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 candidiasis^2,3. 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 therapeutics^4,5. 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 agents^6,7,8 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 activity^10,11,12. AMPs with activity against C. albicans include well-known peptides from the histatin and cecropin families^13,14,15, along with more recently described peptides like ToAP2, NDBP-5.7, and the histatin 5 variant K11R-K17R^16,17. 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 lactoferrin^19,20,21. 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 peptides^{17,22,23,24,25}.

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.

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

1. 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.
2. Prepare sterile liquid yeast-peptone-dextrose (YPD, see Table of Materials) medium (10 g/L yeast extract,   20 g/L dextrose, 20 g/L peptone). Preparing 100 mL of YPD will be more than sufficient for most applications of this protocol.
3. Prepare sterile ultrapure water. Preparing 100 mL of water will be more than sufficient for most applications of this protocol.
   ​NOTE: The buffers, media, and water are sterilized by autoclaving at 121 °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.

2. Inoculation, culturing, and subculturing of C. 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.

1. Inoculate the desired C. albicans strain 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.
2. Grow the culture overnight (~12-16 h) at 30 °C and 230 rpm on a rotary shaker.
3. Subculture the overnight culture of C. albicans, and grow to an OD₆₀₀ of ~1.0-1.2.
   1. Measure the OD₆₀₀ of the overnight culture using a UV spectrophotometer (see Table of Materials).
   2. 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.
   3. Grow the subculture at 30 °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) while the subculture is growing; the subculture will be diluted for use in the assay in step 4.

3. Preparation of the peptide solutions in a 96-well plate

NOTE: This step may be done ahead of time, if the peptide stock solutions are stable during storage. Typically, the peptide solutions are stored at −20 °C until use. They can be thawed in a room temperature water bath before proceeding to the next step.

1. 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 µM.
   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.
2. Dissolve each peptide in sterile water at twice the desired highest concentration. For the data presented in the representative results section, 150 µL of 100 µM 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.
3. Add 40 µL 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.
4. Add 20 µL 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.
5. 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.
   1. Remove 20 µL from Column 1, transfer it to Column 2, and mix by pipetting up and down. Column 2 now contains 40 µL of peptide solution at a 1:2 dilution of the concentration in Column 1.
   2. Remove 20 µL from Column 2, transfer it to Column 3, and mix by pipetting up and down, so Column 3 now contains 40 µL of peptide solution at a 1:4 dilution of the concentration in Column 1.
   3. Repeat this process until Column 10 contains 40 µL of peptide solution at a 1:512 dilution of the concentration in Column 1.
   4. Remove 20 µL of peptide solution from Column 10 and discard it. Each column now contains 20 µL of peptide solution (Columns 1-10) or water (Column 11 and Column 12).
      ​NOTE: The wells in Column 11 serve as sterility controls, and the wells in Column 12 serve as controls containing no peptide.

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).

1. 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.
2. Resuspend the pellet with 1 mL of 2 mM NaPB (step 1.1), and transfer the suspension to a 1.7 mL centrifuge tube.
3. Pellet the cells (as in step 4.1), discard the supernatant, and again resuspend the pellet in 1 mL of 2 mM NaPB.
4. Repeat step 4.3 two additional times to leave the washed cells in 1 mL of 2 mM NaPB.
5. 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.
6. 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.

5. Incubation of C. albicans with peptide solutions and preparation of the cells for the quantification of viability

1. Add 20 µL 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 µM 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.
2. Add 20 µL 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.
3. Cover Plate 1 (containing both cells and peptide), and incubate it at 30 °C for 30 min.
   NOTE: An incubation time of 30 min is sufficient for many peptides^17,21,22,30, 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 activity^25,31,32.
4. 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.
   1. Add 100 µL of YPD (step 1.2) to all the wells of the plate.
   2. Add 100 µL of 2 mM NaPB (Step 1.1) to all the wells of the plate.
5. Dilute the samples in Plate 1 (containing cells and peptides), and transfer to Plate 2 (containing medium and buffer).
   1. Retrieve Plate 1 from the incubator following the 30 min of incubation.
   2. Dilute each well of Plate 1 by adding 280 µL of 1 mM NaPB (step 1.1) for a total volume of 320 µL in each well (Figure 2).
   3. 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.
   4. Transfer 8 µL 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).
6. Cover Plate 2 and incubate on a microtiter plate shaker (see Table of Materials) at 350 rpm for 17 h at 30 °C.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

6. Determination of antifungal activity

1. Obtain OD₆₀₀ readings for each well in Plate 2.
   1. Remove Plate 2 from the incubator after 17 h of incubation.
   2. 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.
   3. Use an absorbance plate reader (see Table of Materials) at a wavelength of 600 nm to obtain the OD₆₀₀ for each well.
2. Calculate the inhibition of growth (as a percentage), and plot it to determine the effect of the peptides on the growth of C. albicans.
   1. For each well, calculate the reduction in viability compared to the control (as a percentage) using the following equation^17,22,23:
      
      where OD_{with peptide} is the OD₆₀₀ of a well containing a given concentration of peptide, OD_background is the OD₆₀₀ of Column 11 in the same row (control containing no cells), and
      OD_{no peptide} is the OD₆₀₀ of Column 12 in the same row (control containing cells and no peptide).
   2. For example, use the following expression to calculate the reduction in viability in Well B5 from the OD₆₀₀ values for Row B:
      
3. Plot the reduction in viability as a function of the peptide concentration.

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. 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. 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 protocol^{17,22,23,24,25}.

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 (B) K5R, (C) K11R, (D) K13R, and (E) K17R (with lysine to arginine modification at the indicated residues) were incubated with C. albicans cells at concentrations of 50 µM to 0.20 µM, 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's multiple comparisons test (α = 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. Please click here to view a larger version of this figure.

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'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 vitro^8,32,33,34, 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 peptides^6,7,34,36. 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 °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' 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 peptide^17,22. 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.