Mechanisms of azole resistance in a clinical isolate of Candida tropicalis

Abstract

Azole resistance has been insufficiently investigated in the yeast Candida tropicalis. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of C. tropicalis MDR1 (CtMDR1), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the CtERG11 gene coding for lanosterol 14alpha-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of CtERG11 showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of CtERG11 associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.

FIG. 1.

Oxygen consumption by C. tropicalis isolates 21234 (A, gray line), 21232 (A, black line), 10264 (B, gray line), and 21233 (B, black line). Comparison of the respiration rates showed an increase in oxygen consumption by resistant cells (42.9 nmol · ml⁻¹ · min⁻¹ versus 33, 18.9, and 30.6 nmol · ml⁻¹ · min⁻¹ for susceptible cells 21232, 10264, and 21233, respectively).

FIG. 2.

Flow cytometric analysis of rhodamine 6G uptake and efflux. Uptake of fluorochrome was quantified by incubating cells of C. tropicalis isolates 21234 (A) and 21232 (B) in YEPD broth for 30 min with 100 μM rhodamine 6G (black line). Then, efflux was evaluated by quantifying the residual fluorescence of the cells after removal of the free dye and an additional incubation of 15 min in YEPD broth (gray area).

FIG. 3.

CtERG11 gene sequence alignment of five C. tropicalis isolates (10264, 21232, 21233, and 21234 [this study] and GenBank strain 750). Only parts of DNA sequences showing differences between isolates are presented, and the number of the corresponding isolate is indicated on the left of each line. Numbers on the top of each five-line group indicate the position relative to the open reading frame. Start (+1) and stop codons are also indicated. Each mutation is positioned below the alignment by an asterisk, and corresponding nucleotides are in bold characters. The point mutation (A393T) leading to an amino acid substitution in the deduced protein sequence of the CtERG11 gene of the azole-resistant isolate 21234 is highlighted by a gray background.

FIG. 4.

Quantification of the relative transcript levels of CtERG11 and CtMDR1 genes from C. tropicalis isolates 21234, 10264, and 21233 compared to those of isolate 21232. Measured quantities of each mRNA were normalized using the expression level of the CtACT gene. Results, which are mean values from triplicate experiments, represent the numbers of times the genes are expressed compared to that in isolate 21232.