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. 2007 Mar;51(3):982-90.
doi: 10.1128/AAC.01510-06. Epub 2006 Dec 11.

Reduced susceptibility to polyenes associated with a missense mutation in the ERG6 gene in a clinical isolate of Candida glabrata with pseudohyphal growth

Patrick Vandeputte  1 , Guy Tronchin, Thierry Bergès, Christophe Hennequin, Dominique Chabasse, Jean-Philippe Bouchara
Affiliations

Reduced susceptibility to polyenes associated with a missense mutation in the ERG6 gene in a clinical isolate of Candida glabrata with pseudohyphal growth

Patrick Vandeputte et al. Antimicrob Agents Chemother. 2007 Mar.

Abstract

Little information is available about the molecular mechanisms responsible for polyene resistance in pathogenic yeasts. A clinical isolate of Candida glabrata with a poor susceptibility to polyenes, as determined by disk diffusion method and confirmed by determination of MIC, was recovered from a patient treated with amphotericin B. Quantitative analysis of sterols revealed a lack of ergosterol and an accumulation of late sterol intermediates, suggesting a defect in the final steps of the ergosterol pathway. Sequencing of CgERG11, CgERG6, CgERG5, and CgERG4 genes revealed exclusively a unique missense mutation in CgERG6 leading to the substitution of a cysteine by a phenylalanine in the corresponding protein. In addition, real-time reverse transcription-PCR demonstrated an overexpression of genes encoding enzymes involved in late steps of the ergosterol pathway. Moreover, this isolate exhibited a pseudohyphal growth whatever the culture medium used, and ultrastructural changes of the cell wall of blastoconidia were seen consisting in a thinner inner layer. Cell wall alterations were also suggested by the higher susceptibility of growing cells to Calcofluor white. Additionally, complementation of this isolate with a wild-type copy of the CgERG6 gene restored susceptibility to polyenes and a classical morphology. Together, these results demonstrated that mutation in the CgERG6 gene may lead to a reduced susceptibility to polyenes and to a pseudohyphal growth due to the subsequent changes in sterol content of the plasma membrane.

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Figures

FIG. 1.
FIG. 1.
Variations in sterol profiles of C. glabrata isolates 21231 and 21229. Sterols of the heptanic fraction were analyzed by gas chromatography. As highlighted by the dashed line, ergosterol, which was the major sterol species for isolate 21231 (A), was not detectable on the chromatogram of isolate 21229 (B). (C) Percentages of the ergosterol biosynthesis intermediates determined from the corresponding peak areas and retention times.
FIG. 2.
FIG. 2.
Gene expression level in isolate 21229 compared with isolate 21231. The expression levels of genes coding for ABC transporter (CgCDR1 and CgCDR2; black bars), a MAP-activated transcription factor involved in pseudohyphal growth (STE12; white bar), or enzymes involved in ergosterol biosynthesis (ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG9, ERG11; gray bars) were determined by RT-PCR. The relative increase (RI) in expression of the studied genes in isolate 21229 was determined as follows: RI = 2 exp[(Ct gene − Ct actin)isolate 21229 − (Ct gene − Ct actin)isolate 21231]. Ct (cycle threshold) is defined as the number of cycles for which the curve representing the fluorescence intensity according to the number of cycles cuts a baseline arbitrarily defined as one fluorescence unit. Results correspond to mean values of results from three independent experiments (±standard deviation).
FIG. 3.
FIG. 3.
Morphology of C. glabrata isolate 21229 on different agar-based culture media: Shadomy (A), YEPD (B), yeast extract-peptone-glycerol (C), RPMI-glucose (D), rice cream-Tween 80 (E), malt (F), and Casitone (G). Fungal cells were suspended in lactic blue dye, mounted on glass slides, and examined by light microscopy. A pseudohyphal growth was seen for isolate 21229 regardless of the culture medium. (H) Isolate 21231 grown on YEPD agar showing solitary blastoconidia and some budding cells. Bars, 20 μm.
FIG. 4.
FIG. 4.
Transmission electron micrographs of C. glabrata isolates 21229 (A) and 21231 (B). Transmission electron microscopy confirmed the pseudohyphal growth of isolate 21229, with cells presenting up to three daughter cells of similar size, and revealed the ultrastructural changes of their cell wall with a thinner inner layer compared with cells of control isolate. N, nucleus; mt, mitochondrion; CW, cell wall; DC, daughter cell.
FIG. 5.
FIG. 5.
Susceptibility of C. glabrata isolates 21231 (A) and 21229 (B) to calcofluor white. Susceptibility was evaluated by inoculation of various quantities of cells (from 2 × 106 to 2 × 102) on YEPD agar plates containing increasing concentrations of the dye (from 0.1 to 4 mg/ml). Calcofluor white inhibited the growth of isolate 21229 at a concentration as low as 0.1 mg/ml, whereas a concentration of 2 mg/ml was required to inhibit the growth of isolate 21231.
FIG. 6.
FIG. 6.
Growth curves of C. glabrata isolates 21231 (black) and 21229 (gray). Growth curves were drawn by monitoring the absorbance at 590 nm of cultures in YEPD broth incubated at 37°C for 30 h. Results correspond to mean absorbances of three independent cultures. For each value, the standard deviation did not exceed 10%.
FIG. 7.
FIG. 7.
Susceptibility to amphotericin B (A, B, and C) and microscopic morphology (D, E, and F) of the cells for C. glabrata isolate 21229 (A and D), its ura3 derivative 21229F34 (B and E), and the complemented strain 21229C218 (C and F). The complementation of isolate 21229 with a wild-type copy of the ERG6 gene restored the susceptibility to amphotericin B as well as a classical morphology, consisting of solitary blastoconidia.
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References

    1. Arthington, B. A., L. G. Bennett, P. L. Skatrud, C. J. Guynn, R. J. Barbuch, C. E. Ulbright, and M. Bard. 1991. Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase. Gene 102:39-44. - PubMed
    1. Ashman, W. H., R. J. Barbuch, C. E. Ulbright, H. W. Jarrett, and M. Bard. 1991. Cloning and disruption of the yeast C-8 sterol isomerase gene. Lipids 26:628-632. - PubMed
    1. Barker, K. S., S. Crisp, N. Wiederhold, R. E. Lewis, B. Bareither, J. Eckstein, R. Barbuch, M. Bard, and P. D. Rogers. 2004. Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans. J. Antimicrob. Chemother. 54:376-385. - PubMed
    1. Brun, S., C. Aubry, O. Lima, R. Filmon, T. Bergès, D. Chabasse, and J. P. Bouchara. 2003. Relationships between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 47:847-853. - PMC - PubMed
    1. Brun, S., T. Bergès, P. Poupard, C. Vauzelle-Moreau, G. Renier, D. Chabasse, and J. P. Bouchara. 2004. Mechanisms of azole resistance in petite mutants of Candida glabrata. Antimicrob. Agents Chemother. 48:1788-1796. - PMC - PubMed

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