Molecular mechanisms associated with the resistance of Rhizoctonia solani AG-4 isolates to the succinate dehydrogenase inhibitor, thifluzamide

2021 ◽  
Author(s):  
Can Zhao ◽  
Yuting Li ◽  
Zhijian Liang ◽  
Lihong Gao ◽  
Chenggui Han ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Molecular Mechanisms ◽  
Major Facilitator Superfamily ◽  
Cross Resistance ◽  
Active Efflux ◽  
Fitness Evaluation ◽  
Reverse Transcription Pcr ◽  
Major Facilitator ◽  
Median Effective Concentration

Thifluzamide, a succinate dehydrogenase (SDH) inhibitor, possesses high activity against Rhizoctonia. In this study, 144 R. solani AG-4 (4HGI, 4HGII, and 4HGIII) isolates, the predominate pathogen associated with sugar beet seedling damping-off, were demonstrated to be sensitive to thifluzamide with a calculated mean median effective concentration of 0.0682 ± 0.0025 μg/mL. Thifluzamide-resistant isolates were generated using fungicide-amended media, resulting in four AG-4HGI isolates and eight AG-4HGII isolates with stable resistance and almost no loss in fitness. Evaluation of cross-resistance of the twelve thifluzamide-resistant isolates and their corresponding parental-sensitive isolates revealed a moderately positive correlation between thifluzamide resistance and the level of resistance to eight other fungicides from three groups, the exception being fludioxonil. An active efflux of fungicide through ATP-binding cassette and major facilitator superfamily transporters was found to be correlated to the resistance of R. solani AG-4HGII isolates to thifluzamide based on RNA-sequencing and quantitative reverse transcription-PCR analyses. Sequence analysis of sdhA, sdhB, sdhC, and sdhD revealed replacement of isoleucine by phenylalanine at position 61 in SDHC in nine of the twelve generated thifluzamide-resistant isolates. No other mutations were found in any of the other genes. Collectively, the data indicate that the active efflux of fungicide and a point mutation in sdhC may contribute to the resistance of R. solani AG-4HGI and AG-4HGII isolates to thifluzamide in vitro. This is the first characterization of the potential molecular mechanism associated with the resistance of R. solani AG-4 isolates to thifluzamide, and provides practical guidance for the use of this fungicide.

scholarly journals The BaeSR Two-Component Regulatory System Activates Transcription of the yegMNOB (mdtABCD) Transporter Gene Cluster in Escherichia coli and Increases Its Resistance to Novobiocin and Deoxycholate

2002 ◽  
Vol 184 (15) ◽  
pp. 4168-4176 ◽  
Author(s):  
Natalya Baranova ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Regulatory System ◽  
Major Facilitator Superfamily ◽  
Multicopy Plasmid ◽  
Active Efflux ◽  
Membrane Fusion Protein ◽  
Two Component ◽  
Major Facilitator

ABSTRACT Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible ΔacrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the ΔacrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.

scholarly journals Structural and Functional Study of the Phenicol-Specific Efflux Pump FloR Belonging to the Major Facilitator Superfamily

2005 ◽  
Vol 49 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
Martine Braibant ◽  
Jacqueline Chevalier ◽  
Elisabeth Chaslus-Dancla ◽  
Jean-Marie Pagès ◽  
Axel Cloeckaert
Keyword(s):  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Efflux Transporters ◽  
Chloramphenicol Resistance ◽  
Active Efflux ◽  
Transmembrane Segments ◽  
Efflux Mechanism ◽  
Major Facilitator

ABSTRACT The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

The mycobacterial efflux pump EfpA can induce high drug tolerance to many anti-tuberculosis drugs, including moxifloxacin, in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Deepika Rai ◽  
Sarika Mehra
Keyword(s):  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Drug Tolerance ◽  
Inducible Promoter ◽  
Major Facilitator Superfamily ◽  
Expression Level ◽  
Efflux Pump Inhibitor ◽  
Gene Encoding ◽  
Active Efflux ◽  
Major Facilitator

