scholarly journals The TonB system in Aeromonas hydrophila NJ-35 is essential for MacA2B2 efflux pump-mediated macrolide resistance

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Yuhao Dong ◽  
Qing Li ◽  
Jinzhu Geng ◽  
Qing Cao ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Efflux Pump ◽  
Fold Increase ◽  
Activity Level ◽  
Macrolide Resistance ◽  
Wild Type ◽  
Iron Deprivation ◽  
Pump Activity ◽  
Tonb System ◽  
Increased Sensitivity

AbstractThe TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis

2006 ◽  
Vol 52 (11) ◽  
pp. 1027-1035 ◽  
Author(s):  
Hyesuk Kong ◽  
Cheryl D Patterson ◽  
Robin E Mitchell ◽  
Jeffrey S Buyer ◽  
M Catherine Aime ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Chain Reaction ◽  
Sequence Identity ◽  
Mutant Strains ◽  
Tonb System ◽  
Polymerase Chain ◽  
High Degree

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription – polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.Key words: Pseudomonas syringae pv. tagetis, Pseudomonas putida, tagetitoxin, exbD, exbB, tonB, TonB system, Helianthus annuus L.

scholarly journals Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

2002 ◽  
Vol 46 (11) ◽  
pp. 3370-3380 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Resistant Mutants ◽  
Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

Efflux pump inhibition controls growth and enhances antifungal susceptibility of Fusarium solani species complex

2020 ◽  
Vol 15 (1) ◽  
pp. 9-20
Author(s):  
Rossana de A Cordeiro ◽  
Fernando VM Portela ◽  
Lívia MG Pereira ◽  
Ana RC de Andrade ◽  
José K de Sousa ◽  
...  
Keyword(s):  
Fusarium Solani ◽  
Species Complex ◽  
Efflux Pump ◽  
Antifungal Susceptibility ◽  
Indirect Evidence ◽  
Efflux Pumps ◽  
Fusarium Solani Species Complex ◽  
Efflux Pump Inhibition ◽  
Pump Activity ◽  
Increased Sensitivity

Aim: To evaluate the inhibition of efflux pumps by using promethazine (PMZ) as a strategy to control Fusarium solani species complex (FSSC). Materials & methods: The susceptibility of FSSC strains to PMZ and the interaction between PMZ and antifungals were evaluated. The efflux pump activity was confirmed by flow cytometry with rhodamine 6G. Finally, PMZ was tested against FSSC biofilms. Results: PMZ inhibited FSSC planktonic growth and showed synergism with antifungals. PMZ reduced R6G efflux and inhibited cell adhesion, impaired the development of biofilms and disrupted mature biofilms. PMZ-challenged biofilms showed increased sensitivity to amphotericin B. Conclusion: The study provides indirect evidence of the occurrence of efflux pumps in FSSC and opens a perspective for this target in the control of fusariosis.

scholarly journals Functional Analysis of the Lactococcus lactis galU and galE Genes and Their Impact on Sugar Nucleotide and Exopolysaccharide Biosynthesis

2001 ◽  
Vol 67 (7) ◽  
pp. 3033-3040 ◽  
Author(s):  
Ingeborg C. Boels ◽  
Ana Ramos ◽  
Michiel Kleerebezem ◽  
Willem M. de Vos
Keyword(s):  
Lactococcus Lactis ◽  
Sole Carbon Source ◽  
Northern Blot Analysis ◽  
Fold Increase ◽  
Activity Level ◽  
Wild Type ◽  
Degenerate Primers ◽  
Poor Growth ◽  
Wild Type Level ◽  
Exopolysaccharide Biosynthesis

