Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candidaalbicans strains

Maesaki, Shigefumi; Marichal, Patrick; Bossche, H. Vanden; Sanglard, Dominique; Kohno, Shigeru

doi:10.1093/jac/44.1.27

Cited by 142 publications

(122 citation statements)

References 11 publications

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“…31,39) In the present study, we used a fluorescent dye, rhoamine 6 G (R6G), which is a substrate of CDR1, to identify CDR activity because a previous study showed that the efflux of rhoamine 6 G is positively correlated with the expression of CDR1. 40) Our results for rhodamine 6 G active efflux also showed that with the susceptibilities of the strains to FLC decreased, and the extrusion of rhodamine 6 G by C. albicans increased in the presence of glucose providing energy for rhodamine 6 efflux (Fig. 6), which suggested CDR1 overexpression contributed to the development of the reduction susceptibilities to FLC by FLC exposure in this study.…”

Section: Discussionsupporting

confidence: 72%

Fcr1p Inhibits Development of Fluconazole Resistance in Candida albicans by Abolishing CDR1 Induction

Shen

Wang

et al. 2007

Biological & Pharmaceutical Bulletin

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Candida albicans (C. albicans), the major opportunistic fungal pathogen in humans, causes diseases varying from superficial mucosal complaints to life-threatening systemic disorders. [1][2][3] Coincident with the increased use of antifungal drugs, the incidences of drug resistance have also increased. [4][5][6] In recent years, the development of multidrug resistance in clinical isolates has challenged effective treatment of the infections. Specifically, the extensive and repetitive use of antifungal azole derivatives such as fluconazole (FLC) has allowed C. albicans to utilize many mechanisms of resistance in order to ensure its survival. 7,8) This situation highlights the need for elucidating the mechanism of drug resistance in C. albicans to develop new antifungal agents.Overexpression of efflux pumps, either ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters, has been shown to be one of the major mechanisms of drug resistance in clinical isolates because if causes active extrusion of the drug out of the cell. 9-11) The Candida drug resistance 1 (CDR1) gene, which encodes an ABC efflux pump, is identified by complementation of the pleiotropic drug resistance 5 (pdr5) mutant, which is hypersensitive to cycloheximide, chloramphenicol, and azole drugs, in Saccharomyces cerevisiae (S. cerevisiae). 12) C. albicans cdr1 mutant resulted in increasing susceptibilities to azole drugs, 13) which is consistent with the observation that overexpression of CDR1 contributes to the drug resistance of clinical isolates of C. albicans. 6,14) Furthermore, the existence of trans-regulatory factors of CDR1 has also been suggested. 15) However, the molecular mechanism and the gene network regulating the expression of CDR1 and drug resistance are poorly understood.C. albicans Fcr1p (for fluconazole resistance 1 protein), a member of the family of zinc cluster proteins, is characterized by a highly conserved Zn(II) 2 Cys 6 zinc finger motif within the N-terminal DNA binding domain (DBD). 16) Well known Pdr1p and Pdr3p (pleiotropic drug resistance protein) zinc cluster proteins regulate the expression of several multidrug ABC transporter genes including PDR5, SNQ2, and YOR1, causing azole resistance in S. cerevisiae. 17-28) C. albicans Fcr1p displays significant sequence homology with S. cerevisiae Pdr1p and Pdr3p, and it could confer azole resistance in S. cerevisiae pdr1 pdr3 mutant by regulating PDR5. 16) We hypothesized that Fcr1p maybe involved in the development of C. albicans azole resistance as well as Pdr1p and Pdr3p in S. cerevisiae.In the present study we investigated the role of Fcr1p in the development of C. albicans azole resistance and possible mechanisms in vitro and in vivo. The results showed that Fcr1p inhibited development of fluconazole resistance in C. albicans through abolishing the induction of CDR1 expression by FLC, and in contrast FLC resistance development was accelerated resulting from the deletion of FCR1. (Table 1). MATERIALS AND METHODS Strains and MediaThe strains were cul...

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Section: Discussionsupporting

confidence: 72%

Fcr1p Inhibits Development of Fluconazole Resistance in Candida albicans by Abolishing CDR1 Induction

Shen

Wang

et al. 2007

Biological & Pharmaceutical Bulletin

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“…We similarly investigated R6G, a fluorescent substrate of many ABC transporters that has been used to study the functions of Cdr1p and Cdr2p in S. cerevisiae (12,15,31) and C. albicans (19,20). Our results showed that strain 5674 was only twofold more resistant to R6G than was strain 5457 (Fig.…”

