Quantifying the Antifungal Activity of Peptides Against Candida albicans

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
96-well plates (round bottom)	VWR	10062-902
Absorbance microplate reader	N/A	N/A	Any available microplate reader is sufficient
C. albicans strain SC5314	ATCC	MYA-2876	Other C. albicans may also be used
Hemocytometer	N/A	N/A	Can be used to make a standard curve relating cell number to OD600
Microplate shaker	VWR	2620-926
Peptide(s)	N/A	N/A	Peptides can be commercially synthesized by any reliable vendor; a purity of ≥95% and trifluoroacetic acid salt removal to hydrochloride salt are recommended
Reagent reservoirs for multichannel pipettors	VWR	18900-320	Simplifies pipetting into multiwell plates with multichannel pipettor
Sodium phosphate, dibasic	Fisher Scientific	BP332-500	For making NaPB
Sodium phosphate, monobasic	Fisher Scientific	BP329-500	For making NaPB
UV spectrophotometer	N/A	N/A	Any available UV spectrophotometer is sufficient
YPD medium powder	BD Life Sciences	242820	May also be made from yeast extract, peptone, and dextrose