scholarly journals Investigation of the MFS drug transporter family in Candida parapsilosis using CRISPR-Cas9

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Fang Zhao ◽  
Geraldine Butler
Keyword(s):  
Major Facilitator Superfamily ◽  
Open Reading Frames ◽  
Sequencing Analysis ◽  
Fluconazole Resistance ◽  
Multidrug Transporters ◽  
Transporter Family ◽  
Polyamine Transport ◽  
Major Facilitator ◽  
Mfs Transporters ◽  
Type Sequence

The function of specific transporters is a key feature underlying drug resistance in Candida species. Drug transporters fall into two main classes – ATP-binding cassette (ABC) transporters, and the major facilitator superfamily (MFS) transporters. Some members of the drug/H (+) antiporter (DHA1) of the MFS superfamily have been shown to function as multidrug transporters. We targeted 16 genes belonging to five families that compose one branch of the DHA1 transporter group. These include MDR1/FLR1, associated with multidrug resistance in C. albicans (3 members); TPO4, associated with polyamine transport (1 member); NAG3/4, associated with transport of N-acetyl glucosamine (2 members); TPO2/3, associated with polyamine transport (1 member); and TPO1/FLU1, possibly associated with fluconazole resistance (9 members). We used CRISPR-Ca9 based gene editing to explore the function of of the five families in C. parapsilosis. All 16 members were individually disrupted by introducing stop codons in the first third of the open reading frames (editing), or by deleting the whole gene. In addition, members of each family were disrupted together, including all 9 members of the TPO1/FLU1 family. CPAR2_603010, CPAR2_207540, and CPAR2_301760 all belonged to the MDR1 family. Editing CPAR2_603010 conferred sensitivity to fluconazole and voriconazole, though disrupting the other two genes had no effect. The azole sensitivity of the CPAR2_603010 edited strain was reverted by introducing the wild type sequence. Disrupting CPAR2_603010 or CPAR2_301760 individually did not affect sensitivity to 4-nitroquinoline 1-oxide. However, the double disruptant was sensitive. Disrupting CPAR2_300760, a member of the TPO1/FLU1 family, resulted in sensitivity to mycophenolic acid. Whole genome sequencing analysis of a strain in which all nine TPO1 genes were disrupted revealed that few off-target effects introduced by the CRISPR-Cas9 system, as few unexpected changes were found after eight rounds of transformation.

scholarly journals Curcumin Modulates Efflux Mediated by Yeast ABC Multidrug Transporters and Is Synergistic with Antifungals

2009 ◽  
Vol 53 (8) ◽  
pp. 3256-3265 ◽  
Author(s):  
Monika Sharma ◽  
Raman Manoharlal ◽  
Suneet Shukla ◽  
Nidhi Puri ◽  
Tulika Prasad ◽  
...  
Keyword(s):  
Abc Transporters ◽  
Curcuma Longa ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Antifungal Drugs ◽  
Major Facilitator Superfamily ◽  
Multidrug Transporters ◽  
Abc Protein ◽  
Major Facilitator ◽  
Mfs Transporters

ABSTRACT Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa, is a known agent of reversal of drug resistance phenotypes in cancer cells overexpressing ATP-binding cassette (ABC) transporters, viz., ABCB1, ABCG2, and ABCC1. In the present study, we evaluated whether CUR could also modulate multidrug transporters of yeasts that belong either to the ABC family or to the major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was demonstrated by examining rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells overexpressing the Candida albicans ABC transporters Cdr1p and Cdr2p (CaCdr1p and CaCdr2p, respectively) and the MFS transporters CaMdr1p and S. cerevisiae Pdr5p. CUR decreased the extracellular concentration of R6G in ABC transporter-expressing cells but had no effect on methotrexate efflux mediated through the MFS transporter CaMdr1p. CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by [125I]iodoarylazidoprazosin, a drug analogue of the substrate prazosin (50% inhibitory concentration, 14.2 μM). Notably, the mutant variants of CaCdr1p that displayed abrogated efflux of R6G also showed reduced modulation by CUR. Drug susceptibility testing of ABC protein-expressing cells by spot assays and checkerboard tests revealed that CUR was selectively synergistic with drug substrates such as R6G, ketoconazole, itraconazole, and miconazole but not with fluconazole, voriconazole, anisomycin, cycloheximide, or FK520. Taken together, our results provide the first evidence that CUR modulates only ABC multidrug transporters and could be exploited in combination with certain conventional antifungal drugs to reverse multidrug resistance in Candida cells.

scholarly journals The Multidrug Transporters Belonging to Major Facilitator Superfamily (MFS) in Mycobacterium tuberculosis

2002 ◽  
Vol 8 (11) ◽  
pp. 714-724 ◽  
Author(s):  
Edda De Rossi ◽  
Patrizio Arrigo ◽  
Marco Bellinzoni ◽  
Pedro E. A. Silva ◽  
Carlos Martin ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Major Facilitator Superfamily ◽  
Multidrug Transporters ◽  
Major Facilitator

