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.
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.
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.
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
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.
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.
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).
5. Incubation of C. albicans with peptide solutions and preparation of the cells for the quantification of viability
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
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 protocol17,22,23,24,25.
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 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 °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 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.
The authors have nothing to disclose.
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.
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 |