The dual loss and gain of function of the FPN1 iron exporter results in the ferroportin disease phenotype.

Heterozygous mutations in SLC40A1, encoding a multi-pass membrane protein of the major facilitator superfamily known as ferroportin 1 (FPN1), are responsible for two distinct hereditary iron-overload diseases: ferroportin disease, which is associated with reduced FPN1 activity (i.e., decrease in cellular iron export), and SLC40A1-related hemochromatosis, which is associated with abnormally high FPN1 activity (i.e., resistance to hepcidin). Here, we report three SLC40A1 missense variants with opposite functional consequences. In cultured cells, the p.Arg40Gln and p.Ser47Phe substitutions partially reduced the ability of FPN1 to export iron and also partially reduced its sensitivity to hepcidin. The p.Ala350Val substitution had more profound effects, resulting in low FPN1 iron egress and weak FPN1/hepcidin interaction. Structural analyses helped to differentiate the first two substitutions, which are predicted to cause local instabilities, and the third, which is thought to prevent critical rigid-body movements that are essential to the iron transport cycle. The phenotypic traits observed in a total of 12 affected individuals are highly suggestive of ferroportin disease. Our findings dismantle the classical dualism of FPN1 loss versus gain of function, highlight some specific and unexpected functions of FPN1 transmembrane helices in the molecular mechanism of iron export and its regulation by hepcidin, and extend the spectrum of rare genetic variants that may cause ferroportin disease.

Insights into the role of glycerophospholipids on the iron export function of SLC40A1 and the molecular mechanisms of ferroportin disease.

SLC40A1 is the sole iron export protein reported in mammals. In humans, its dysfunction is responsible for ferroportin disease, an inborn error of iron metabolism transmitted as an autosomal dominant trait and observed in different ethnic groups. As a member of the major facilitator superfamily, SLC40A1 requires a series of conformational changes to enable iron translocation across the plasma membrane. The influence of lipids on protein stability and its conformational changes has been little investigated to date. Here, we combine molecular dynamics simulations of SLC40A1 embedded in membrane bilayers with experimental alanine scanning mutagenesis to analyze the specific role of glycerophospholipids. We identify four basic residues (Lys90, Arg365, Lys366, and Arg371) that are located at the membrane-cytosol interface and consistently interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) molecules. These residues surround a network of salt bridges and hydrogens bonds that play a critical role in stabilizing SLC40A1 in its basal outward-facing conformation. More deeply embedded in the plasma membrane, we identify Arg179 as a charged amino acid residue also tightly interacting with lipid polar heads. This results in a local deformation of the lipid bilayer. Interestingly, Arg179 is adjacent to Arg178, which forms a functionally important salt-bridge with Asp473 and is a recurrently associated with ferroportin disease when mutated to glutamine. We demonstrate that the two p.Arg178Gln and p.Arg179Thr missense variants have similar functional behaviors. These observations provide insights into the role of phospholipids in the formation/disruption of the SLC40A1 inner gate, and give a better understanding of the diversity of molecular mechanisms of ferroportin disease.

Multiple and multidrug resistance in Botrytis cinerea: molecular mechanisms of MLR/MDR strains in Greece and effects of co-existence of different resistance mechanisms on fungicide sensitivity.

