Overexpression of the MDR1 Gene Is Sufficient To Confer Increased Resistance to Toxic Compounds in Candida albicans

ABSTRACT Overexpression of MDR1, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen Candida albicans can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical C. albicans isolates, constitutive MDR1 overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether MDR1 overexpression is sufficient to confer a drug-resistant phenotype in C. albicans, we expressed the MDR1 gene from the strong ADH1 promoter in C. albicans laboratory strains that did not express the endogenous MDR1 gene as well as in a fluconazole-resistant clinical C. albicans isolate in which the endogenous MDR1 alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced MDR1 overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the MDR1 gene from the ADH1 promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that MDR1 overexpression in C. albicans is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.

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MDR1-Mediated Drug Resistance inCandida dubliniensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3416-3421.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3416-3421 ◽

Cited By ~ 70

Author(s):

Stephanie Wirsching ◽

Gary P. Moran ◽

Derek J. Sullivan ◽

David C. Coleman ◽

Joachim Morschhäuser

Keyword(s):

Drug Resistance ◽

Efflux Pump ◽

Brefeldin A ◽

Acid Resistance ◽

Amino Acid Sequences ◽

Candida Dubliniensis ◽

Major Facilitator Superfamily ◽

Multidrug Efflux Pump ◽

Major Facilitator

ABSTRACT Candida dubliniensis is a recently described opportunistic fungal pathogen that is closely related to Candida albicans. Candida dubliniensis readily develops resistance to the azole antifungal agent fluconazole, both in vitro and in infected patients, and this resistance is usually associated with upregulation of the CdMDR1 gene, encoding a multidrug efflux pump of the major facilitator superfamily. To determine the role ofCdMDR1 in drug resistance in C. dubliniensis, we constructed an mdr1 null mutant from the fluconazole-resistant clinical isolate CM2, which overexpressed the CdMDR1 gene. Sequential deletion of both CdMDR1 alleles was performed by theMPA R-flipping method, which is based on the repeated use of a dominant mycophenolic acid resistance marker for selection of integrative transformants and its subsequent deletion from the genome by FLP-mediated, site-specific recombination. In comparison with its parental strain, the mdr1 mutant showed decreased resistance to fluconazole but not to the related drug ketoconazole. In addition, we found that CdMDR1 confers resistance to the structurally unrelated drugs 4-nitroquinoline-N-oxide, cerulenin, and brefeldin A, since the enhanced resistance to these compounds of the parent strain CM2 compared with the matched susceptible isolate CM1 was abolished in the mdr1 mutant. In contrast, CdMDR1inactivation did not cause increased susceptibility to amorolfine, terbinafine, fluphenazine, and benomyl, although overexpression ofCdMDR1 in a hypersusceptible Saccharomyces cerevisiae strain had previously been shown to confer resistance to these compounds. The effect of CdMDR1inactivation was identical to that seen in two similarly constructedC. albicans mdr1 mutants. Therefore, despite species-specific differences in the amino acid sequences of the Mdr1 proteins, overexpression of CaMDR1 andCdMDR1 in clinical C. albicans andC. dubliniensis strains seems to confer the same drug resistance profile in both species.

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Systematic truncations of chromosome 4 and their responses to antifungals in Candida albicans

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00197-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Wasim Uddin ◽

Darshan Dhabalia ◽

S. M. Udaya Prakash ◽

M. Anaul Kabir

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Resistant Mutant ◽

Wild Type ◽

Human Fungal Pathogen ◽

Zones Of Inhibition ◽

Life Threatening ◽

Fluconazole Resistant ◽

Diffusion Assay ◽

Type Strains

Abstract Background Candida albicans is an opportunistic human fungal pathogen responsible for superficial and systemic life-threatening infections. Treating these infections is challenging as many clinical isolates show increased drug resistance to antifungals. Chromosome (Chr) 4 monosomy was implicated in a fluconazole-resistant mutant. However, exposure to fluconazole adversely affects Candida cells and can generate numerous mutations. Hence, the present study aimed to truncate Chr4 and challenge the generated Candida strains to antifungals and evaluate their role in drug response. Results Herein, Chr4 was truncated in C. albicans using the telomere-mediated chromosomal truncation method. The resulting eight Candida strains carrying one truncated homolog of Chr4 were tested for response to multiple antifungals. The minimal inhibitory concentration (MIC) for these strains was determined against three classes of antifungals. The MIC values against fluconazole, amphotericin B, and caspofungin were closer to that of the wild type strain. Microdilution assay against fluconazole showed that the mutants and wild type strains had similar sensitivity to fluconazole. The disc diffusion assay against five azoles and two polyenes revealed that the zones of inhibition for all the eight strains were similar to those of the wild type. Thus, none of the generated strains showed any significant resistance to the tested antifungals. However, spot assay exhibited a reasonably high tolerance of a few generated strains with increasing concentrations of fluconazole. Conclusion This analysis suggested that Chr4 aneuploidy might not underlie drug resistance but rather drug tolerance in Candida albicans.

