Inhibitor Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated twenty Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (eight mutants), enniatin B (eight) or beauvericin (four). The twenty mutations were in just nine residues at the centres of transmembrane segment 1 (TMS1) (six mutations), TMS4 (four), TMS5 (four), TMS8 (one) and TMS11 (two) and in A713P (three), a previously reported FK506-resistant ‘hotspot 1’ mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) isolates were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25 resistant mutants were altered in four amino acids: G521R, M639I, A713P and T1355N. These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N (TMS11) possibly cause inhibitor-resistance by altering TMS-contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

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Characterization and Molecular Determinants for β-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium

Antibiotics ◽

10.3390/antibiotics10121494 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1494

Author(s):

Jenifer Cuesta Bernal ◽

Jasmin El-Delik ◽

Stephan Göttig ◽

Klaas M. Pos

Keyword(s):

Substrate Specificity ◽

Salmonella Typhimurium ◽

Efflux Pump ◽

Binding Pocket ◽

Salt Resistance ◽

Drug Binding ◽

Multidrug Efflux Pump ◽

E Coli ◽

Molecular Determinants ◽

Groove Region

Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.

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Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis

Canadian Journal of Microbiology ◽

10.1139/w08-019 ◽

2008 ◽

Vol 54 (5) ◽

pp. 411-416 ◽

Cited By ~ 14

Author(s):

Sanela Begic ◽

Elizabeth A. Worobec

Keyword(s):

Antibiotic Resistance ◽

Substrate Specificity ◽

Serratia Marcescens ◽

Efflux Pump ◽

Gene Knockout ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Active Efflux ◽

Major Mechanism

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance–Nodulation–Cell Division family pump with limited substrate specificity.

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SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03065-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5102-5110 ◽

Cited By ~ 16

Author(s):

Bernardo Ramírez-Zavala ◽

Selene Mogavero ◽

Eva Schöller ◽

Christoph Sasse ◽

P. David Rogers ◽

...

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Efflux Pump ◽

Wild Type ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gain Of Function ◽

Content Type ◽

Zinc Cluster ◽

Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

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Antifungal curcumin induces reactive oxygen species and triggers an early apoptosis but prevents hyphae development by targeting the global repressor TUP1 in Candida albicans

Bioscience Reports ◽

10.1042/bsr20090151 ◽

2010 ◽

Vol 30 (6) ◽

pp. 391-404 ◽

Cited By ~ 73

Author(s):

Monika Sharma ◽

Raman Manoharlal ◽

Nidhi Puri ◽

Rajendra Prasad

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Candida Albicans ◽

Efflux Pump ◽

Reactive Oxygen ◽

Thymidine Uptake ◽

Oxygen Species ◽

Inhibitory Effects ◽

Multidrug Efflux Pump ◽

Early Apoptosis

In the present study, we have investigated the antifungal effects of a natural polyphenol, CUR (curcumin), against albicans and non-albicans species of Candida and have shown its ability to inhibit the growth of all the tested strains. The inhibitory effects of CUR were independent of the status of the multidrug efflux pump proteins belonging to either ABC transporter (ATP-binding cassette transporter) or MFS (major facilitator) superfamilies of transporters. By using a systemic murine model of infection, we established that CUR and piperine, when administered together, caused a significant fungal load reduction (1.4log10) in kidneys of Swiss mice. Additionally, CUR raised the levels of ROS (reactive oxygen species), which, as revealed by annexin V–FITC labelling, triggered early apoptosis in Candida cells. Coincident with the raised ROS levels, mRNAs of tested oxidative stress-related genes [CAP1 (Candida albicans AP-1), CaIPF7817 (putative NADH-dependent flavin oxidoreductase), SOD2 (superoxide dismutase 2), GRP2 (NADPH-dependent methyl glyoxal reductase) and CAT1 (catalase 1)] were also elevated. The growth inhibitory effects of CUR could be reversed by the addition of natural and synthetic antioxidants. Notably, independent of ROS status, polyphenol CUR prevented hyphae development in both liquid and solid hypha-inducing media by targeting the global suppressor TUP1 (thymidine uptake 1). Taken together, our results provide the first evidence that CUR acts as an antifungal agent, via generation of oxidative stress, and inhibits hyphae development by targeting TUP1.