Botrytis cinerea is a high-risk pathogen for fungicide resistance development. Within the fungal populations, strains have developed multiple mutations in different target genes leading to multiple resistance (MLR) or mutations associated with overexpression of efflux transporters leading to multidrug resistance (MDR). These types of resistance are a major threat, and their successful management is a major challenge. The current study was initiated to a) determine frequencies of MLR/MDR strains in populations originating from several crops, b) identify the types of MDR that occur in Greece, and c) determine interactions between MLR and MDR at the level of sensitivity to botryticides. The frequencies of MLR/MDR phenotypes were determined in 515 isolates subjected to bioassays using discriminatory concentrations of thiophanate-methyl, iprodione, cyprodinil, fenhexamid, boscalid, fluopyram, fludioxonil, pyraclostrobin, and tolnaftate. Interestingly, 7.8% and 31.3% of isolates from strawberry and rootstock seedlings were resistant to every single fungicide class, while MDR phenotypes from strawberries, rootstocks, and tomatoes accounted for 26%, 87%, and 13.4%, respectively. The MLR and MDR isolates were further molecularly analyzed regarding genes erg27, sdhB, Bcpos5, and Mrr1, responsible for resistance to fenhexamid, boscalid and fluopyram, cyprodinil, and MDR, respectively. The different mutations' presence was determined along with a new mutation in Mrr1 leading to MDR. MDR isolates were characterized as MDR1 or MDR1h based on the presence of a 3-bp deletion in Mrr1. MDR1h was predominant in isolates from rootstocks and MDR1 from tomatoes and strawberries, whereas the most frequent target-site mutations were F412S (erg27), H272R (sdhB), and L412F (Bcpos5). To determine whether the accumulation of target-site mutations along with MDR mutations exhibits an additive effect concerning fungicide resistance, the sensitivity of isolates possessing the predominant target-site mutations was calculated in both the presence and the absence of MDR-associated mutations. EC50 in cyprodinil and boscalid increased to about twofold in the presence of MDR mutations, while there was no difference for fenhexamid. In conclusion, MLR/MDR frequencies are notably high in heavily treated crops in Greece, and the combination of MLR and MDR mutations leads to even higher fungicide resistance levels, highlighting the importance of resistance management.

Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Do mitochondria use efflux pumps to protect their ribosomes from antibiotics?

Fungal environments are rich in natural and engineered antimicrobials, and this, combined with the fact that fungal genomes are rich in coding sequences for transporters, suggests that fungi are an intriguing group in which to search for evidence of antimicrobial efflux pumps in mitochondria. Herein, the range of protective mechanisms used by fungi against antimicrobials is introduced, and it is hypothesized, based on the susceptibility of mitochondrial and bacterial ribosomes to the same antibiotics, that mitochondria might also contain pumps that efflux antibiotics from these organelles. Preliminary evidence of ethidium bromide efflux is presented and several candidate efflux pumps are identified in fungal mitochondrial proteomes.

Mutagenesis and functional analysis of SotB: A multidrug transporter of the major facilitator superfamily from Escherichia coli.

Dysfunction of the major facilitator superfamily multidrug (MFS Mdr) transporters can lead to a variety of serious diseases in human. In bacteria, such membrane proteins are often associated with bacterial resistance. However, as one of the MFS Mdr transporters, the physiological function of SotB from Escherichia coli is poorly understood to date. To better understand the function and mechanism of SotB, a systematic study on this MFS Mdr transporter was carried out. In this study, SotB was found to directly efflux L-arabinose in E. coli by overexpressing sotB gene combined with cell based radiotracer uptake assay. Besides, the surface plasmon resonance (SPR) studies, the L-arabinose inhibition assays, together with precise molecular docking analysis, reveal the following: (i) the functional importance of E29 (protonation), H115/N343 (substrate recognition), and W119/S339 (substrate efflux) in the SotB mediated export of L-arabinose, and (ii) for the first time find that D-xylose, an isomer of L-arabinose, likely hinders the binding of L-arabinose with SotB as a competitive inhibitor. Finally, by analyzing the structure of SotB2 (shares 62.8% sequence similarity with SotB) predicted by AlphaFold 2, the different molecular mechanism of substrate recognition between SotB and SotB2 is explained. To our knowledge, this is the first systematic study of MFS Mdr transporter SotB. The structural information, together with the biochemical inspections in this study, provide a valuable framework for further deciphering the functional mechanisms of the physiologically important L-arabinose transporter SotB and its family.

Genomic landscape of the DHA1 family in Candida auris and mapping substrate repertoire of CauMdr1.

