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.

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.

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.

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.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Characterization of hexose transporters in Yarrowia lipolytica reveals new groups of Sugar Porters involved in yeast growth.

Sugar assimilation has been intensively studied in the model yeast S. cerevisiae, and for two decades, it has been clear that the homologous HXT genes, which encode a set of hexose transporters, play a central role in this process. However, in the yeast Yarrowia lipolytica, which is well-known for its biotechnological applications, sugar assimilation is only poorly understood, even though this yeast exhibits peculiar intra-strain differences in fructose uptake: some strains (e.g., W29) are known to be slow-growing in fructose while others (e.g., H222) grow rapidly under the same conditions. Here, we retrieved 24 proteins of the Sugar Porter family from these two strains, and determined that at least six of these proteins can function as hexose transporters in the heterologous host Saccharomyces cerevisiae EBY.VW4000. Transcriptional studies and deletion analysis in Y. lipolytica indicated that two genes, YHT1 and YHT4, are probably the main players in both strains, with a similar role in the uptake of glucose, fructose, and mannose at various concentrations. The other four genes appear to constitute a set of 'reservoir' hexose transporters with an as-yet unclear physiological role. Furthermore, through examining Sugar Porters of the entire Yarrowia clade, we show that they constitute a dynamic family, within which hexose transport genes have been duplicated and lost several times. Our phylogenetic analyses support the existence of at least three distinct evolutionary groups of transporters which allow yeasts to grow on hexoses. In addition to the well-known and widespread Hxt-type transporters (which are not essential in Y. lipolytica), we highlight a second group of transporters, represented by Yht1, which are phylogenetically related to sensors that play a regulatory role in S. cerevisiae, and a third group, represented by Yht4, previously thought to contain only high-affinity glucose transporters related to Hgt1of Kluyveromyces lactis.

MFS transporters of Candida species and their role in clinical drug resistance.

ABC (ATP-binding cassette) and MFS (major facilitator superfamily) exporters, belonging to two different superfamilies, are one of the most prominent contributors of multidrug resistance (MDR) in yeast. While the role of ABC efflux pump proteins in the development of MDR is well documented, the MFS transporters which are also implicated in clinical drug resistance have not received due attention. The MFS superfamily is the largest known family of secondary active membrane carriers, and MFS exporters are capable of transporting a host of substrates ranging from small molecules, including organic and inorganic ions, to complex biomolecules, such as peptide and lipid moieties. A few of the members of the drug/H(+) antiporter family of the MFS superfamily function as multidrug transporters and employ downhill transport of protons to efflux their respective substrates. This review focuses on the recent developments in MFS of Candida and highlights their role in drug transport by using the example of the relatively well characterized promiscuous Mdr1 efflux pump of the pathogenic yeast C. albicans.

A novel major facilitator superfamily transporter in Penicillium digitatum (PdMFS2) is required for prochloraz resistance, conidiation and full virulence.

OBJECTIVES: To clone a novel major facilitator superfamily (MFS, a large protein family with diverse physiological functions in all kingdoms) transporter gene, Pdmfs2, and characterize its function in Penicillium digitatum. RESULTS: A novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. The full-length DNA of Pdmfs2 had a 1590 bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7 µg prochloraz/ml. The deletion of Pdmfs2 (ΔPdmfs2) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value (the concentration of prochloraz producing 50 % growth inhibition) compared with the PdHS-F6 or complementation strain (COPdmfs2). The ΔPdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent. CONCLUSIONS: Pdmfs2 is the second MFS transporter gene in P. digitatum and is required for prochloraz resistance, conidiation and full virulence.

Candida Efflux ATPases and Antiporters in Clinical Drug Resistance.

An enhanced expression of genes encoding ATP binding cassette (ABC) and major facilitator superfamily (MFS) transport proteins are known to contribute to the development of tolerance to antifungals in pathogenic yeasts. For example, the azole resistant (AR) clinical isolates of the opportunistic human fungal pathogen Candida albicans show an overexpression of CDR1 and/or CaMDR1 belonging to ABC and MFS, superfamilies, respectively. The reduced accumulation (due to rapid efflux) of drugs in AR isolates confirms the role of efflux pump proteins in the development of drug tolerance. Considering the importance of major multidrug transporters, the focus of recent research has been to understand the structure and function of these proteins which could help to design inhibitors/modulators of these pump proteins. This chapter focuses on some aspects of the structure and function of yeast transporter proteins particularly in relation to MDR in Candida.

Phylogenetic and syntenic analyses of the 12-spanner drug:H(+) antiporter family 1 (DHA1) in pathogenic Candida species: evolution of MDR1 and FLU1 genes.

Candida albicans and other pathogenic Candida species can develop resistance to clinical fungicides through active drug export mediated by multidrug efflux pumps, in particular by members of the drug:H(+) antiporter family 1 (DHA1). The DHA1 proteins encoded in the genomes of 31 hemiascomycetous strains from 25 species were identified and homology relationships between these proteins and the functionally characterised DHA1 in the model yeast Saccharomyces cerevisiae were established. Gene neighbourhood analysis allowed the reconstruction of sixteen DHA1 lineages conserved during the CTG complex species evolution. The evolutionary history of C. albicans MDR1 and FLU1 genes and Candida dubliniensis, Candida tropicalis and Candida parapsilosis MDR1 genes was detailed. Candida genomes show an abundant number of MDR1 and FLU1 homologues but the chromosome environment where MDR1 homologues reside was poorly conserved during evolution. Gene duplication and loss are major mechanisms underlying the evolution of the DHA1 genes in Candida species.

Transcriptomic and functional analyses on a Botrytis cinerea multidrug-resistant (MDR) strain provides new insights into the potential molecular mechanisms of MDR and fitness.

Botrytis cinerea is a notorious pathogen causing pre- and post-harvest spoilage in many economically important crops. Excessive application of site-specific fungicides to control the pathogen has led to the selection of strains possessing target site alterations associated with resistance to these fungicides and/or strains overexpressing efflux transporters associated with multidrug resistance (MDR). MDR in B. cinerea has been correlated with the overexpression of atrB and mfsM2, encoding an ATP-binding cassette (ABC) and a major facilitator superfamily (MFS) transporter, respectively. However, it remains unknown whether other transporters may also contribute to the MDR phenotype. In the current study, the transcriptome of a B. cinerea multidrug-resistant (MDR) field strain was analysed upon exposure to the fungicide fludioxonil, and compared to the B05.10 reference strain. The transcriptome of this field strain displayed significant differences as compared to B05.10, including genes involved in sugar membrane transport, toxin production and virulence. Among the induced genes in the field strain, even before exposure to fludioxonil, were several putatively encoding ABC and MFS transmembrane transporters. Overexpression of a highly induced MFS transporter gene in the B05.10 strain led to an increased tolerance to the fungicides fluopyram and boscalid, indicating an involvement in efflux transport of these compounds. Overall, the data from this study give insights towards better understanding the molecular mechanisms involved in MDR and fitness cost, contributing to the development of more efficient control strategies against this pathogen.

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.

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.

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.

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.

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.

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.

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.