Biomarkers of caspofungin resistance in Candida albicans isolates: A proteomic approach

Abstract

Candida albicans is a clinically important polymorphic fungal pathogen that causes life-threatening invasive infections in immunocompromised patients. Antifungal therapy failure is a substantial clinical problem, due to the emergence of an increasing number of drug-resistant isolates. Caspofungin is a common antifungal drug, often used as first-line therapy that inhibits cell wall β-(1,3)-glucan synthesis. In this work, the cell surface of different echinocandin-resistant C. albicans clinical isolates was compared with sensitive isolates and their responses to echinocandin treatment analyzed. Proteomic analysis detected changes in the repertoire of proteins involved in cell wall organization and maintenance, in drug-resistant strains compared to susceptible isolates and after incubation with caspofungin. Moreover, an interaction network was created from the differential expression results. Our findings suggest drug resistance may involve not only a different cell wall architecture, but also a different response to drugs.

Keywords: Candida albicans; caspofungin; drug resistance; interaction network; proteomics.

Figure 1.

Biofilm formation by caspofungin-susceptible isolates of C. albicans. Absorbance from crystal violet staining of C. albicans Ca1, Ca2, Ca3, Ca4, Ca5, Ca6 isolates grown at 37 C in RPMI-1640 + 20% FCS grown either without drug (magenta) or the addition of 2 μg/ml CAS (yellow) or 4 μg/ml CAS (cyan) and measured at: (a) 6 h; (b) 24 h; (c) 48 h; (d) 72 h. The values are expressed as absorbance at 570 nm. The statistical analysis performed was one-way ANOVA (n = 1 (3 replicates), *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005).

Figure 2.

Biofilm formation assay for caspofungin-resistant isolates of C. albicans. Absorbance from crystal violet staining of C. albicans Car1, Car2, and Car3 isolates grown at 37°C in RPMI-1640 + 20% FCS grown either without drug (magenta) or the addition of 2 μg/ml CAS (yellow) or 4 μg/ml CAS (cyan) and measured at: (a) 6 h; (b) 24 h; (c) 48 h; (d) 72 h. The values are expressed as absorbance at 570 nm. The statistical analysis performed was one-way ANOVA (n = 1 (3 replicates), *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005).

Figure 3.

Proteomic analysis of cell wall fractions from C. albicans resistant and susceptible isolates exposed to caspofungin. (a, b) Total number of proteins identified by LC MS/MS in susceptible and resistant isolates in absence (a) and presence (b) of caspofungin in RPMI 1640 medium. (c) Differential expression of relevant proteins identified by LC MS/MS in susceptible Ca1 and resistant Car1 isolates in absence (blue) and presence (orange) of caspofungin in RPMI 1640 medium. The values displayed are the ratios of the averages (n=3) of the peak areas from the LC MS/MS analysis of the two isolates (Car1 and Ca1). The values displayed are the ratios of the log 10 of the averages of the peak areas from the LC MS/MS analysis of the two groups (resistant and susceptible). (d) Differential protein expression for the resistant compared to the susceptible isolates with (orange) and without (blue) caspofungin. The full list is available in File S1 in Supplementary Material. Venn diagrams were created using Venny software (n=1).

Figure 4.

Volcano plots of cell wall proteome comparison of caspofungin -resistant and -susceptible isolate of C. albicans performed by differential expression (DE) analysis. The plots compare fold change and statistical significance of DE for caspofungin-resistant and -sensitive isolates of C. albicans (a) without drug and (b) with drug. The DE analysis was carried out using limma package, part of Bioconductor software. The plots compare fold change and statistical significance of DE for caspofungin-resistant and -sensitive isolates of C. albicans (a) without drug and (b) with drug. The DE analysis was carried out using limma package, part of Bioconductor software.

Figure 5a.

Interaction network of cell wall proteome from C. albicans isolates. The network was created using LC-MS/MS data from cell wall fractions of C. albicans isolates grown in absence and in presence of caspofungin. The green edges of the circles indicate the significant proteins for the DE analysis (adjusted p-value ≤0.1). The lines connecting the circles indicate the positive (red, both increase or decrease+/- caspofungin) or negative interaction (blue, one decreases and the other increases or vice versa). The thickness of the line indicates the strength of the interaction. The histograms inside the circles indicate the expression of the proteins related to the normalized average between the two groups of isolates in the two conditions: from left to right, the columns represent susceptible, susceptible+caspofungin, resistant, resistant+caspofungin. Proteins were clustered in seven groups and the GO analysis performed: (a) general view of the network; (b) ribosomal proteins; (c) cell wall organization; (d) modulation of the host response; (e) G0; (f) adhesion to the host; (g) germ-tube formation; (h) glucose transporters.

Figure 5b.

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Figure 5c.

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