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Crystal Structures of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01134-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 9

Author(s):

Mikhail V. Keniya ◽

Manya Sabherwal ◽

Rajni K. Wilson ◽

Matthew A. Woods ◽

Alia A. Sagatova ◽

...

Keyword(s):

Candida Albicans ◽

Crystal Structures ◽

Candida Glabrata ◽

Fungal Pathogens ◽

Catalytic Domain ◽

Fungal Infections ◽

Binding Pocket ◽

Full Length ◽

Content Type ◽

Cure Rates

ABSTRACT Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

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AcrB Multidrug Efflux Pump of Escherichia coli: Composite Substrate-Binding Cavity of Exceptional Flexibility Generates Its Extremely Wide Substrate Specificity

Journal of Bacteriology ◽

10.1128/jb.185.19.5657-5664.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5657-5664 ◽

Cited By ~ 121

Author(s):

Edward W. Yu ◽

Julio R. Aires ◽

Hiroshi Nikaido

Keyword(s):

Escherichia Coli ◽

Substrate Specificity ◽

Efflux Pump ◽

Substrate Binding ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Composite Substrate ◽

Binding Cavity ◽

Wide Substrate Specificity

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Differential Requirement of the Transcription Factor Mcm1 for Activation of the Candida albicans Multidrug Efflux PumpMDR1by Its Regulators Mrr1 and Cap1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01467-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2061-2066 ◽

Cited By ~ 31

Author(s):

Selene Mogavero ◽

Arianna Tavanti ◽

Sonia Senesi ◽

P. David Rogers ◽

Joachim Morschhäuser

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gain Of Function ◽

Pathogenic Yeast ◽

Content Type

ABSTRACTOverexpression of the multidrug efflux pump Mdr1 causes increased fluconazole resistance in the pathogenic yeastCandida albicans. The transcription factors Mrr1 and Cap1 mediateMDR1upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutiveMDR1overexpression. The essential MADS box transcription factor Mcm1 also binds to theMDR1promoter, but its role in inducible or constitutiveMDR1upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, we investigated the importance of Mcm1 forMDR1expression. We found that Mcm1 was dispensable forMDR1upregulation by H2O2but was required for fullMDR1induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulateMDR1expression both in the presence and in the absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to causeMDR1overexpression. These results demonstrate a differential requirement for the coregulator Mcm1 for Cap1- and Mrr1-mediatedMDR1upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 can induceMDR1expression independently of Mcm1, whereas Mrr1 requires either Mcm1 or an active Cap1 to cause overexpression of theMDR1efflux pump. Our findings provide more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen.

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AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1419939112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3511-3516 ◽

Cited By ~ 91

Author(s):

Jessica M. A. Blair ◽

Vassiliy N. Bavro ◽

Vito Ricci ◽

Niraj Modi ◽

Pierpaolo Cacciotto ◽

...

Keyword(s):

Substrate Specificity ◽

Bacterial Infections ◽

Drug Transport ◽

Efflux Pump ◽

Treatment Strategies ◽

Binding Pocket ◽

Drug Binding ◽

Hydration Properties ◽

Pump System ◽

Increased Susceptibility

The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

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Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525143113 ◽

2016 ◽

Vol 113 (5) ◽

pp. 1405-1410 ◽

Cited By ~ 41

Author(s):

Alfred D. Kinana ◽

Attilio V. Vargiu ◽

Thithiwat May ◽

Hiroshi Nikaido

Keyword(s):

Efflux Pump ◽

Hydrolysis Product ◽

Binding Pocket ◽

Multidrug Resistant ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Short Period ◽

Resistance Nodulation Division ◽

Dynamics Simulations

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.

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