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Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

Costa C, Ribeiro J, Miranda IM, Silva-Dias A, Cavalheiro M, Costa-de-Oliveira S, Rodrigues AG, Teixeira MC - Front Microbiol (2016)

Bottom Line: For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters.The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance.The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Instituto Superior Técnico, University of LisbonLisboa, Portugal; Institute for Bioengineering and Biosciences, Biological Sciences Research GroupLisboa, Portugal.

ABSTRACT
For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H(+) Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

No MeSH data available.


Related in: MedlinePlus

Comparison of the susceptibility to (A) clotrimazole and (B) fluconazole, at the indicated concentrations displayed by the clinical isolate 51800 and the derived 51800_Δcgtpo3 deletion mutant, in BM agar plates by spot assays. Cell suspensions used to prepare the spots were (b) 1:5 and (c) 1:25 of the cell suspension used in (a). The displayed images are representative of at least three independent experiments.
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Figure 4: Comparison of the susceptibility to (A) clotrimazole and (B) fluconazole, at the indicated concentrations displayed by the clinical isolate 51800 and the derived 51800_Δcgtpo3 deletion mutant, in BM agar plates by spot assays. Cell suspensions used to prepare the spots were (b) 1:5 and (c) 1:25 of the cell suspension used in (a). The displayed images are representative of at least three independent experiments.

Mentions: CgTPO3 had previously been shown to contribute to resistance to clotrimazole and fluconazole in a laboratorial strain (Costa et al., 2014b). Given the observation that this gene was found to be up-regulated in clotrimazole resistant clinical isolates, when compared to susceptible ones, it seemed important to assess if its absence could affect azole resistance in clinical isolates as well. Therefore, this gene was deleted in the azole-resistant isolate 51800, found to exhibit high levels of CgTPO3 expression. The deletion of this gene in the 51800 isolate was found to significantly decrease its resistance to clotrimazole and fluconazole (Figures 4A,B), thus reinforcing the notion that this transporter contributes to azole resistance in the clinical context. The MIC50 values of clotrimazole and fluconazole for the 51800 and 51800_Δcgtpo3 were further evaluated. MIC50 values were found to be in all cases higher in the parental 51800 strain (8 and >128 for clotrimazole and fluconazole, respectively) than for the derived Δcgtpo3 deletion mutant (4 and 64, respectively).


Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

Costa C, Ribeiro J, Miranda IM, Silva-Dias A, Cavalheiro M, Costa-de-Oliveira S, Rodrigues AG, Teixeira MC - Front Microbiol (2016)

Comparison of the susceptibility to (A) clotrimazole and (B) fluconazole, at the indicated concentrations displayed by the clinical isolate 51800 and the derived 51800_Δcgtpo3 deletion mutant, in BM agar plates by spot assays. Cell suspensions used to prepare the spots were (b) 1:5 and (c) 1:25 of the cell suspension used in (a). The displayed images are representative of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835504&req=5

Figure 4: Comparison of the susceptibility to (A) clotrimazole and (B) fluconazole, at the indicated concentrations displayed by the clinical isolate 51800 and the derived 51800_Δcgtpo3 deletion mutant, in BM agar plates by spot assays. Cell suspensions used to prepare the spots were (b) 1:5 and (c) 1:25 of the cell suspension used in (a). The displayed images are representative of at least three independent experiments.
Mentions: CgTPO3 had previously been shown to contribute to resistance to clotrimazole and fluconazole in a laboratorial strain (Costa et al., 2014b). Given the observation that this gene was found to be up-regulated in clotrimazole resistant clinical isolates, when compared to susceptible ones, it seemed important to assess if its absence could affect azole resistance in clinical isolates as well. Therefore, this gene was deleted in the azole-resistant isolate 51800, found to exhibit high levels of CgTPO3 expression. The deletion of this gene in the 51800 isolate was found to significantly decrease its resistance to clotrimazole and fluconazole (Figures 4A,B), thus reinforcing the notion that this transporter contributes to azole resistance in the clinical context. The MIC50 values of clotrimazole and fluconazole for the 51800 and 51800_Δcgtpo3 were further evaluated. MIC50 values were found to be in all cases higher in the parental 51800 strain (8 and >128 for clotrimazole and fluconazole, respectively) than for the derived Δcgtpo3 deletion mutant (4 and 64, respectively).

Bottom Line: For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters.The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance.The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Instituto Superior Técnico, University of LisbonLisboa, Portugal; Institute for Bioengineering and Biosciences, Biological Sciences Research GroupLisboa, Portugal.

ABSTRACT
For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H(+) Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

No MeSH data available.


Related in: MedlinePlus