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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

(A) Time course accumulation ratio of [3H]-Clotrimazole in non-adapted cells of the parental strain 51800 () or the mutant strain 51800_Δcgtpo3 (), during cultivation in BM liquid medium in the presence of 30 mg/L unlabeled clotrimazole. (B) Transcript levels of CgCDR1 (in black) and CgCDR2 (in white) in the C. glabrata clinical isolate 51800, when compared to those determined in the 51800_Δcgtpo3 derived deletion mutant. Transcript levels were assessed through quantitative RT-PCR, as described in the section “Materials and Methods.” The obtained values are the average of at least three independent experiments. Error bars represent the corresponding standard deviations. ∗p-value < 0.05, ∗∗p-value < 0.01.
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Figure 6: (A) Time course accumulation ratio of [3H]-Clotrimazole in non-adapted cells of the parental strain 51800 () or the mutant strain 51800_Δcgtpo3 (), during cultivation in BM liquid medium in the presence of 30 mg/L unlabeled clotrimazole. (B) Transcript levels of CgCDR1 (in black) and CgCDR2 (in white) in the C. glabrata clinical isolate 51800, when compared to those determined in the 51800_Δcgtpo3 derived deletion mutant. Transcript levels were assessed through quantitative RT-PCR, as described in the section “Materials and Methods.” The obtained values are the average of at least three independent experiments. Error bars represent the corresponding standard deviations. ∗p-value < 0.05, ∗∗p-value < 0.01.

Mentions: Given this observation and the previous demonstration that CgTPO3 mediates 3H-clotrimazole efflux in C. glabrata (Costa et al., 2014b), CgTpo3 ability to reduce the accumulation of radiolabeled clotrimazole in the C. glabrata clinical isolate 51800 was evaluated. The accumulation of 3H-labeled clotrimazole in non-adapted C. glabrata 51800 and 51800_Δcgtpo3 cells, suddenly exposed to the presence of 30 mg/L of cold clotrimazole, was assessed (Figure 6A). In these conditions, cells devoid of CgTPO3 accumulate twofold higher levels of 3H-clotrimazole when compared to the parental strain. Since it is possible that the observed moderate role played by CgTpo3 in clotrimazole resistance could result from an indirect effect in the expression of CgCDR1 or CgCDR2, the effect of CgTPO3 deletion in the 51800 clinical isolate on the expression of CgCDR1 and CgCDR2 was assessed. The deletion of CgTPO3 was found to have only a slight effect on CgCDR2 expression and no effect on CgCDR1 (Figure 6B). This result strongly suggests that CgTpo3 activity increases C. glabrata resistance to clotrimazole in clinical isolates by directly reducing its accumulation inside the cell.


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)

(A) Time course accumulation ratio of [3H]-Clotrimazole in non-adapted cells of the parental strain 51800 () or the mutant strain 51800_Δcgtpo3 (), during cultivation in BM liquid medium in the presence of 30 mg/L unlabeled clotrimazole. (B) Transcript levels of CgCDR1 (in black) and CgCDR2 (in white) in the C. glabrata clinical isolate 51800, when compared to those determined in the 51800_Δcgtpo3 derived deletion mutant. Transcript levels were assessed through quantitative RT-PCR, as described in the section “Materials and Methods.” The obtained values are the average of at least three independent experiments. Error bars represent the corresponding standard deviations. ∗p-value < 0.05, ∗∗p-value < 0.01.
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Related In: Results  -  Collection

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

Figure 6: (A) Time course accumulation ratio of [3H]-Clotrimazole in non-adapted cells of the parental strain 51800 () or the mutant strain 51800_Δcgtpo3 (), during cultivation in BM liquid medium in the presence of 30 mg/L unlabeled clotrimazole. (B) Transcript levels of CgCDR1 (in black) and CgCDR2 (in white) in the C. glabrata clinical isolate 51800, when compared to those determined in the 51800_Δcgtpo3 derived deletion mutant. Transcript levels were assessed through quantitative RT-PCR, as described in the section “Materials and Methods.” The obtained values are the average of at least three independent experiments. Error bars represent the corresponding standard deviations. ∗p-value < 0.05, ∗∗p-value < 0.01.
Mentions: Given this observation and the previous demonstration that CgTPO3 mediates 3H-clotrimazole efflux in C. glabrata (Costa et al., 2014b), CgTpo3 ability to reduce the accumulation of radiolabeled clotrimazole in the C. glabrata clinical isolate 51800 was evaluated. The accumulation of 3H-labeled clotrimazole in non-adapted C. glabrata 51800 and 51800_Δcgtpo3 cells, suddenly exposed to the presence of 30 mg/L of cold clotrimazole, was assessed (Figure 6A). In these conditions, cells devoid of CgTPO3 accumulate twofold higher levels of 3H-clotrimazole when compared to the parental strain. Since it is possible that the observed moderate role played by CgTpo3 in clotrimazole resistance could result from an indirect effect in the expression of CgCDR1 or CgCDR2, the effect of CgTPO3 deletion in the 51800 clinical isolate on the expression of CgCDR1 and CgCDR2 was assessed. The deletion of CgTPO3 was found to have only a slight effect on CgCDR2 expression and no effect on CgCDR1 (Figure 6B). This result strongly suggests that CgTpo3 activity increases C. glabrata resistance to clotrimazole in clinical isolates by directly reducing its accumulation inside the cell.

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