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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.


Transcript levels of (A)CgAQR1, (B)CgQDR2, (C)CgTPO1_1, (D)CgTpo1_2, and (E)CgTPO3 in both susceptible (S) and resistant (R) C. glabrata clinical isolates. 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. The average of the expression values in each group of clinical isolates is represented by a red line (). ∗p-value < 0.05.
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Figure 2: Transcript levels of (A)CgAQR1, (B)CgQDR2, (C)CgTPO1_1, (D)CgTpo1_2, and (E)CgTPO3 in both susceptible (S) and resistant (R) C. glabrata clinical isolates. 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. The average of the expression values in each group of clinical isolates is represented by a red line (). ∗p-value < 0.05.

Mentions: A much higher variability in terms of the DHA gene expression was found in the azole-resistant strains, when compared to the susceptible ones. Despite this variability, the transcript level of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 genes was found to be significantly higher in resistant isolates, when compared to the susceptible ones, considering more than 70% of the tested strains (Figures 2A–C,E). In the case of CgTPO1_2, no statistically significant correlation could be observed (Figure 2D). As expected (Miyazaki et al., 1998; Sanglard et al., 1999; Bennett et al., 2004), the expression of CgCDR1 and CgCDR2 was found to correlate with azole drug resistance in the clinical isolates (Figure 3). It is important to notice that, the level of correlation between the expression of CgCDR2 and azole drug resistance was found to be similar to that observed for the DHA genes (p-value < 0.05). Consistent with a more relevant role in this context, a higher degree of correlation between gene expression and azole drug resistance was found for CgCDR1 (p-value < 0.01).


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)

Transcript levels of (A)CgAQR1, (B)CgQDR2, (C)CgTPO1_1, (D)CgTpo1_2, and (E)CgTPO3 in both susceptible (S) and resistant (R) C. glabrata clinical isolates. 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. The average of the expression values in each group of clinical isolates is represented by a red line (). ∗p-value < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Transcript levels of (A)CgAQR1, (B)CgQDR2, (C)CgTPO1_1, (D)CgTpo1_2, and (E)CgTPO3 in both susceptible (S) and resistant (R) C. glabrata clinical isolates. 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. The average of the expression values in each group of clinical isolates is represented by a red line (). ∗p-value < 0.05.
Mentions: A much higher variability in terms of the DHA gene expression was found in the azole-resistant strains, when compared to the susceptible ones. Despite this variability, the transcript level of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 genes was found to be significantly higher in resistant isolates, when compared to the susceptible ones, considering more than 70% of the tested strains (Figures 2A–C,E). In the case of CgTPO1_2, no statistically significant correlation could be observed (Figure 2D). As expected (Miyazaki et al., 1998; Sanglard et al., 1999; Bennett et al., 2004), the expression of CgCDR1 and CgCDR2 was found to correlate with azole drug resistance in the clinical isolates (Figure 3). It is important to notice that, the level of correlation between the expression of CgCDR2 and azole drug resistance was found to be similar to that observed for the DHA genes (p-value < 0.05). Consistent with a more relevant role in this context, a higher degree of correlation between gene expression and azole drug resistance was found for CgCDR1 (p-value < 0.01).

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.