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

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.

The PathoYeastract database: an information system for the analysis of gene and genomic transcription regulation in pathogenic yeasts

“…It is important to point out, however, that the amount of data collected for the regulation of QDR2 in C. glabrata is resumed to a single bibliographic reference, suggesting that many more regulators of the C. glabrata QDR2 may still be uncovered. Nonetheless, the observed variability in terms of QDR2 regulation within the three species appears consistent with the fact that the function of the QDR2 gene appears also to have diverged within these yeasts (25,26,28–30). …”

Azole Antifungal Resistance in Candida albicans and Emerging Non-albicans Candida Species

“…Recent work has shown increased expression of four MFS transporters in clotrimazole resistant isolates compared to clotrimazole susceptible clinical isolates. Disruption of one of these, TPO3 , moderately increased susceptibility to clotrimazole and fluconazole (Costa et al, 2016). These findings suggest MFS transporters may have a minor role in azole resistance in C. glabrata .…”

The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogenCandida glabrata

“…The Drug:H + antiporter (DHA) family of the Major Facilitator Superfamily (MFS) has been demonstrated to play a key role in drug resistance in Saccharomyces cerevisiae (Sá‐Correia, dos Santos, Teixeira, Cabrito, & Mira, ; Santos, Teixeira, Dias, & Sá‐correia, ) and, more recently, in C. glabrata (Costa, Dias, et al, ). Indeed, the DHA transporters CgAqr1, CgQdr2, CgTpo1_1, CgTpo1_2, and CgTpo3 were found to confer resistance to drugs and other stress factors, such as azoles, flucytosine, acetic acid, and polyamines, mostly by contributing to decrease the intracellular accumulation of those molecules (Costa, Henriques, et al, ; Costa, Pires, et al, ; Costa, Nunes, et al, ; Pais et al, ; Costa et al, ). Interestingly, however, the C. albicans DHA transporters CaMdr1, CaNag3, CaNag4 and, more recently, CaQdr1, CaQdr2, and CaQdr3 were found to further play a role in its virulence, although the molecular mechanisms behind this observation are still to be fully elucidated (Becker, Henry, Jiang, & Koltin, ; Yamada‐Okabe & Yamada‐Okabe, ; Shah et al, ).…”