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump EfpA, from the major facilitator superfamily, that is highly conserved among both slow growing and fast-growing mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis . The gene encoding EfpA from Mycobacterium smegmatis was over-expressed under both constitutive and an inducible promoter. Expression of efpA gene under both the promoters resulted in greater than 32-fold increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid and streptomycin) and second-line (amikacin) anti-tuberculosis drugs. Notably, drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was over-expressed. The increase in minimum inhibitory concentration (MIC) correlated with the decreased uptake of drugs including norfloxacin, moxifloxacin and ethidium bromide and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MIC of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose.The efpA gene is conserved across both pathogenic and non-pathogenic mycobacteria. The efpA gene from the Mycobacterium bovis BCG/ M. tuberculosis , which is 80% identical to efpA from M. smegmatis , also led to decreased antimicrobial efficacy to many drugs, although the fold-change was lower. When over-expressed in M. bovis BCG, an 8-fold higher drug tolerance to moxifloxacin was observed . This is the first report of an efflux pump from mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

scholarly journals Transcriptomic Evidence of Molecular Mechanisms Underlying the Response of Lactobacillus plantarum WCFS1 to Hydroxytyrosol

Antioxidants ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 442
Author(s):  
Inés Reverón ◽  
Laura Plaza-Vinuesa ◽  
Laura Santamaría ◽  
Juan Carlos Oliveros ◽  
Blanca de las Rivas ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Antioxidant Response ◽  
Major Facilitator Superfamily ◽  
Subcellular Compartment ◽  
Cell Wall Biogenesis ◽  
Expression Of Genes ◽  
Major Facilitator ◽  
Genome Scale

This study was aimed to gain new insights into the molecular mechanisms used by Lactobacillus plantarum WCFS1 to respond to hydroxytyrosol (HXT), one of the main and health-relevant plant phenolics present in olive oil. To this goal, whole genome transcriptomic profiling was used to better understand the contribution of differential gene expression in the adaptation to HXT by this microorganism. The transcriptomic profile reveals an HXT-triggered antioxidant response involving genes from the ROS (reactive oxygen species) resistome of L. plantarum, genes coding for H2S-producing enzymes and genes involved in the response to thiol-specific oxidative stress. The expression of a set of genes involved in cell wall biogenesis was also upregulated, indicating that this subcellular compartment was a target of HXT. The expression of several MFS (major facilitator superfamily) efflux systems and ABC-transporters was differentially affected by HXT, probably to control its transport across the membrane. L. plantarum transcriptionally reprogrammed nitrogen metabolism and involved the stringent response (SR) to adapt to HXT, as indicated by the reduced expression of genes involved in cell proliferation or related to the metabolism of (p)ppGpp, the molecule that triggers the SR. Our data have identified, at genome scale, the antimicrobial mechanisms of HXT action as well as molecular mechanisms that potentially enable L. plantarum to cope with the effects of this phenolic compound.

scholarly journals Antimicrobial Activity of a Library of Thioxanthones and Their Potential as Efflux Pump Inhibitors

2021 ◽  
Vol 14 (6) ◽  
pp. 572
Author(s):  
Fernando Durães ◽  
Andreia Palmeira ◽  
Bárbara Cruz ◽  
Joana Freitas-Silva ◽  
Nikoletta Szemerédi ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Mammalian Cells ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Major Facilitator Superfamily ◽  
Design And Synthesis ◽  
Resistance Nodulation Division ◽  
Efflux Pump Inhibitors ◽  
Major Facilitator

The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 µM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation.

scholarly journals γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

2015 ◽  
Vol 81 (21) ◽  
pp. 7656-7665 ◽  
Author(s):  
Daisuke Kasai ◽  
Naoto Araki ◽  
Kota Motoi ◽  
Shota Yoshikawa ◽  
Toju Iino ◽  
...  
Keyword(s):  
Major Facilitator Superfamily ◽  
Pcr Analysis ◽  
Decarboxylase Activity ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Intergenic Regions ◽  
Major Facilitator Superfamily Transporter ◽  
Maleylacetate Reductase ◽  
Major Facilitator ◽  
Rhodococcus Jostii