ABSTRACT We studied the UDP-glucose pyrophosphorylase (galU) and UDP-galactose epimerase (galE) genes of Lactococcus lactis MG1363 to investigate their involvement in biosynthesis of UDP-glucose and UDP-galactose, which are precursors of glucose- and galactose-containing exopolysaccharides (EPS) in L. lactis. The lactococcal galU gene was identified by a PCR approach using degenerate primers and was found by Northern blot analysis to be transcribed in a monocistronic RNA. The L. lactis galU gene could complement an Escherichia coli galU mutant, and overexpression of this gene in L. lactis under control of the inducible nisA promoter resulted in a 20-fold increase in GalU activity. Remarkably, this resulted in approximately eightfold increases in the levels of both UDP-glucose and UDP-galactose. This indicated that the endogenous GalE activity is not limiting and that the GalU activity level in wild-type cells controls the biosynthesis of intracellular UDP-glucose and UDP-galactose. The increased GalU activity did not significantly increase NIZO B40 EPS production. Disruption of the galE gene resulted in poor growth, undetectable intracellular levels of UDP-galactose, and elimination of EPS production in strain NIZO B40 when cells were grown in media with glucose as the sole carbon source. Addition of galactose restored wild-type growth in the galE disruption mutant, while the level of EPS production was approximately one-half the wild-type level.

scholarly journals Mechanisms and Frequency of Resistance to Gatifloxacin in Comparison to AM-1121 and Ciprofloxacin inStaphylococcus aureus

2001 ◽  
Vol 45 (10) ◽  
pp. 2755-2764 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Efflux Pump ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Single Step ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Genetic Studies ◽  
Clinical Breakpoints ◽  
Methoxy Substituent ◽  
Selection Of

ABSTRACT Gatifloxacin, an 8-methoxyfluoroquinolone, was found to be two- to fourfold more active against wild-typeStaphylococcus aureus ISP794 than its desmethoxy derivative, AM-1121, and ciprofloxacin, another desmethoxy fluoroquinolone. Single grlBA mutations caused two- to fourfold increases in the MIC of gatifloxacin, and a single gyrase mutation was silent. Double mutations in gyrA andgrlA or grlB caused a 32-fold increase in the MIC of gatifloxacin, in contrast to a 128-fold increase for ciprofloxacin and AM-1121. Overexpression of the NorA efflux pump had minimal effect on the MIC of gatifloxacin. The bactericidal activity of the three quinolones at four times the MIC differed only for a double mutant, with gatifloxacin exhibiting a killing pattern similar to that for ISP794, whereas ciprofloxacin and AM-1121 failed to show any killing. With gatifloxacin, selection of resistant mutants at twice the MIC was 100- to 1,000-fold less frequent than with the comparison quinolones, and mutants could rarely be selected at four times the MIC. The limit resistance in ISP74 was 512 times the MIC of gatifloxacin and 1,024 times the MICs of ciprofloxacin and AM-1121. Novel mutations in topoisomerase IV were selected in five of the six single-step mutants, three of which were shown to cause quinolone resistance by genetic studies. In conclusion, topoisomerase IV is the primary target of gatifloxacin. In contrast to comparison quinolones, mutations in both topoisomerase IV and gyrase are required for resistance to gatifloxacin by clinical breakpoints and do not abolish bactericidal effect, further supporting the benefit of the 8-methoxy substituent in gatifloxacin.

scholarly journals Phenotypic Characterization of acopAMutant of Neisseria gonorrhoeae Identifies a Link between Copper and Nitrosative Stress

2011 ◽  
Vol 80 (3) ◽  
pp. 1065-1071 ◽  
Author(s):  
Karrera Y. Djoko ◽  
Jessica A. Franiek ◽  
Jennifer L. Edwards ◽  
Megan L. Falsetta ◽  
Stephen P. Kidd ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Nitrosative Stress ◽  
Efflux Pump ◽  
Copper Ions ◽  
Copper Ion ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Content Type ◽  
Increased Sensitivity

NGO0579 is annotatedcopAin theNeisseria gonorrhoeaechromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, acopAmutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. ThecopAmutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, thecopAmutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system ofNeisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species.

Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

1987 ◽  
Vol 45 ◽  
pp. 908-909
Author(s):  
Alain R. Trudel ◽  
M. Trudel
Keyword(s):  
Electron Microscopy ◽  
Fold Increase ◽  
Observation Time ◽  
Clinical Samples ◽  
Maximum Speed ◽  
Viral Particles ◽  
Structural Details ◽  
Latex Spheres ◽  
Increased Sensitivity ◽  
Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

scholarly journals Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1507-1512 ◽  
Author(s):  
H Zhu ◽  
H Conrad-Webb ◽  
X S Liao ◽  
P S Perlman ◽  
R A Butow
Keyword(s):  
Genetic Analysis ◽  
Rna Processing ◽  
Fold Increase ◽  
Functional Expression ◽  
Rrna Gene ◽  
Yeast Mitochondria ◽  
Wild Type ◽  
Site Specific ◽  
Var1 Gene ◽  
A Site

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-I, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the var1 gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit1, a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. The fit1 dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-type fit1 dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit1 gene is essential for fit1 expression.

scholarly journals New Trimethoprim-Like Molecules: Bacteriological Evaluation and Insights into Their Action

Antibiotics ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 709
Author(s):  
Marta Jorba ◽  
Marina Pedrola ◽  
Ouldouz Ghashghaei ◽  
Rocío Herráez ◽  
Lluis Campos-Vicens ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Detailed Characterization ◽  
Low Concentrations ◽  
Pump Activity ◽  
Bacteriological Evaluation ◽  
Kinetics Of

This work reports a detailed characterization of the antimicrobial profile of two trimethoprim-like molecules (compounds 1a and 1b) identified in previous studies. Both molecules displayed remarkable antimicrobial activity, particularly when combined with sulfamethoxazole. In disk diffusion assays on Petri dishes, compounds 1a and 1b showed synergistic effects with colistin. Specifically, in combinations with low concentrations of colistin, very large increases in the activities of compounds 1a and 1b were determined, as demonstrated by alterations in the kinetics of bacterial growth despite only slight changes in the fractional inhibitory concentration index. The effect of colistin may be to increase the rate of antibiotic entry while reducing efflux pump activity. Compounds 1a and 1b were susceptible to extrusion by efflux pumps, whereas the inhibitor phenylalanine arginyl β-naphthylamide (PAβN) exerted effects similar to those of colistin. The interactions between the target enzyme (dihydrofolate reductase), the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), and the studied molecules were explored using enzymology tools and computational chemistry. A model based on docking results is reported.

scholarly journals Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 150
Author(s):  
Kimberly J. Nelson ◽  
Terri Messier ◽  
Stephanie Milczarek ◽  
Alexis Saaman ◽  
Stacie Beuschel ◽  
...  
Keyword(s):  
Oxygen Species ◽  
Mitochondrial Reactive Oxygen Species ◽  
Wild Type ◽  
Cellular Models ◽  
Chemotherapeutic Approach ◽  
Promising Target ◽  
Redox Responsive ◽  
Catalytic Cysteine ◽  
Increased Sensitivity ◽  
Peroxiredoxin 3

A central hallmark of tumorigenesis is metabolic alterations that increase mitochondrial reactive oxygen species (mROS). In response, cancer cells upregulate their antioxidant capacity and redox-responsive signaling pathways. A promising chemotherapeutic approach is to increase ROS to levels incompatible with tumor cell survival. Mitochondrial peroxiredoxin 3 (PRX3) plays a significant role in detoxifying hydrogen peroxide (H2O2). PRX3 is a molecular target of thiostrepton (TS), a natural product and FDA-approved antibiotic. TS inactivates PRX3 by covalently adducting its two catalytic cysteine residues and crosslinking the homodimer. Using cellular models of malignant mesothelioma, we show here that PRX3 expression and mROS levels in cells correlate with sensitivity to TS and that TS reacts selectively with PRX3 relative to other PRX isoforms. Using recombinant PRXs 1–5, we demonstrate that TS preferentially reacts with a reduced thiolate in the PRX3 dimer at mitochondrial pH. We also show that partially oxidized PRX3 fully dissociates to dimers, while partially oxidized PRX1 and PRX2 remain largely decameric. The ability of TS to react with engineered dimers of PRX1 and PRX2 at mitochondrial pH, but inefficiently with wild-type decameric protein at cytoplasmic pH, supports a novel mechanism of action and explains the specificity of TS for PRX3. Thus, the unique structure and propensity of PRX3 to form dimers contribute to its increased sensitivity to TS-mediated inactivation, making PRX3 a promising target for prooxidant cancer therapy.

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