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Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00861-07 ◽

2007 ◽

Vol 52 (2) ◽

pp. 694-704 ◽

Cited By ~ 77

Author(s):

Ritu Pasrija ◽

Sneh Lata Panwar ◽

Rajendra Prasad

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Plasma Membranes ◽

Fluorescent Protein ◽

Membrane Lipid ◽

Major Facilitator Superfamily ◽

Pathogenic Yeast ◽

Atp Binding Cassette Transporter ◽

Major Facilitator ◽

Green Fluorescent

ABSTRACT In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called ‘rafts.’ Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (Δsur4 or Δfen1 or Δipt1) or ergosterol (Δerg24 or Δerg6 or Δerg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

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Activation of the Multiple Drug Resistance GeneMDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in atrans-Regulatory Factor

Journal of Bacteriology ◽

10.1128/jb.182.2.400-404.2000 ◽

2000 ◽

Vol 182 (2) ◽

pp. 400-404 ◽

Cited By ~ 68

Author(s):

Stephanie Wirsching ◽

Sonja Michel ◽

Gerwald Köhler ◽

Joachim Morschhäuser

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Multiple Drug Resistance ◽

Regulatory Region ◽

Major Facilitator Superfamily ◽

Selection Marker ◽

Regulatory Factor ◽

Altered Expression ◽

Fluconazole Resistant

ABSTRACT Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicansisolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stableMDR1 activation were analyzed. Sequence analysis of theMDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement oftrans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. ThisMDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPA R developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutiveMDR1 expression.

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Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p

10.1101/2020.06.26.173484 ◽

2020 ◽

Author(s):

Yaojun Tong ◽

Nuo Sun ◽

Xiangming Wang ◽

Qi Wei ◽

Yu Zhang ◽

...

Keyword(s):

Candida Albicans ◽

Multidrug Resistance ◽

Efflux Pump ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Major Facilitator Superfamily ◽

Drug Efflux ◽

Multidrug Efflux Pump ◽

Drug Efflux Pumps ◽

Major Facilitator

AbstractClinical use of antimicrobials faces great challenges from the emergence of multidrug resistant (MDR) pathogens. The overexpression of drug efflux pumps is one of the major contributors to MDR. It is considered as a promising approach to overcome MDR by reversing the function of drug efflux pumps. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to multidrug resistance. Here we report a counterintuitive case of reversing multidrug resistance in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria, and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. It results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time (MST) of mice with a blood-borne dissemination of Mdr1p overexpressed multidrug resistant candidiasis. This study provided a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

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Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2835 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2835-2841 ◽

Cited By ~ 189

Author(s):

G D Albertson ◽

M Niimi ◽

R D Cannon ◽

H F Jenkinson

Keyword(s):

Candida Albicans ◽

Sodium Azide ◽

Resistant Mutant ◽

Major Facilitator Superfamily ◽

Facilitated Diffusion ◽

Drug Efflux ◽

Fluconazole Resistance ◽

Fluconazole Resistant ◽

Major Facilitator ◽

Energy Dependent

Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.

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Multiple cis-Acting Sequences Mediate Upregulation of the MDR1 Efflux Pump in a Fluconazole-Resistant Clinical Candida albicans Isolate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00196-06 ◽

2006 ◽

Vol 50 (7) ◽

pp. 2300-2308 ◽

Cited By ~ 28

Author(s):

Davina Hiller ◽

Stephanie Stahl ◽

Joachim Morschhäuser

Keyword(s):

Candida Albicans ◽

Transcription Factors ◽

Efflux Pump ◽

Regulatory Region ◽

Laboratory Strain ◽

Major Facilitator Superfamily ◽

Metabolic Inhibitors ◽

Multidrug Efflux Pump ◽

Promoter Deletion Analysis ◽

Major Facilitator

ABSTRACT Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.

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Overexpression of Candida albicans CDR1, CDR2, or MDR1 Does Not Produce Significant Changes in Echinocandin Susceptibility

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1148-1155.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1148-1155 ◽

Cited By ~ 92

Author(s):

K. Niimi ◽

K. Maki ◽

F. Ikeda ◽

A. R. Holmes ◽

E. Lamping ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Abc Transporters ◽

Acquired Resistance ◽

Agar Plate ◽

Medium Composition ◽

Major Facilitator Superfamily ◽

Major Facilitator ◽

Mfs Transporters ◽

Increased Susceptibility

ABSTRACT The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.

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Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00853-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4673-4677 ◽

Cited By ~ 24

Author(s):

James J. Vecchione ◽

Blair Alexander ◽

Jason K. Sello

Keyword(s):

Efflux Pump ◽

Streptomyces Coelicolor ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Close Relative ◽

Gram Positive ◽

Antibacterial Drugs ◽

Chloramphenicol Resistance ◽

Drug Activity ◽

Major Facilitator

ABSTRACT Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.

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CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.12.4505-4512.2004 ◽

2004 ◽

Vol 48 (12) ◽

pp. 4505-4512 ◽

Cited By ~ 66

Author(s):

Chia-Geun Chen ◽

Yun-Liang Yang ◽

Hsin-I Shih ◽

Chia-Li Su ◽

Hsiu-Jung Lo

Keyword(s):

Saccharomyces Cerevisiae ◽

Drug Resistance ◽

Candida Albicans ◽

Type Strain ◽

Antifungal Agents ◽

Wild Type Strain ◽

Efflux Pump ◽

Antifungal Drugs ◽

Galactosidase Activity ◽

Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

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