Name: quantifying the antifungal activity of peptides against candida albicans
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Description: Watch this Scientific Journal Video about Quantifying the Antifungal Activity of Peptides Against Candida albicans at JoVE.com

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</...

<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. 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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. 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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. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>C. albicans strain SC5314</td>
			<td>ATCC&#160;</td>
			<td>MYA-2876</td>
			<td>Other C. albicans 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 OD600</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 &#8805;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 subcult...

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 ...

Peptides are being studied as novel antifungal molecules. This protocol can be used to study the antifungal efficacy against the human fungal pathogen Candida albicans. This technique provides quantitative data on antifungal activity while reducing experimental time and plastic waste compared to the other methods that require creating on agar.In addition to testing the activity against Candida albicans, this protocol can be used to test the activity against other yeast-forming cells and to test the activity of small-molecule antifungal agents Begin by inoculating the desired Candida albicans strain into 10 milliliters of liquid yeast extract-peptone-dextrose, or YPD medium, in a culture tube. Grow the culture overnight at 30 degrees Celsius and 230 rpm on a rotary shaker. Subculture the overnight culture of C.albicans and grow to an optical density of approximately 1.0 to 1.2 at 600 nanometer wavelength.Solubilize each peptide in sterile water and dilute it to twice the desired highest concentration, then add 40 microliters of the desired peptide stock solutions to the first well of each row in a round-bottomed 96-well plate. Next, add 20 microliters of sterile ultra-pure water to columns 2 to 12 of the rows containing the peptide. To serially dilute the peptide stock solutions across the plate up to column 10, transfer 20 microliters from column 1 to column 2 and mix it by pipetting up and down.Repeat the dilution process up to column 10 which will contain 40 microliters of peptide solution at a 1-to-512 dilution of the column 1 concentration. Once done, remove 20 microliters of peptide solution from column 10 and discard it. Transfer the C.albicans subculture to a 15-milliliter centrifuge tube and centrifuge at 3, 900 g for 3 minutes at room temperature.Remove the supernatant by pipetting or decanting. Resuspend the pellet with 1 milliliter of 2-millimolar sodium phosphate buffer and transfer the suspension to a 1.7-milliliter centrifuge tube. Pellet the cells by centrifuging and discard the supernatant.Again, resuspend the pellet in 1 milliliter of 2-millimolar sodium phosphate buffer and wash 2 more times to leave the washed cells in 1 milliliter of sodium phosphate buffer. Determine the cell density of the washed cell suspension and dilute the suspension to 5 times 10 to the 5th cells per milliliter in a 2-millimolar sodium phosphate buffer. Take diluted C.albicans cell suspension and add 20 microliters to columns 1 to 10 and column 12 of each row, then add 20 microliters of 2-millimolar sodium phosphate buffer to column 11.Cover plate 1 and place it into an incubator at 30 degrees Celsius for 30 minutes. Prepare a new 96-well culture plate to quantify viability by adding 100 microliters of YPD to all the wells of plate number 2, then add 100 microliters of 2-millimolar sodium phosphate buffer to all the wells of plate 2. To dilute the samples in plate 1, retrieve plate 1 from the incubator, and add 280 microliters of 1-millimolar sodium phosphate buffer in each well.After mixing the contents well with a pipette, transfer 8 microliters from plate 1 to the corresponding well of plate 2. Once done, place the covered plate 2 on a microtiter plate shaker at 350 rpm for 17 hours at 30 degrees Celsius. Mix all the wells in plate 2 by pipetting up and down, ensuring all the cells are resuspended.Obtain optical density readings at a 600 nanometer wavelength for each well in plate 2 using an absorbance plate reader. Plot the reduction in viability as a function of the peptide concentration to calculate the growth inhibition percentage and to determine the effect of the peptides on the C.albicans growth. After incubating the peptides with the C.albicans cells, the antifungal activity was quantified using the colony-forming units, or CFU counting method and the described protocol incorporating optical density measurements.The data using the optical density readings were highly reproducible as indicated by the small standard deviations of the replicates. In performing this protocol, maintain the Candida albicans cells in the yeast form by incubating the cells at 30 degrees Celsius since optical density measurements are not reliable in the hyphal cells. The efficiency of this method has allowed researchers to perform high triple studies on peptide activity by testing larger numbers of peptides and peptide concentrations.

Research

JoVE Journal

Biology

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