Section: Vol 53 2009mentioning

confidence: 88%

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

Tsao

Rahkhoodaee

Raymond

2009

Antimicrob Agents Chemother

115

101

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Candida albicans frequently develops resistance to treatment with azole drugs due to the acquisition of gain-of-function mutations in the transcription factor Tac1p. Tac1p hyperactivation in azole-resistant isolates results in the constitutive overexpression of several genes, including CDR1 and CDR2, which encode two homologous transporters of the ATP-binding cassette family. Functional studies of Cdr1p and Cdr2p have been carried out so far by heterologous expression in the budding yeast Saccharomyces cerevisiae and by gene deletion or overexpression in azole-sensitive C. albicans strains in which CDR1 expression is low and CDR2 expression is undetectable. Thus, the direct demonstration that CDR1 and CDR2 overexpression causes azole resistance in clinical strains is still lacking, as is our knowledge of the relative contribution of each transporter to clinical azole resistance. In the present study, we used the SAT1 flipper system to delete the CDR1 and CDR2 genes from clinical isolate 5674. This strain is resistant to several azole derivatives due to a strong hyperactive mutation in Tac1p and expresses high levels of Cdr1p and Cdr2p. We found that deleting CDR1 had a major effect, reducing resistance to fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC) by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a much weaker effect, reducing FLC or KTC resistance by 1.5-fold, and had no effect on ITC resistance. These results demonstrate that Cdr1p is a major determinant of azole resistance in strain 5674 and potentially in other clinical strains overexpressing Cdr1p and Cdr2p, while Cdr2p plays a more minor role.Candida albicans is one of the leading causes of fungal infections affecting immunocompromised individuals. Candida infections range from chronic superficial infections of the skin and mucosal surfaces to invasive, life-threatening systemic infections (21, 38). Many antifungal drugs used to treat Candida infections target the biosynthesis of ergosterol, the major sterol in the fungal cell membranes (24). Polyenes, such as amphotericin B (AMB), directly bind to ergosterol and form pores in the cell membrane, resulting in low selectivity and high toxicity (24). Azoles, a class of well-tolerated antifungal drugs that includes fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), and new-generation derivatives such as voriconazole and posaconazole, target the enzyme lanosterol 14␣-demethylase (Erg11p), which is involved in ergosterol biosynthesis, blocking the production of ergosterol and causing the accumulation of toxic intermediate sterol species (24). As a consequence, the fluidity and permeability of the fungal cell membrane are changed and the activity of membrane-bound proteins, such as enzymes involved in cell wall synthesis, is altered (24).However, the fungistatic rather than fungicidal action of azole drugs leads to the frequent emergence of azole-resistant (A r ) C. albicans strains (1, 44). One mechanism of azole resistance consists of increased levels of ERG11 RNA, resulting in i...

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“…To test this possibility in the GPI2 heterozygote, we studied the efflux of the fluorescent dye, R6G. Although R6G and azoles do not share a common pathway for influx (20), they are effluxed out via the same mechanism (21). R6G influx was first monitored to confirm that variation in R6G influx does not lead to any observed difference in the efflux pattern of R6G.…”

Section: Gpi2 Mutant Shows An Up-regulation Of Ergosterol Biosynthesimentioning

confidence: 99%

“…Panel ii, R6G efflux assay was done to see if the efflux of R6G varied in the two strains. R6G is a good substrate of the multidrug pumps that also efflux out azoles in C. albicans (21). The two strains exhibited similar levels of R6G efflux in the absence as well as presence of glucose (Glc).…”

Section: Gpi2 Disruption Leads To Defective Hyphal Morphogenesis Becamentioning

confidence: 99%

First Step of Glycosylphosphatidylinositol (GPI) Biosynthesis Cross-talks with Ergosterol Biosynthesis and Ras Signaling in Candida albicans

Yadav¹,

Bhatnagar

Ahmad

et al. 2014

Journal of Biological Chemistry

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Background: GPI anchor is essential for virulence of C. albicans. Little is known about its GPI biosynthetic pathway. We explore roles of two GPI-N-acetylglucosaminyltransferase subunits catalyzing the first step. Results: Subunits GPI2 and GPI19 are negatively co-regulated, affecting Ras1 activity and ERG11 levels, respectively. Conclusion: GPI2/GPI19 levels affect morphogenesis and ergosterol biosynthesis. Significance: C. albicans can be targeted by modulation of cross-talk among major pathways.

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Rhodamine 6G efflux for the detection of CDR1-overexpressing azole-resistant Candidaalbicans strains

Cited by 142 publications

References 11 publications

Fcr1p Inhibits Development of Fluconazole Resistance in Candida albicans by Abolishing CDR1 Induction

Fcr1p Inhibits Development of Fluconazole Resistance in Candida albicans by Abolishing CDR1 Induction

Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

First Step of Glycosylphosphatidylinositol (GPI) Biosynthesis Cross-talks with Ergosterol Biosynthesis and Ras Signaling in Candida albicans

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