Curcumin targets cell wall integrity via calcineurin-mediated signaling in Candida albicans

Abstract

Curcumin (CUR) shows antifungal activity against a range of pathogenic fungi, including Candida albicans. The reported mechanisms of action of CUR include reactive oxygen species (ROS) generation, defects in the ergosterol biosynthesis pathway, decrease in hyphal development, and modulation of multidrug efflux pumps. Reportedly, each of these pathways is independently linked to the cell wall machinery in C. albicans, but surprisingly, CUR has not been previously implicated in cell wall damage. In the present study, we performed transcriptional profiling to identify the yet-unidentified targets of CUR in C. albicans. We found that, among 348 CUR-affected genes, 51 were upregulated and 297 were downregulated. Interestingly, most of the cell wall integrity pathway genes were downregulated. The possibility of the cell wall playing a critical role in the mechanism of CUR required further validation; therefore, we performed specific experiments to establish if there was any link between the two. The fractional inhibitory concentration index values of 0.24 to 0.37 show that CUR interacts synergistically with cell wall-perturbing (CWP) agents (caspofungin, calcofluor white, Congo red, and SDS). Furthermore, we could observe cell wall damage and membrane permeabilization by CUR alone, as well as synergistically with CWP agents. We also found hypersusceptibility in calcineurin and mitogen-activated protein (MAP) kinase pathway mutants against CUR, which confirmed that CUR also targets cell wall biosynthesis in C. albicans. Together, these data provide strong evidence that CUR disrupts cell wall integrity in C. albicans. This new information on the mechanistic action of CUR could be employed in improving treatment strategies and in combinatorial drug therapy.

FIG 1

Microarray results showing percent distributions of various categories of genes that were differentially expressed in CUR-treated C. albicans. The gene names in boldface indicate upregulation in response to CUR treatment, while those in gray type indicate downregulation.

FIG 2

(A) Time-kill curve of CUR in combination with CWP agents: 173.7 μM CUR (63.98 μg/ml) plus 1.95 μM (1.79 μg/ml) CFW, 173.7 μM (63.98 μg/ml) CUR plus 1.90 μM (1.36 μg/ml) CR, and 86.8 μM (31.97 μg/ml) CUR plus 31.25 μM (9 μg/ml) SDS. (B) Percent growth of C. albicans at the same synergy concentrations of CUR with CWP agents as for panel A. (C) Solid-medium spotting assay. The wild-type (SC5314) strain was tested by spotting decreasing numbers of cells on YPD agar plates with CUR and CWP agents alone and in combination.

FIG 3

Confocal microscopy analysis of membrane permeabilization assay by PI uptake. C. albicans was incubated with CUR alone (251 μM [92.45 μg/ml] and 502 μM [184.9 μg/ml]) and at synergistic concentrations with CWP agents (1.95 μM [1.79 μg/ml] CFW plus 173.7 μM [63.98 μg/ml] CUR, 1.95 μM [1.36 μg/ml] CR plus 173.7 μM [63.98 μg/ml] CUR, and 31.25 μM [9 μg/ml] SDS plus 86.8 μM [31.97 μg/ml] CUR) for 4 h.

FIG 4

Scanning electron micrographs showing corrugation of the cell surface, squeezing of the cell, and leakage of cytoplasmic content due to cell wall damage (arrows) in C. albicans after 24-h treatment with CUR and CWP agents at synergistic concentrations. (A) SEM of C. albicans treated or not with CUR (695 μM; 256 μg/ml) for 24 h showing corrugation of the cell wall and squeezing of cells. (B-i, C-i, and D-i) Controls; cells are intact and evenly shaped. (B-ii to -iv) Cells after treatment with 1.95 μM (1.79 μg/ml) CFW (B-ii), 173.7 μM (63.98 μg/ml) CUR (B-iii), and 1.95 μM (1.79 μg/ml) CFW plus 173.7 μM (63.98 μg/ml) CUR (B-iv). (C-ii to -iv) Cells after incubation with 1.90 μM (1.36 μg/ml) CR (C-ii), 173.7 μM (63.98 μg/ml) CUR (C-iii), and 1.90 μM (1.36 μg/ml) CR plus 173.7 μM (63.98 μg/ml) CUR (C-iv). (D-ii to -iv) Cells during incubation with 31.25 μM SDS (D-ii), 86.8 μM CUR (D-iii), and 31.25 μM (9 μg/ml) SDS plus 86.8 μM (31.97 μg/ml) CUR (C-iv).

FIG 5

Calcineurin, MAP kinase, and stress mutants showed enhanced fungicidal activity of CUR in C. albicans. Cells were grown overnight in YPD at 30°C, 5-fold serially diluted, and spotted onto YPD medium containing CFW and CUR at the concentrations indicated. The plates were incubated at 30°C for 48 h.

FIG 6

Summary of altered mechanisms in C. albicans cells after CUR treatment. These mechanisms are crucial for the development of drug tolerance and virulence in C. albicans.