The ATP Binding Cassette Transporter GeneCgCDR1 from Candida glabrata Is Involved in the Resistance of Clinical Isolates to Azole Antifungal Agents

1999 ◽  
Vol 43 (11) ◽  
pp. 2753-2765 ◽  
Author(s):  
Dominique Sanglard ◽  
Françoise Ischer ◽  
David Calabrese ◽  
Paul A. Majcherczyk ◽  
Jacques Bille
Keyword(s):  
Clinical Isolate ◽  
Abc Transporter ◽  
Candida Glabrata ◽  
Antifungal Agents ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Atp Binding ◽  
Transporter Gene ◽  
Azole Antifungals ◽  
Susceptible Isolate

ABSTRACT The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiaestrain in which the ATP binding cassette (ABC) transporter genePDR5 was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporterCDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion ofCgCDR1 in an azole-resistant C. glabrataclinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrata clinical isolates.

scholarly journals Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata

2001 ◽  
Vol 45 (4) ◽  
pp. 1174-1183 ◽  
Author(s):  
Dominique Sanglard ◽  
Francoise Ischer ◽  
Jacques Bille
Keyword(s):  
Abc Transporter ◽  
High Frequency ◽  
Candida Glabrata ◽  
Antifungal Agents ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Atp Binding ◽  
Transporter Gene ◽  
Atp Binding Cassette

ABSTRACT Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation ofCgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, calledCgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.

Effect of antifungals on itraconazole resistant Candida glabrata

2010 ◽  
Vol 5 (3) ◽  
pp. 318-323 ◽  
Author(s):  
Soňa Kucharíková ◽  
Patrick Dijck ◽  
Magdaléna Lisalová ◽  
Helena Bujdáková
Keyword(s):  
Amphotericin B ◽  
Biofilm Formation ◽  
Candida Glabrata ◽  
Antifungal Agents ◽  
Inhibitory Concentration ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Low Concentrations ◽  
Reduction Assay ◽  
Very High

AbstractIn the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC100 values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.

scholarly journals Azole Resistance in Candida glabrata: Coordinate Upregulation of Multidrug Transporters and Evidence for a Pdr1-Like Transcription Factor

2004 ◽  
Vol 48 (10) ◽  
pp. 3773-3781 ◽  
Author(s):  
John-Paul Vermitsky ◽  
Thomas D. Edlind
Keyword(s):  
Candida Glabrata ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Common Mechanism ◽  
Azole Resistance ◽  
Multidrug Transporter ◽  
Activation Domain ◽  
Common Cause ◽  
Multidrug Transporters ◽  
Azole Antifungals

ABSTRACT Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

scholarly journals Mechanisms of Azole Resistance in Clinical Isolates of Candida glabrata Collected during a Hospital Survey of Antifungal Resistance

2005 ◽  
Vol 49 (2) ◽  
pp. 668-679 ◽  
Author(s):  
Maurizio Sanguinetti ◽  
Brunella Posteraro ◽  
Barbara Fiori ◽  
Stefania Ranno ◽  
Riccardo Torelli ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Candida Glabrata ◽  
Molecular Mechanisms ◽  
Efflux Pumps ◽  
Antifungal Resistance ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Azole Resistance ◽  
Relative Level ◽  
Fluconazole Resistant

ABSTRACT The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.

scholarly journals Molecular biological characterization of an azole-resistant Candida glabrata isolate.

1997 ◽  
Vol 41 (10) ◽  
pp. 2229-2237 ◽  
Author(s):  
P Marichal ◽  
H Vanden Bossche ◽  
F C Odds ◽  
G Nobels ◽  
D W Warnock ◽  
...  
Keyword(s):  
Copy Number ◽  
Candida Glabrata ◽  
Fold Increase ◽  
Resistant Isolate ◽  
Protein Patterns ◽  
Gene Encoding ◽  
Two Dimensional Gel Electrophoresis ◽  
Azole Antifungals ◽  
Susceptible Isolate ◽  
Lanosterol Demethylase

Two isolates of Candida glabrata, one susceptible and one resistant to azole antifungals, were previously shown to differ in quantity and activity of the cytochrome P-450 14alpha-lanosterol demethylase which is the target for azole antifungals. The resistant isolate also had a lower intracellular level of fluconazole, but not of ketoconazole or itraconazole, than the susceptible isolate. In the present study a 3.7-fold increase in the copy number of the CYP51 gene, encoding the 14alpha-lanosterol demethylase, was found. The amount of CYP51 mRNA transcript in the resistant isolate was eight times greater than it was in the susceptible isolate. Hybridization experiments on chromosomal blots indicated that this increase in copy number was due to duplication of the entire chromosome containing the CYP51 gene. The phenotypic instability of the resistant isolate was demonstrated genotypically: a gradual loss of the duplicated chromosome was seen in successive subcultures of the isolate in fluconazole-free medium and correlated with reversion to susceptibility. The greater abundance of the amplified chromosome induced pronounced differences in the protein patterns of the susceptible and revertant isolates versus that of the resistant isolate, as demonstrated by two-dimensional gel electrophoresis (2D-GE). Densitometry of the 2D-GE product indicated upregulation of at least 25 proteins and downregulation of at least 76 proteins in the resistant isolate.

scholarly journals In Silico Genome-Wide Analysis of the ATP-Binding Cassette Transporter Gene Family in Soybean (Glycine max L.) and Their Expression Profiling

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Awdhesh Kumar Mishra ◽  
Jinhee Choi ◽  
Muhammad Fazle Rabbee ◽  
Kwang-Hyun Baek
Keyword(s):  
Gene Family ◽  
Abc Transporter ◽  
Abc Transporters ◽  
In Silico ◽  
Gene Families ◽  
Protein Domain ◽  
Atp Binding ◽  
Transporter Gene ◽  
Genome Wide ◽  
Atp Binding Cassette

ATP-binding cassette (ABC) transporters constitute one of the largest gene families in all living organisms, most of which mediate transport across biological membranes by hydrolyzing ATP. However, detailed studies of ABC transporter genes in the important oil crop, soybean, are still lacking. In the present study, we carried out genome-wide identification and phylogenetic and transcriptional analyses of the ABC gene family in G. max. A total of 261 G. max ABC (GmABCs) genes were identified and unevenly localized onto 20 chromosomes. Referring to protein-domain orientation and phylogeny, the GmABC family could be classified into eight (ABCA-ABCG and ABCI) subfamilies and ABCG were the most abundantly present. Further, investigation of whole genome duplication (WGD) signifies the role of segmental duplication in the expansion of the ABC transporter gene family in soybean. The Ka/Ks ratio indicates that several duplicated genes are governed by intense purifying selection during evolution. In addition, in silico expression analysis based on RNA-sequence using publicly available database revealed that ABC transporters are differentially expressed in tissues and developmental stages and in dehydration. Overall, we provide an extensive overview of the GmABC transporter gene family and it promises the primary basis for the study in development and response to dehydration tolerance.

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