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 infecti....

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....

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