scholarly journals Function of a Mycobacterial Major Facilitator Superfamily Pump Requires a Membrane-Associated Lipoprotein

2007 ◽  
Vol 190 (5) ◽  
pp. 1783-1791 ◽  
Author(s):  
Mary F. Farrow ◽  
Eric J. Rubin
Keyword(s):  
Drug Resistance ◽  
Cell Surface ◽  
Small Molecules ◽  
Ethidium Bromide ◽  
Mycobacterium Smegmatis ◽  
Normal Function ◽  
Cell Wall Composition ◽  
Major Facilitator Superfamily ◽  
Wild Type ◽  
Major Facilitator

ABSTRACT The lprG-Rv1410c operon is critical for the survival of Mycobacterium tuberculosis during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in Mycobacterium smegmatis is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from M. tuberculosis. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type M. smegmatis and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the lprG-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.

scholarly journals Structural and Drug Targeting Insights on Mutant p53

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3344
Author(s):  
Ana Sara Gomes ◽  
Helena Ramos ◽  
Alberto Inga ◽  
Emília Sousa ◽  
Lucília Saraiva
Keyword(s):  
Small Molecules ◽  
Drug Targeting ◽  
Normal Function ◽  
Structure Stability ◽  
Mutant P53 ◽  
Structural Studies ◽  
Wild Type ◽  
Tumor Onset ◽  
Long Time ◽  
Stability Dynamics

p53 is a transcription factor with a pivotal role in cell homeostasis and fate. Its impairment is a major event in tumor onset and development. In fact, about half of human cancers bear TP53 mutations that not only halt the normal function of p53, but also may acquire oncogenic gain of functions that favor tumorigenesis. Although considered undruggable for a long time, evidence has proven the capability of many compounds to restore a wild-type (wt)-like function to mutant p53 (mutp53). However, they have not reached the clinic to date. Structural studies have strongly contributed to the knowledge about p53 structure, stability, dynamics, function, and regulation. Importantly, they have afforded relevant insights into wt and mutp53 pharmacology at molecular levels, fostering the design and development of p53-targeted anticancer therapies. Herein, we provide an integrated view of mutp53 regulation, particularly focusing on mutp53 structural traits and on targeting agents capable of its reactivation, including their biological, biochemical and biophysical features. With this, we expect to pave the way for the development of improved small molecules that may advance precision cancer therapy by targeting p53.

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 Cysteine Is Exported from theEscherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly

2002 ◽  
Vol 277 (51) ◽  
pp. 49841-49849 ◽  
Author(s):  
Marc S. Pittman ◽  
Hazel Corker ◽  
Guanghui Wu ◽  
Marie B. Binet ◽  
Arthur J. G. Moir ◽  
...  
Keyword(s):  
Redox Homeostasis ◽  
Membrane Vesicles ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Major Protein ◽  
Wild Type ◽  
Independent Manner ◽  
Periplasmic Transport ◽  
Major Facilitator ◽  
Atp Binding Cassette

Assembly ofEscherichia colicytochromebdand periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type andcydDmutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in thecydDperiplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropiccydDphenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmicc-type cytochromes incydDand wild-type strains but did not restore cytochromed. Consistent with CydDC being a cysteine exporter,cydDmutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective inorf299, encoding a transporter of the major facilitator superfamily. AcydD orf299double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

scholarly journals Efflux Pump-Mediated Intrinsic Drug Resistance in Mycobacterium smegmatis

2004 ◽  
Vol 48 (7) ◽  
pp. 2415-2423 ◽  
Author(s):  
Xian-Zhi Li ◽  
Li Zhang ◽  
Hiroshi Nikaido
Keyword(s):  
Drug Resistance ◽  
Ethidium Bromide ◽  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Upstream Region ◽  
Drug Efflux ◽  
Intact Cells ◽  
Intrinsic Drug Resistance ◽  
Multiple Drugs ◽  
Increased Susceptibility

ABSTRACT The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc2155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic β-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain β-lactams in M. smegmatis.

scholarly journals CFP, the Putative Cercosporin Transporter of Cercospora kikuchii, Is Required for Wild Type Cercosporin Production, Resistance, and Virulence on Soybean

1999 ◽  
Vol 12 (10) ◽  
pp. 901-910 ◽  
Author(s):  
Terrence M. Callahan ◽  
Mark S. Rose ◽  
Maura J. Meade ◽  
Marilyn Ehrenshaft ◽  
Robert G. Upchurch
Keyword(s):  
Major Facilitator Superfamily ◽  
Cdna Clones ◽  
Membrane Transporter ◽  
Wild Type ◽  
Drastic Reduction ◽  
Targeted Disruption ◽  
Reading Frame ◽  
Mrna Transcripts ◽  
Increased Sensitivity ◽  
Major Facilitator

