scholarly journals Mechanisms and Frequency of Resistance to Premafloxacin in Staphylococcus aureus: Novel Mutations Suggest Novel Drug-Target Interactions

2000 ◽  
Vol 44 (12) ◽  
pp. 3344-3350 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Fold Increase ◽  
Single Step ◽  
The Novel ◽  
Novel Mutations ◽  
Topoisomerase Iv ◽  
Double Mutation ◽  
Fourfold Increase ◽  
Novel Drug

ABSTRACT Premafloxacin is a novel 8-methoxy fluoroquinolone with enhanced activity against Staphylococcus aureus. We found premafloxacin to be 32-fold more active than ciprofloxacin against wild-type S. aureus. Single mutations in either subunit of topoisomerase IV caused a four- to eightfold increase in the MICs of both quinolones. A double mutation (gyrA and eithergrlA or grlB) caused a 32-fold increase in the MIC of premafloxacin, while the MIC of ciprofloxacin increased 128-fold. Premafloxacin appeared to be a poor substrate for NorA, with NorA overexpression causing an increase of twofold or less in the MIC of premafloxacin in comparison to a fourfold increase in the MIC of ciprofloxacin. The frequency of selection of resistant mutants was 6.4 × 10−10 to 4.0 × 10−7 at twofold the MIC of premafloxacin, 2 to 4 log10 less than that with ciprofloxacin. Single-step mutants could not be selected at higher concentrations of premafloxacin. In five single-step mutants, only one previously described uncommon mutation (Ala116Glu), and four novel mutations (Arg43Cys, Asp69Tyr, Ala176Thr, and Pro157Leu), three of which were outside the quinolone resistance-determining region (QRDR) were found. Genetic linkage studies, in which incross ofgrlA + and outcross of mutations were performed, showed a high correlation between the mutations and the resistance phenotypes, and allelic exchange experiments confirmed the role of the novel mutations in grlA in resistance. Our results suggest that although topoisomerase IV is the primary target of premafloxacin, premafloxacin appears to interact with topoisomerase IV in a manner different from that of other quinolones and that the range of the QRDR of grlA should be expanded.

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.

scholarly journals Mutations in Topoisomerase IV and DNA Gyrase of Staphylococcus aureus: Novel Pleiotropic Effects on Quinolone and Coumarin Activity

1998 ◽  
Vol 42 (1) ◽  
pp. 121-128 ◽  
Author(s):  
Bénédicte Fournier ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Gyrase ◽  
Single Step ◽  
Pleiotropic Effects ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Chromosomal Dna ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Double Mutation

ABSTRACT Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB andgrlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlBallele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, butgrlB + appeared dominant over grlB(Asp-470) for coumarin resistance. Finally, the gyrA(Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB(Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.

scholarly journals Topoisomerase Targeting with and Resistance to Gemifloxacin in Staphylococcus aureus

2003 ◽  
Vol 47 (1) ◽  
pp. 274-282 ◽  
Author(s):  
Dilek Ince ◽  
Xiamei Zhang ◽  
L. Christine Silver ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Low Frequency ◽  
Fold Increase ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
Fourfold Increase ◽  
Insight Into ◽  
Selection Of

ABSTRACT Gemifloxacin, a novel quinolone with potent activity against Staphylococcus aureus, was 8- to 16-fold more active against wild-type S. aureus than ciprofloxacin. The two- to fourfold increase in the MIC of gemifloxacin in genetically defined grlBA mutants and the twofold increase in a single gyrA mutant, supported by the low frequency of selection of resistant mutants at twice the MIC (7.4 × 10−11 to 1.1 × 10−10), suggested similar targeting of the two enzymes by gemifloxacin. Dual mutations in both gyrase and topoisomerase IV caused a 64- to 128-fold increase in the MIC of gemifloxacin, similar to that seen with ciprofloxacin. Gemifloxacin also had similar activity in vitro against topoisomerase IV and gyrase purified from S. aureus (50% inhibitory concentrations of 0.25 and 0.31 μg/ml, respectively). This activity was 10- to 20-fold higher than that of ciprofloxacin for topoisomerase IV and 33-fold higher than that for gyrase. In contrast to the in vitro findings, only topoisomerase IV mutants were selected in first-step mutants. Overexpression of the NorA efflux pump had a minimal effect on resistance to gemifloxacin, and a mutation in the promoter region of the gene for NorA was selected only in the sixth step of serial selection of mutants. Our data show that although gemifloxacin targets purified topoisomerase IV and gyrase similarly in vitro, topoisomerase IV is the preferred target in the bacteria. Selection of novel resistance mutations in grlA requires further expansion of quinolone-resistance-determining regions, and their study may provide increased insight into enzyme-quinolone interactions.