The last decade has witnessed the rise of an extremely threatening healthcare-associated multidrug-resistant non-albicans Candida (NAC) species, Candida auris. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the major facilitator superfamily (MFS) of efflux pumps, drug/H+ antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against most of the tested substrates, the ortholog when overexpressed in a minimal background Saccharomyces cerevisiae strain, AD1-8u-, showed significant enhancement in the minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms. KEY POINTS: • Fourteen putative DHA1 transporters are encoded in the Candida auris genome. • Deletion of the CauMDR1 gene does not lead to major changes in drug resistance. • CauMdr1 recognizes and effluxes numerous xenobiotics, including prominent azoles.

Improving Drug Sensitivity of HIV-1 Protease Inhibitors by Restriction of Cellular Efflux System in a Fission Yeast Model.

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

A State-of-the-art Review and Prospective Therapeutic Applications of Prenyl Flavonoids as Chemosensitizers against Antifungal Multidrug Resistance in Candida albicans.

Multidrug resistance (MDR) in the opportunistic pathogen Candida albicans is defined as non-susceptibility to at least one agent in two or more drug classes. This phenomenon has been increasingly reported since the rise in the incidence of fungal infections in immunocompromised patients at the end of the last century. After the discovery of efflux pump overexpression as a principal mechanism causing MDR in Candida strains, drug discovery targeting fungal efflux transporters has had a growing impact. Chemosensitization aims to enhance azole intracellular concentrations through combination therapy with transporter inhibitors. Consequently, the use of drug efflux inhibitors combined with the antifungal agent will sensitize the pathogen. As a result, the use of lower drug concentrations will reduce possible adverse effects on the host. Through an extensive revision of the literature, this review aims to provide an exhaustive and critical analysis of the studies carried out in the past two decades regarding the chemosensitization strategy to cope with multidrug resistance in C. albicans. This work provides a deep analysis of the research on the inhibition of drug-efflux membrane transporters by prenylated flavonoids and the interactions of these phytocompounds with azole antifungals as an approach to chemosensitize multidrug-resistant C. albicans strains. We highlight the importance of prenylflavonoids and their particular chemical and pharmacological characteristics that make them excellent candidates with therapeutic potential as chemosensitizers. Finally, we propose the need for further research on prenyl flavonoids as inhibitors of drug-efflux mediated fungal resistance.

MFS1, a Pleiotropic Transporter in Dermatophytes That Plays a Key Role in Their Intrinsic Resistance to Chloramphenicol and Fluconazole.

A recently identified Trichophyton rubrum major facilitator superfamily (MFS)-type transporter (TruMFS1) has been shown to give resistance to azole compounds and cycloheximide (CYH) when overexpressed in Saccharomyces cerevisiae. We investigated the roles of MFS1 in the intrinsic resistance of dermatophytes to CYH and chloramphenicol (CHL), which are commonly used to isolate these fungi, and to what extent MFS1 affects the susceptibility to azole antifungals. Susceptibility to antibiotics and azoles was tested in S. cerevisiae overexpressing MFS1 and ΔMFS1 mutants of Trichophyton benhamiae, a dermatophyte that is closely related to T. rubrum. We found that TruMFS1 functions as an efflux pump for CHL in addition to CYH and azoles in S. cerevisiae. In contrast, the growth of T. benhamiae ΔMFS1 mutants was not reduced in the presence of CYH but was severely impaired in the presence of CHL and thiamphenicol, a CHL analog. The suppression of MFS1 in T. benhamiae also increased the sensitivity of the fungus to fluconazole and miconazole. Our experiments revealed a key role of MFS1 in the resistance of dermatophytes to CHL and their high minimum inhibitory concentration for fluconazole. Suppression of MFS1 did not affect the sensitivity to CYH, suggesting that another mechanism was involved in resistance to CYH in dermatophytes.

Insights into the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, the pathogen Botrytis cinerea and their plant host.

