.Naast het testen van de activiteit tegen Candida albicans, kan dit protocol worden gebruikt om de acti...

Video: Quantifying the Antifungal Activity of Peptides Against Candida albicans

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

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Environment

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts ...

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 ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Source: Chandley, P., et al. <a href="https://app.jove.com/v/64425">A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms.</a> J. Vis. Exp. (2022).
This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

1. C. tropicalis&#160;biofilm formation

	Prepare&#160;Candida&#160;biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier ...

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Samenvatting

Meer video's verkennen