ABSTRACTTheRhodococcus jostiiRHA1 gene cluster required for γ-resorcylate (GRA) catabolism was characterized. The cluster includestsdA,tsdB,tsdC,tsdD,tsdR,tsdT, andtsdX, which encode GRA decarboxylase, resorcinol 4-hydroxylase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, an IclR-type regulator, a major facilitator superfamily transporter, and a putative hydrolase, respectively. ThetsdAgene conferred GRA decarboxylase activity onEscherichia coli. Purified TsdB oxidized NADH in the presence of resorcinol, suggesting thattsdBencodes a unique NADH-specific single-component resorcinol 4-hydroxylase. Mutations in eithertsdAortsdBresulted in growth deficiency on GRA. ThetsdCandtsdDgenes conferred hydroxyquinol 1,2-dioxygenase and maleylacetate reductase activities, respectively, onE. coli. Inactivation oftsdTsignificantly retarded the growth of RHA1 on GRA. The growth retardation was partially suppressed under acidic conditions, suggesting the involvement oftsdTin GRA uptake. Reverse transcription-PCR analysis revealed that thetsdgenes constitute three transcriptional units, thetsdBADCandtsdTXoperons andtsdR. Transcription of thetsdBADCandtsdTXoperons was induced during growth on GRA. Inactivation oftsdRderepressed transcription of thetsdBADCandtsdTXoperons in the absence of GRA, suggesting thattsdgene transcription is negatively regulated by thetsdR-encoded regulator. Binding of TsdR to thetsdR-tsdBandtsdT-tsdRintergenic regions was inhibited by the addition of GRA, indicating that GRA interacts with TsdR as an effector molecule.

Novel DNA-Damaging Linker Payloads Are Active in Models of Acquired Resistance to Calicheamicin and Standard of Care

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3522-3522
Author(s):  
Jennifer Kahler ◽  
Maureen Dougher ◽  
Jia Lu ◽  
Jane Xu ◽  
Tasneem Kausar ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Crosslinking Agent ◽  
Standard Of Care ◽  
Cross Resistance ◽  
Continuous Exposure ◽  
Next Generation ◽  
Multidrug Resistance Proteins ◽  
Clinical Resistance

Abstract Gemtuzumab ozogamicin (GO) is an FDA-approved antibody-drug conjugate that has shown clinical benefit in acute myeloid leukemia (AML) patients but has been voluntarily withdrawn from the market in the US. GO targets CD33 on AML tumor cells and delivers its cytotoxic payload, namely calicheamicin. Despite promising initial responses to GO, many patients relapsed while on therapy, and the development of clinical resistance to GO hampers its effectiveness. Initial investigations into the mechanisms responsible for clinical resistance to GO implicated the activity of multidrug resistance proteins (MDR) as a major contributor to the lack of clinical efficacy. To better understand the molecular mechanisms that may drive resistance to the payload used in GO, as well as standard of care agents in AML, we created calicheamicin-resistant and cytarabine-resistant AML cell lines. HL60 cells were continuously exposed to increasing concentrations of calicheamicin or cytarabine until resistant populations emerged. Calicheamicin-resistant HL60 (HL60-CAL-R) cells are ~40-fold resistant to calicheamicin and cytarabine-resistant HL60 (HL60-CYTAR-R) are ~2000- fold resistant to cytarabine compared to their isogenic parental counterparts as determined by in vitro cytotoxicity assays. Initial model characterization of HL60-CAL-R suggests that continuous exposure to calicheamicin induces the acquisition of a chemo-resistant phenotype highlighted by increased expression of MDR1 and MRP1/2. Furthermore, functional assays by flow cytometry revealed high levels of efflux activity in MDR1 and MRP1/2, but not BCRP, in HL60-CAL-R as compared to parental HL60. HL60-CAL-R could be re-sensitized to calicheamicin with the addition of verapamil (MDR1 inhibitor) or reversan (MDR1 and MRP1/2 inhibitor). HL60-CAL-R shows cross-resistance to daunorubicin (MDR1 substrate), but not cytarabine (non-MDR1 substrate), supporting MDR1 upregulation as one major mechanism of resistance to calicheamicin in this cell line. HL60-CAL-R cells are also cross-resistant to GO. In contrast, HL60-CYTAR-R do not have an induction of MDR1 and MRP1/2 protein levels or activity and retain sensitivity to GO in vitro and in vivo. Initial RNA-Seq profiling of HL60-CYTAR-R cells suggest they have decreased DCK expression, the loss of which was previously shown to mediate cytarabine resistance in other models. Intriguingly, HL60-CAL-R and HL60-CYTAR-R cells retained sensitivity to a proprietary next-generation DNA alkylating- and crosslinking- agent being developed at Pfizer, cyclopropylpyrrolo[e]indolone (CPI). These data demonstrate the utility of generating and characterizing drug-resistant cell lines to uncover clinically relevant mechanisms of resistance and identify next-generation compounds that can overcome them. Disclosures Kahler: Pfizer: Employment. Dougher:Pfizer: Employment. Lu:Pfizer: Other: Ex-Pfizer employee. Xu:Pfizer: Employment. Kausar:Pfizer: Other: Ex-Pfizer Employee. Lemon:Pfizer: Employment. Zhong:Pfizer: Employment. Lucas:Pfizer: Employment. Sung:Pfizer: Employment. Sapra:Pfizer: Employment, Equity Ownership.