Botrytis cinerea is a plant pathogen causing the gray mold disease in a plethora of host plants. The control of the disease is based mostly on chemical pesticides, which are responsible for environmental pollution, while they also pose risks for human health. Furthermore, B. cinerea resistant isolates have been identified against many fungicide groups, making the control of this disease challenging. The application of biocontrol agents can be a possible solution, but requires deep understanding of the molecular mechanisms in order to be effective. In this study, we investigated the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, a new commercialized biopesticide, the pathogen B. cinerea and their plant host. Our analysis showed that this biocontrol agent reduced B. cinerea mycelial growth in vitro, and was able to suppress the disease incidence on cucumber plants. Moreover, treatment with B. subtilis led to induction of genes involved in plant immunity. RNA-seq analysis of B. cinerea transcriptome upon exposure to bacterial secretome, showed that genes coding for MFS and ABC transporters were highly induced. Deletion of the Bcmfs1 MFS transporter gene, using a CRISP/Cas9 editing method, affected its virulence and the tolerance of B. cinerea to bacterial secondary metabolites. These findings suggest that specific detoxification transporters are involved in these interactions, with crucial role in different aspects of B. cinerea physiology.

Directed Mutational Strategies Reveal Drug Binding and Transport by the MDR Transporters of Candida albicans.

Multidrug resistance (MDR) transporters belonging to either the ATP-Binding Cassette (ABC) or Major Facilitator Superfamily (MFS) groups are major determinants of clinical drug resistance in fungi. The overproduction of these proteins enables the extrusion of incoming drugs at rates that prevent lethal effects. The promiscuity of these proteins is intriguing because they export a wide range of structurally unrelated molecules. Research in the last two decades has used multiple approaches to dissect the molecular basis of the polyspecificity of multidrug transporters. With large numbers of drug transporters potentially involved in clinical drug resistance in pathogenic yeasts, this review focuses on the drug transporters of the important pathogen Candida albicans. This organism harbors many such proteins, several of which have been shown to actively export antifungal drugs. Of these, the ABC protein CaCdr1 and the MFS protein CaMdr1 are the two most prominent and have thus been subjected to intense site-directed mutagenesis and suppressor genetics-based analysis. Numerous results point to a common theme underlying the strategy of promiscuity adopted by both CaCdr1 and CaMdr1. This review summarizes the body of research that has provided insight into how multidrug transporters function and deliver their remarkable polyspecificity.

Synergistic interactions between ß-lapachone and fluconazole in the inhibition of CaCdr2p and CaMdr1p in Candida albicans.

BACKGROUND: Mortality rate of invasive Candida infections is raising mainly amongst immunocompromised patients. These infections are hard-to-treat mainly due to the increasing incidence of resistance. The overexpression of ATP-binding cassette and major facilitator superfamily transporters is the main responsible for the failure of antifungal therapies. In a Saccharomyces cerevisiae model, ß-lapachone inhibited Pdr5p, a transporter homologous to those found in Candida albicans. AIMS: To determine whether ß-lapachone reverses the resistance phenotype mediated by efflux transporters in C. albicans clinical isolates. METHODS: The antifungal activity of ß-lapachone combined with fluconazole was measured by agarose chemosensitization and microdilution assays. CaCdr2p and CaMdr1p activities were evaluated through fluorescent dyes accumulation. ATPase activity was assessed using transporter-enriched plasma membranes. RESULTS: ß-lapachone reverted antifungal resistance of S. cerevisiae and C. albicans strains overexpressing CaCdr2p and CaMdr1p transporters by inhibiting these proteins activities. CaCdr2p ATPase activity was not impaired by the compound. CONCLUSIONS: ß-lapachone is a promising drug candidate to be used as an adjuvant in the treatment of candidiasis caused by fluconazole-resistant C. albicans strains.

Multidrug resistance of Penicillium expansum to fungicides: whole transcriptome analysis of MDR strains reveals overexpression of efflux transporter genes.

