Flow Cytometry-Based Method To Detect Persisters in Candida albicans

Abstract

Candida albicans biofilms contain a subpopulation whose members are defined as persisters, displaying great tolerance of fungicides. To directly observe such persisters, an effective method using green fluorescent protein (GFP) strain labeling by mutation of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (TDH3), combined with propidium iodide (PI) staining, was established. Amphotericin B-tolerant persisters harbor the characteristics of both GFP positivity [GFP (+)] and propidium iodide (PI) negativity [PI (-)], which are easily visualized using a fluorescence microscope and measured by flow cytometry.

FIG 1

Differential culture times prior to amphotericin B challenge affect the persister fraction of C. albicans SC5314. C. albicans SC5314 was inoculated into 96-well plates. The cells were treated with 80 μM farnesol [Farnesol (+)] or dimethyl sulfoxide (DMSO) solvent as the control [Farnesol (−)]. After the indicated time of culture, the persister fractions in each treatment were measured. Data are the mean values from the persister fractions, and the bars represent the standard deviations.

FIG 2

Establishment of a model for persister detection in C. albicans. (A) The double stains of FDA and PI for amphotericin B-treated C. albicans SC5314 biofilms. DIC, differential inference contrast. (B) Confocal laser scanning microscopy (CLSM) observation of PI staining cells of the C. albicans TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin B. The images were taken with a 40× objective by CLSM. (C) Flow cytometry detection for the persister fraction. The lateral axis represents the fluorescence of GFP, while the vertical axis indicates PI intensity. (D) CLSM observation of PI staining cells of the C. albicans TDH3-GFP-CAI4 strain exposed to 100 μg/ml of amphotericin B. The images were taken with a 63× oil lens by CLSM.

FIG 3

Persister fraction of C. albicans TDH3-GFP-CAI4 under different culture conditions. (A and B) Persister fraction of C. albicans cultured in SD medium at different pH values (A) or under conditions of different osmotic stresses using NaCl as the regulator (B). (C) Differential culture times prior to amphotericin B challenge affect the persister fraction of C. albicans. Data are the mean values for the persister fractions, and the bars represent the standard deviations.

FIG 4

Persister fraction of the cdr1 mutant strain tagged with GFP in Tdh3 in isolate MG1005 compared with control isolate MG1004. Error bars indicate standard errors of the means.