Structure of renal organic anion and cation transporters

2000 ◽  
Vol 278 (6) ◽  
pp. F853-F866 ◽  
Author(s):  
Gerhard Burckhardt ◽  
Natascha A. Wolff
Keyword(s):  
Transmembrane Domain ◽  
Organic Anion ◽  
Major Facilitator Superfamily ◽  
Anion Transporters ◽  
Transporter Family ◽  
Structural And Functional Properties ◽  
Large Extracellular Loop ◽  
Cation Transporters ◽  
Renal Proximal Tubular ◽  
Major Facilitator

Here we review the structural and functional properties of organic anion transporters (OAT1, OAT2, OAT3) and organic cation transporters (OCTN1, OCTN2, OCT1, OCT2, OCT3), some of which are involved in renal proximal tubular organic anion and cation secretion. These transporters share a predicted 12-transmembrane domain (TMD) structure with a large extracellular loop between TMD1 and TMD2, carrying potential N-glycosylation sites. Conserved amino acid motifs revealed a relationship to the sugar transporter family within the major facilitator superfamily. Following heterologous expression, most OATs transported the model anion p-aminohippurate (PAH). OAT1, but not OAT2, exhibited PAH-α-ketoglutarate exchange. OCT1–3 transported the model cations tetraethylammonium (TEA), N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. OCTNs exhibited transport of TEA and/or preferably the zwitterionic carnitine. Substrate substitution as well as cis-inhibition experiments demonstrated polyspecificity of the OATs, OCTs, and OCTN1. On the basis of comparison of the structurally closely related OATs and OCTs, it may be possible to delineate the binding sites for organic anions and cations in future experiments.

scholarly journals No Single Irreplaceable Acidic Residues in the Escherichia coli Secondary Multidrug Transporter MdfA

2006 ◽  
Vol 188 (15) ◽  
pp. 5635-5639 ◽  
Author(s):  
Nadejda Sigal ◽  
Shahar Molshanski-Mor ◽  
Eitan Bibi
Keyword(s):  
Escherichia Coli ◽  
Critical Role ◽  
Energy Coupling ◽  
Major Facilitator Superfamily ◽  
Multidrug Transporter ◽  
Coupling Mechanism ◽  
Acidic Residues ◽  
Major Facilitator ◽  
Mfs Transporters ◽  
Solute Transporters

ABSTRACT The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.

Identification and functional characterization of Penicillium marneffei major facilitator superfamily (MFS) transporters

2020 ◽  
Vol 84 (7) ◽  
pp. 1373-1383
Author(s):  
Setyowati T. Utami ◽  
Carissa I. Indriani ◽  
Anom Bowolaksono ◽  
Takashi Yaguchi ◽  
Xinyue Chen ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Major Facilitator Superfamily ◽  
Penicillium Marneffei ◽  
Major Facilitator ◽  
Mfs Transporters

scholarly journals Inhibitors of the Candida albicans Major Facilitator Superfamily Transporter Mdr1p Responsible for Fluconazole Resistance

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0126350 ◽  
Author(s):  
Mikhail V. Keniya ◽  
Edmond Fleischer ◽  
Anette Klinger ◽  
Richard D. Cannon ◽  
Brian C. Monk
Keyword(s):  
Candida Albicans ◽  
Major Facilitator Superfamily ◽  
Fluconazole Resistance ◽  
Major Facilitator Superfamily Transporter ◽  
Major Facilitator

scholarly journals Characterization of Bacterial Drug Antiporters Homologous to Mammalian Neurotransmitter Transporters

2005 ◽  
Vol 187 (21) ◽  
pp. 7518-7525 ◽  
Author(s):  
Eyal Vardy ◽  
Sonia Steiner-Mordoch ◽  
Shimon Schuldiner
Keyword(s):  
Halobacterium Salinarum ◽  
Major Facilitator Superfamily ◽  
Control Measurement ◽  
Multidrug Transporters ◽  
Bacterial Proteins ◽  
Distinct Cluster ◽  
The Family ◽  
Major Facilitator ◽  
Dependent Transport

ABSTRACT Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.

scholarly journals Conformational Transition Pathways in Major Facilitator Superfamily Transporters

2019 ◽  
Author(s):  
Dylan Ogden ◽  
Kalyan Immadisetty ◽  
Mahmoud Moradi
Keyword(s):  
Conformational Changes ◽  
Glucose Transporter ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Salt Bridge ◽  
Md Simulations ◽  
Major Facilitator Superfamily ◽  
Glucose Transporter 1 ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of MFS superfamily. We have performed a variety of equilibrium, non-equilibrium, biased, and unbiased all-atom molecular dynamics (MD) simulations of bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

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