Ciclopirox olamine treatment affects the expression pattern of Candida albicans genes encoding virulence factors, iron metabolism proteins, and drug resistance factors

Abstract

The hydroxypyridone ciclopirox olamine belongs to the antimycotic drugs used for the treatment of superficial mycoses. In contrast to the azoles and other antimycotic drugs, its specific mode of action is only poorly understood. To investigate the mode of action of ciclopirox olamine on fungal viability, pathogenicity, and drug resistance, we examined the expression patterns of 47 Candida albicans genes in cells grown in the presence of a subinhibitory concentration (0.6 micro g/ml) of ciclopirox olamine by reverse transcription-PCR. In addition, we used suppression-subtractive hybridization to further identify genes that are up-regulated in the presence of ciclopirox olamine. The expression of essential genes such as ACT1 was not significantly modified in cells exposed to ciclopirox olamine. Most putative and known virulence genes such as genes encoding secreted proteinases or lipases had no or only moderately reduced expression levels. In contrast, exposure of cells to ciclopirox olamine led to a distinct up- or down-regulation of genes encoding iron permeases or transporters (FTR1, FTR2, FTH1), a copper permease (CCC2), an iron reductase (CFL1), and a siderophore transporter (SIT1); these effects resembled those found under iron-limited conditions. Addition of FeCl(3) to ciclopirox olamine-treated cells reversed the effect of the drug. Addition of the iron chelator bipyridine to the growth medium induced similar patterns of expression of distinct iron-regulated genes (FTR1, FTR2). While serum-induced yeast-to-hyphal phase transition of C. albicans was not affected in ciclopirox olamine-treated cells in the presence of subinhibitory conditions, a dramatic increase in sensitivity to oxidative stress was noted, which may indicate the reduced activities of iron-containing gene products responsible for detoxification. Although the Candida drug resistance genes CDR1 and CDR2 were up-regulated, no change in resistance or increased tolerance could be observed even after an incubation period of 6 months. This was in contrast to control experiments with fluconazole, in which the MICs for cells incubated with this drug had noticeably increased after 2 months. These data support the view that the antifungal activity of ciclopirox olamine may at least be partially caused by iron limitation. Furthermore, neither the expression of certain multiple-drug resistance genes nor other resistance mechanisms caused C. albicans resistance to this drug even after long-term exposure.

FIG. 1.

Analysis of RT-PCR products amplified from RNA samples collected from cultures without ciclopirox olamine (−), cultures with 0.6 μg of ciclopirox olamine/ml (+), and control PCRs with genomic DNA (G) of C. albicans SC5314. Results are shown for primer pairs amplifying cDNA from the housekeeping actin gene ACT1, the high-affinity iron permease gene FTR1, the low-affinity iron permease gene FTR2, the putative iron transporter FTH1, the multicopper oxidase gene FET3, and the copper transporter gene CCC2 after 20, 25, 30, and 35 cycles of PCR. The levels of expression of ACT1 and FET3 remained unchanged in the drug-treated culture, while the levels of expression of FTR1, FTH1, and CCC2 were strongly increased in the presence of a subinhibitory concentration of ciclopirox olamine. In contrast, low-affinity iron permease gene FTR2 was strongly down-regulated in the presence of the drug. Similar data were observed by Northern analysis (Fig. 5).

FIG. 2.

Growth curves for C. albicans SC5314 in 2% Sabouraud glucose medium measured at an OD of 630 nm containing different concentrations of ciclopirox olamine (A), fluconazole (B), and bipyridine (C). In these experiments 0.6 μg of ciclopirox olamine/ml, 2.0 μg of fluconazole/ml, and 100 μM bipyridine turned out to be the optimal subinhibitory concentrations which notably inhibited the cells but still allowed growth.

FIG. 3.

Growth curves for C. albicans SC5314 in 2% Sabouraud glucose medium containing 1.0 μg of ciclopirox olamine/ml supplemented with different concentrations of iron(III) chloride measured at an OD of 630 nm. Iron(III) chloride at concentrations of 20 μM and greater increased the levels of growth of the Candida cultures.

FIG. 4.

Electron microscopy of C. albicans SC5314 cells grown in 2% Sabouraud glucose medium without (A) and with (B) 0.6 μg of ciclopirox olamine/ml for 15 h. Not all cells were damaged to the same extent. Some cells appear normal, while others have severe morphological defects. (C) Detail of a single cell grown without ciclopirox olamine. (D) Detail of a cell grown under the influence of 0.6 μg of ciclopirox olamine/ml with enlargements of the vacuole and numerous invaginations of the cell membrane. In some areas the cytoplasmic margin was separated from the cell wall. In addition, mitochondria appeared bloated and fissures in the inner membrane system were largely widened. With the exception of the cell wall, which looked unaffected, most parts and organelles of the damaged cells were severely affected by the drug.

FIG. 5.

Northern analysis of selected genes involved in iron metabolism. Similar to the data observed by RT-PCR analysis (Fig. 1; Table 4), high transcript levels were detected for FTR1 (lane 1), FTH1 (lane 3), and CCC2 (lane 7) but not FET1 (lane 5) in ciclopirox olamine-treated cells compared with the levels for untreated cells (lanes 2, 4, 6, and 8, respectively). In contrast, FTR2 was up-regulated in untreated cells (lane 2) compared with the regulation in treated cells. mRNA levels were measured relative to the rRNA levels by loading approximately equal amounts of total RNA in each lane of the Northern blots (examples of ethidium bromide-stained membranes for the two samples with the ACT1 gene are shown in lanes 11 and 12). The ACT1 gene was hybridized as an additional internal loading control (lanes 9 and 10). Due to the high degree of similarity of FTR1 and FTR2, transcripts of both genes were detected with the same hybridization probe (lanes 1 and 2). Since this FTR1-FTR2 probe detected two different mRNAs with different lengths and different intensities in samples from nontreated (lane 2) and ciclopirox olamine-treated (lane 1) cells, we concluded that the transcript of FTR1 is smaller than that of FTR2. The lanes are unnumbered but represent lanes 1 to 12, from left to right, respectively.