Penicillium expansum is the most common apple fruit postharvest spoilage agent that causes a disease known as Blue Mold. Disease control is based on fungicide use. However, development of resistance to fungicides hampers the success of this control method. Fungicide sensitivity monitoring studies in Greece revealed the presence of pathogen strains exhibiting simultaneous resistance to different chemically unrelated compounds (multidrug resistance, MDR). This study was initiated aiming primarily to test the hypothesis that the MDR phenotype is associated with overexpression of efflux transporter genes and to determine the fitness of the MDR isolates. The monitoring study (n = 264) and the measurements of sensitivity in terms of EC50 values to 9 different compounds revealed that almost 5% of the population was of the MDR type. In the selected MDR isolates, the highest resistant factors were calculated for fludioxonil and pyraclostrobin, while the same isolates were moderately resistant to cyprodinil, thiophanate methyl and fluxapyroxad. In the resistant strains no target site mutations were detected in the target genes of each fungicide class, while in addition, a synergistic activity was observed between fungicides and the drug transporter modulator verapamil in some isolates. To obtain a direct insight on the resistance mechanism, the transcriptome of 2 MDR and 1 sensitive isolates was sequenced using Illumina HiSeq 2500 and differences in efflux transporter gene expression profile were figured out. Gene expression profiling analysis was performed before and after the exposure of fungal mycelia to fludioxonil. This analysis revealed the up-regulation of several MFS transporter genes and a limited number of ABC transporter genes either before or after the exposure to fludioxonil in the MDR isolates. Expression results for genes with the highest expression levels were verified by qRT-PCR assays. Fitness components measurements revealed that MDR isolates were of lower mycelial growth and pathogenicity compared to sensitive strains but they were producing higher number of conidia. The above mentioned data represent the first report of MDR in P. expansum associated with overexpression of drug efflux transporters and contribute to our knowledge in the mechanisms associated with fungicide resistance development in this fungal species.

Transport systems, intracellular traffic of intermediates and secretion of ß-lactam antibiotics in fungi.

Fungal secondary metabolites are synthesized by complex biosynthetic pathways catalized by enzymes located in different subcellular compartments, thus requiring traffic of precursors and intermediates between them. The ß-lactam antibiotics penicillin and cephalosporin C serve as an excellent model to understand the molecular mechanisms that control the subcellular localization of secondary metabolites biosynthetic enzymes. Optimal functioning of the ß-lactam biosynthetic enzymes relies on a sophisticated temporal and spatial organization of the enzymes, the intermediates and the final products. The first and second enzymes of the penicillin pathway, ACV synthetase and IPN synthase, in Penicillium chrysogenum and Aspergillus nidulans are cytosolic. In contrast, the last two enzymes of the penicillin pathway, phenylacetyl-CoA ligase and isopenicillin N acyltransferase, are located in peroxisomes working as a tandem at their optimal pH that coincides with the peroxisomes pH. Two MFS transporters, PenM and PaaT have been found to be involved in the import of the intermediates isopenicillin N and phenylacetic acid, respectively, into peroxisomes. Similar compartmentalization of intermediates occurs in Acremonium chrysogenum; two enzymes isopenicillin N-CoA ligase and isopenicillin N-CoA epimerase, that catalyse the conversion of isopenicillin N in penicillin N, are located in peroxisomes. Two genes encoding MFS transporters, cefP and cefM, are located in the early cephalosporin gene cluster. These transporters have been localized in peroxisomes by confocal fluorescence microscopy. A third gene of A. chrysogenum, cefT, encodes an MFS protein, located in the cell membrane involved in the secretion of cephalosporin C, although cefT-disrupted mutants are still able to export cephalosporin by redundant transporters. The secretion of penicillin from peroxisomes to the extracellular medium is still unclear. Attempts have been made to identify a gene encoding the penicillin secretion protein among the 48 ABC-transporters of P. chrysogenum. The highly efficient secretion system that exports penicillin against a concentration gradient may involve active penicillin extrusion systems mediated by vesicles that fuse to the cell membrane. However, there is no correlation of pexophagy with penicillin or cephalosporin formation since inactivation of pexophagy leads to increased penicillin or cephalosporin biosynthesis due to preservation of peroxisomes. The penicillin biosynthesis finding shows that in order to increase biosynthesis of novel secondary metabolites it is essential to adequately target enzymes to organelles.

Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

Ferroportin 1 (FPN1) is a major facilitator superfamily transporter that is essential for proper maintenance of human iron homeostasis at the systemic and cellular level. FPN1 dysfunction leads to the progressive accumulation of iron in reticuloendothelial cells, causing hemochromatosis type 4A (or ferroportin disease), an autosomal dominant disorder that displays large phenotypic heterogeneity. Although crystal structures have unveiled the outward- and inward-facing conformations of the bacterial homolog Bdellovibrio bacteriovorus Fpn (or Bd2019) and calcium has recently been identified as an essential cofactor, our molecular understanding of the iron transport mechanism remains incomplete. Here, we used a combination of molecular modeling, molecular dynamics simulations, and Ala site-directed mutagenesis, followed by complementary in vitro functional analyses, to explore the structural architecture of the human FPN1 intracellular gate. We reveal an interdomain network that involves 5 key amino acids and is likely very important for stability of the iron exporter facing the extracellular milieu. We also identify inter- and intradomain interactions that rely on the 2 Asp84 and Asn174 critical residues and do not exist in the bacterial homolog. These interactions are thought to play an important role in the modulation of conformational changes during the transport cycle. We interpret these results in the context of hemochromatosis type 4A, reinforcing the idea that different categories of loss-of-function mutations exist. Our findings provide an unprecedented view of the human FPN1 outward-facing structure and the particular function of the so-called "gating residues" in the mechanism of iron export.-Guellec, J., Elbahnsi, A., Le Tertre, M., Uguen, K., Gourlaouen, I., Férec, C., Ka, C., Callebaut, I., Le Gac, G. Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

Trichophyton rubrum Azole Resistance Mediated by a New ABC Transporter, TruMDR3.

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.

More Transporters, More Substrates: The Arabidopsis Major Facilitator Superfamily Revisited.

The Major Facilitator Superfamily (MFS) is ubiquitous in living organisms and represents the largest group of secondary active membrane transporters. In plants, significant research efforts have focused on the role of specific families within the MFS, particularly those transporting macronutrients (C, N, and P) that constitute the vast majority of the members of this superfamily. Other MFS families remain less explored, although a plethora of additional substrates and physiological functions have been uncovered. Nevertheless, the lack of a systematic approach to analyzing the MFS as a whole has obscured the high diversity and versatility of these transporters. Here, we present a phylogenetic analysis of all annotated MFS domain-containing proteins encoded in the Arabidopsis thaliana genome and propose that this superfamily of transporters consists of 218 members, clustered in 22 families. In reviewing the available information regarding the diversity in biological functions and substrates of Arabidopsis MFS members, we provide arguments for intensified research on these membrane transporters to unveil the breadth of their physiological relevance, disclose the molecular mechanisms underlying their mode of action, and explore their biotechnological potential.

Efflux pump as alternate mechanism for drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) remains an important global public health issue with an approximate prevalence of 10 million people with TB worldwide in 2015. Since antibiotic treatment is one of the foremost tools for TB control, knowledge of Mycobacterium tuberculosis (MTB) drug resistance is an important component for disease control. Although gene mutations in specific loci of the MTB genomes are reported as the primary basis for drug resistance, additional mechanisms conferring resistance to MTB are thought to exist. Efflux is a ubiquitous mechanism responsible for innate and acquired drug resistance in prokaryotic and eukaryotic cells. MTB presents a large number of putative drug efflux pumps compared to its genome size. Bioinformatics-based evidence has shown an association between drug efflux and innate or acquired resistance in MTB. This review describes the recent understanding of drug efflux in MTB.