scholarly journals Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis

2009 ◽  
Vol 58 (2) ◽  
pp. 163-168 ◽  
Author(s):  
Fernanda C. A. Maranhão ◽  
Fernanda G. Paião ◽  
Ana Lúcia Fachin ◽  
Nilce M. Martinez-Rossi
Keyword(s):  
Abc Transporter ◽  
Trichophyton Rubrum ◽  
Disease Model ◽  
Initial Step ◽  
Functional Expression ◽  
Infection Process ◽  
Major Facilitator Superfamily ◽  
Gene Encoding ◽  
Major Facilitator

Trichophyton rubrum is a dermatophyte responsible for the majority of human superficial mycoses. The functional expression of proteins important for the initial step and the maintenance of the infection process were identified previously in T. rubrum by subtraction suppression hybridization after growth in the presence of keratin. In this study, sequences similar to genes encoding the multidrug-resistance ATP-binding cassette (ABC) transporter, copper ATPase, the major facilitator superfamily and a permease were isolated, and used in Northern blots to monitor the expression of the genes, which were upregulated in the presence of keratin. A sequence identical to the TruMDR2 gene, encoding an ABC transporter in T. rubrum, was isolated in these experiments, and examination of a T. rubrum ΔTruMDR2 mutant showed a reduction in infecting activity, characterized by low growth on human nails compared with the wild-type strain. The high expression levels of transporter genes by T. rubrum in mimetic infection and the reduction in virulence of the ΔTruMDR2 mutant in a disease model in vitro suggest that transporters are involved in T. rubrum pathogenicity.

scholarly journals The Role of fnx1, a Fission Yeast Multidrug Resistance Protein, in the Transition of Cells to a Quiescent G0 State

1998 ◽  
Vol 18 (9) ◽  
pp. 5239-5246 ◽  
Author(s):  
Krassen Dimitrov ◽  
Shelley Sazer
Keyword(s):  
Multidrug Resistance ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Sequence Similarity ◽  
Cell Communication ◽  
Major Facilitator Superfamily ◽  
Natural Habitats ◽  
Resistance Protein ◽  
Major Facilitator

ABSTRACT Most microorganisms live in conditions of nutrient limitation in their natural habitats. When exposed to these conditions they respond with physiological and morphological changes that enable them to survive. To obtain insights into the molecular mechanisms of this response a systematic genetic screen was performed to identify genes that when overexpressed can induce a starvation-like response in the yeast species Schizosaccharomyces pombe. One gene that meets these criteria, fnx1 +, induces, transcriptionally correlates with, and is required for the entry into the quiescent G0 state that is normally induced by nitrogen starvation. fnx1 + encodes a protein with sequence similarity to the proton-driven plasma membrane transporters from the multidrug resistance group of the major facilitator superfamily of proteins. We propose that fnx1 +plays a role in the entry into G0, possibly by facilitating the release of a signaling substance into the environment as a means of cell-to-cell communication.

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