Many species of the fungal genus Cercospora, including the soybean pathogen C. kikuchii, produce the phytotoxic polyketide cercosporin. Cercosporin production is induced by light. Previously, we identified several cDNA clones of mRNA transcripts that exhibited light-enhanced accumulation in C. kikuchii. Targeted disruption of the genomic copy of one of these, now designated CFP (cercosporin facilitator protein), results in a drastic reduction in cercosporin production, greatly reduced virulence of the fungus to soybean, and increased sensitivity to exogenous cercosporin. Sequence analysis of CFP reveals an 1,821-bp open reading frame encoding a 65.4-kDa protein similar to several members of the major facilitator superfamily (MFS) of integral membrane transporter proteins known to confer resistance to various antibiotics and toxins in fungi and bacteria. We propose that CFP encodes a cercosporin transporter that contributes resistance to cercosporin by actively exporting cercosporin, thus maintaining low cellular concentrations of the toxin.

scholarly journals Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

2007 ◽  
Vol 52 (2) ◽  
pp. 694-704 ◽  
Author(s):  
Ritu Pasrija ◽  
Sneh Lata Panwar ◽  
Rajendra Prasad
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Membrane Lipid ◽  
Major Facilitator Superfamily ◽  
Pathogenic Yeast ◽  
Atp Binding Cassette Transporter ◽  
Major Facilitator ◽  
Green Fluorescent

ABSTRACT In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called ‘rafts.’ Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (Δsur4 or Δfen1 or Δipt1) or ergosterol (Δerg24 or Δerg6 or Δerg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

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 Fusarium graminearum Tri12p Influences Virulence to Wheat and Trichothecene Accumulation

2012 ◽  
Vol 25 (11) ◽  
pp. 1408-1418 ◽  
Author(s):  
Jon Menke ◽  
Yanhong Dong ◽  
H. Corby Kistler
Keyword(s):  
Fusarium Graminearum ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Major Facilitator Superfamily ◽  
Induction Medium ◽  
Wild Type ◽  
In Planta ◽  
Major Facilitator ◽  
Green Fluorescent ◽  
Self Protection

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.

The human iron exporter ferroportin. Insight into the transport mechanism by molecular modeling

2016 ◽  
Vol 12 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Valentina Tortosa ◽  
Maria Carmela Bonaccorsi di Patti ◽  
Giovanni Musci ◽  
Fabio Polticelli
Keyword(s):  
Molecular Modeling ◽  
Transport Mechanism ◽  
Structural Models ◽  
Degradation Pathway ◽  
Atomic Level ◽  
Major Facilitator Superfamily ◽  
Wild Type ◽  
Major Facilitator ◽  
First Time ◽  
Insight Into

AbstractFerroportin, a membrane protein belonging to the major facilitator superfamily of transporters, is the only vertebrate iron exporter known so far. Several ferroportin mutations lead to the so-called ferroportin disease or type 4 hemochromatosis, characterized by two distinct iron accumulation phenotypes depending on whether the mutation affects the activity of the protein or its degradation pathway. Through extensive molecular modeling analyses using the structure of all known major facilitator superfamily members as templates, multiple structural models of ferroportin in the three mechanistically relevant conformations (inward open, occluded, and outward open) have been obtained. The best models, selected on the ground of experimental data available on wild-type and mutant ferroportion, provide for the first time a prediction at the atomic level of the dynamics of the transporter. Based on these results, a possible mechanism for iron export is proposed.

scholarly journals A Novel Nitrate/Nitrite Permease in the Marine CyanobacteriumSynechococcus sp. Strain PCC 7002

1999 ◽  
Vol 181 (23) ◽  
pp. 7363-7372 ◽  
Author(s):  
Toshio Sakamoto ◽  
Kaori Inoue-Sakamoto ◽  
Donald A. Bryant
Keyword(s):  
Nitrite Reductase ◽  
Major Facilitator Superfamily ◽  
Wild Type ◽  
Nitrite Uptake ◽  
Genes Encoding ◽  
Major Facilitator ◽  
Mutant Cells ◽  
Nitrate Transporters ◽  
In Cells

ABSTRACT The nrtP and narB genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium Synechococcus sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An nrtP mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The nrtP mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A narB mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the narBmutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the nrtP andnarB genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of thenrtP and narB genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in Synechococcussp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.

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