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 Characterization and Functional Complementation of a Nonlethal Deletion in the Chromosome of a β-Glycosidase Mutant of Sulfolobus solfataricus

2003 ◽  
Vol 185 (13) ◽  
pp. 3948-3957 ◽  
Author(s):  
Simonetta Bartolucci ◽  
Mosè Rossi ◽  
Raffaele Cannio
Keyword(s):  
Insertion Sequence ◽  
Genomic Sequence ◽  
Sulfolobus Solfataricus ◽  
Major Facilitator Superfamily ◽  
Gene Encoding ◽  
Sequence Element ◽  
Plasmid Construct ◽  
Major Facilitator ◽  
Flanking Regions ◽  
Insertion Sequence Element

ABSTRACT LacS− mutants of Sulfolobus solfataricus defective in β-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation. One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the β-glycosidase gene (lacS). Fine mapping performed in comparison to the genomic sequence of S. solfataricus P2 indicated an extended deletion of ∼13 kb. The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058. In order to complement the LacS− phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5′ and 3′ flanking regions into the pEXSs plasmid. Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily. Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the β-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside).

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 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.

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 Bcmfs1, a Novel Major Facilitator Superfamily Transporter from Botrytis cinerea, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides

2002 ◽  
Vol 68 (10) ◽  
pp. 4996-5004 ◽  
Author(s):  
Keisuke Hayashi ◽  
Henk-jan Schoonbeek ◽  
Maarten A. De Waard
Keyword(s):  
Botrytis Cinerea ◽  
Abc Transporter ◽  
Pathogenic Fungus ◽  
Major Facilitator Superfamily ◽  
Defense Compounds ◽  
Toxic Compounds ◽  
Major Facilitator Superfamily Transporter ◽  
Dmi Fungicides ◽  
Increased Sensitivity ◽  
Major Facilitator

ABSTRACT Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

scholarly journals Genetic Loci of Streptococcus mitis That Mediate Binding to Human Platelets

2001 ◽  
Vol 69 (3) ◽  
pp. 1373-1380 ◽  
Author(s):  
Barbara A. Bensing ◽  
Craig E. Rubens ◽  
Paul M. Sullam
Keyword(s):  
Streptococcus Thermophilus ◽  
Human Platelets ◽  
Major Facilitator Superfamily ◽  
Surface Proteins ◽  
Tail Fiber ◽  
Streptococcus Mitis ◽  
Platelet Binding ◽  
Membrane Spanning ◽  
Major Facilitator

ABSTRACT The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis. To identify bacterial components that mediate platelet binding byStreptococcus mitis, we screened a Tn916ΔE-derived mutant library of S. mitisstrain SF100 for reduced binding to human platelets in vitro. Two distinct loci were found to affect platelet binding. The first contains a gene (pblT) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments. This protein resembles members of the major facilitator superfamily of small-molecule transporters. The second platelet binding locus consists of an apparent polycistronic operon. This region includes genes that are highly similar to those of Lactococcus lactis phage r1t andStreptococcus thermophilus phage 01205. Two genes (pblA and pblB) encoding large surface proteins are also present. The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall. The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205. Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA. The combined data indicate that at least two genomic regions contribute to platelet binding by S. mitis.One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.

scholarly journals Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp. yingchengensis Strain 10–22

2004 ◽  
Vol 36 (8) ◽  
pp. 519-528 ◽  
Author(s):  
Lei Qin ◽  
Heng-An Wang ◽  
Zhong-Qin Wu ◽  
Xiao-Feng Zhang ◽  
Mei-Lei Jin ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Similarity ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Streptomyces Hygroscopicus ◽  
Wild Type ◽  
Gene Encoding ◽  
Major Facilitator

Abstract The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10–22, a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–20 fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22. While with the addition of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

scholarly journals Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

2021 ◽  
Author(s):  
Elisabeth Lambert ◽  
Ahmad Reza Mehdipour ◽  
Alex Schmidt ◽  
Gerhard Hummer ◽  
Camilo Perez
Keyword(s):  
Lipid Binding ◽  
Major Facilitator Superfamily ◽  
Common Mechanism ◽  
Lipid Transporters ◽  
Dynamics Simulations ◽  
Major Facilitator ◽  
Lipid Transporter ◽  
Lipid Translocation

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a trap-and-flip mechanism that might be shared among MFS lipid transporters.

scholarly journals Deletion of the Major Facilitator Superfamily TransporterfptBAlters Host Cell Interactions and Attenuates Virulence of Type AFrancisella tularensis

2018 ◽  
Vol 86 (3) ◽  
Author(s):  
Phillip M. Balzano ◽  
Aimee L. Cunningham ◽  
Christen Grassel ◽  
Eileen M. Barry
Keyword(s):  
Mouse Model ◽  
Francisella Tularensis ◽  
Severe Disease ◽  
Type A ◽  
Major Facilitator Superfamily ◽  
Human Macrophage ◽  
Intracellular Replication ◽  
Content Type ◽  
Major Facilitator

ABSTRACTFrancisella tularensisis a Gram-negative, facultative, intracellular coccobacillus that can infect a wide variety of hosts. In humans,F. tularensiscauses the zoonosis tularemia following insect bites, ingestion, inhalation, and the handling of infected animals. The fact that a very small inoculum delivered by the aerosol route can cause severe disease, coupled with the possibility of its use as an aerosolized bioweapon, has led to the classification ofFrancisella tularensisas a category A select agent and has renewed interest in the formulation of a vaccine. To this end, we engineered a type A strain SchuS4 derivative containing a targeted deletion of the major facilitator superfamily (MFS) transporterfptB. Based on the attenuating capacity of this deletion in theF. tularensisLVS background, we hypothesized that the deletion of this transporter would alter the intracellular replication and cytokine induction of the type A strain and attenuate virulence in the stringent C57BL/6J mouse model. Here we demonstrate that the deletion offptBsignificantly alters the intracellular life cycle ofF. tularensis, attenuating intracellular replication in both cell line-derived and primary macrophages and inducing a novel cytosolic escape delay. Additionally, we observed prominent differences in thein vitrocytokine profiles in human macrophage-like cells. The mutant was highly attenuated in the C57BL/6J mouse model and provided partial protection against virulent type AF. tularensischallenge. These results indicate a fundamental necessity for this nutrient transporter in the timely progression ofF. tularensisthrough its replication cycle and in pathogenesis.

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