Influence of C-ANF receptor and neutral endopeptidase on pharmacokinetics of ANF in rats

1991 ◽  
Vol 260 (1) ◽  
pp. R208-R216 ◽  
Author(s):  
P. J. Chiu ◽  
G. Tetzloff ◽  
M. T. Romano ◽  
C. J. Foster ◽  
E. J. Sybertz
Keyword(s):  
High Performance ◽  
Fold Increase ◽  
Neutral Endopeptidase ◽  
Volume Of Distribution ◽  
Fourfold Increase ◽  
Control Value ◽  
Enzymatic Pathways ◽  
Hydrolysis Of

The role of C-atrial natriuretic factor (ANF) receptors and neutral endopeptidase (NEP) in the pharmacokinetics and hydrolysis of 125I-labeled ANF was evaluated in rats by using C-ANF and SCH 39370 to block the nonenzymatic and enzymatic pathways, respectively. After a bolus injection of 125I-ANF, the resulting area under the plasma concentration curve (AUC) with C-ANF treatment was seven times the control value with regard to trichloroacetic acid-precipitable (TCA-ppt) radioactivity (intact ANF). SCH 39370 tended to increase AUC, but the changes were not significant. Nevertheless, SCH 39370 suppressed the appearance of TCA-soluble radioactivity (hydrolytic products), indicating that in vivo inhibition of ANF degradation had occurred. SCH 39370 plus C-ANF produced a 15-fold increase in AUC for TCA-ppt radioactivity and a reduction in plasma TCA-soluble radioactivity. High-performance liquid chromatography (HPLC) analysis confirmed that combination treatment increased intact ANF and reduced hydrolytic products in the plasma. SCH 39370 reduced clearance (C) without altering volume of distribution in steady state (Vss) and half-life (t1/2). C-ANF decreased both C and Vss leading to a fourfold increase in t1/2, which was further prolonged by SCH 39370 (7.5 times control). Bilateral nephrectomy caused a proportionally similar decrease in Vss and C without changing t1/2, suggesting significant extrarenal metabolism of ANF. SCH 39370 systemically inhibits ANF hydrolysis; the resulting increase in ANF, however, is masked by the great capacity of ANF clearance receptors but can be revealed with excess C-ANF, suggesting that the plasma ANF concentrations are determined by the interplay of the C-ANF receptor and NEP systems.

scholarly journals The Teicoplanin-Associated Locus Regulator (TcaR) and the Intercellular Adhesin Locus Regulator (IcaR) Are Transcriptional Inhibitors of the ica Locus in Staphylococcus aureus

2004 ◽  
Vol 186 (8) ◽  
pp. 2449-2456 ◽  
Author(s):  
Kimberly K. Jefferson ◽  
Danielle B. Pier ◽  
Donald A. Goldmann ◽  
Gerald B. Pier
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Negative Regulator ◽  
Regulatory Proteins ◽  
Polysaccharide Intercellular Adhesin ◽  
Topoisomerase Iv ◽  
Exogenous Factors ◽  
Dna Affinity Chromatography

ABSTRACT Infections involving Staphylococcus aureus are often more severe and difficult to treat when the organism assumes a biofilm mode of growth. The polysaccharide poly-N-acetylglucosamine (PNAG), also known as polysaccharide intercellular adhesin, is synthesized by the products of the intercellular adhesin (ica) locus and plays a key role in biofilm formation. Numerous conditions and exogenous factors influence ica transcription and PNAG synthesis, but the regulatory factors and pathways through which these environmental stimuli act have been only partially characterized. We developed a DNA affinity chromatography system to purify potential regulatory proteins that bind to the ica promoter region. Using this technique, we isolated four proteins, including the staphylococcal gene regulator SarA, a MarR family transcriptional regulator of the teicoplanin-associated locus TcaR, DNA-binding protein II, and topoisomerase IV, that bound to the ica promoter. Site-directed deletion mutagenesis of tcaR indicated that TcaR was a negative regulator of ica transcription, but deletion of tcaR alone did not induce any changes in PNAG production or in adherence to polystyrene. We also investigated the role of IcaR, encoded within the ica locus but divergently transcribed from the biosynthetic genes. As has been shown previously in Staphylococcus epidermidis, we found that IcaR was also a negative regulator of ica transcription in S. aureus. We also demonstrate that mutation of icaR augmented PNAG production and adherence to polystyrene. Transcription of the ica locus, PNAG production, and adherence to polystyrene were further increased in a tcaR icaR double mutant. In summary, TcaR appeared to be a weak negative regulator of transcription of the ica locus, whereas IcaR was a strong negative regulator, and in their absence PNAG production and biofilm formation were enhanced.

scholarly journals ParC and GyrA May Be Interchangeable Initial Targets of Some Fluoroquinolones in Streptococcus pneumoniae

1999 ◽  
Vol 43 (2) ◽  
pp. 302-306 ◽  
Author(s):  
Emmanuelle Varon ◽  
Claire Janoir ◽  
Marie-Dominique Kitzis ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Susceptible Strain ◽  
Quinolone Resistance ◽  
Topoisomerase Iv ◽  
Double Mutation ◽  
Active Efflux ◽  
Single Target ◽  
Resistant Mutants ◽  
The Individual

ABSTRACT To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae, we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC orgyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.

Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3423-3428 ◽  
Author(s):  
Mirko Pinotti ◽  
Raffaella Toso ◽  
Domenico Girelli ◽  
Debora Bindini ◽  
Paolo Ferraresi ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Fold Increase ◽  
Factor Vii ◽  
The Novel ◽  
Expression Studies ◽  
Sequence Variations ◽  
Fvii Deficiency ◽  
The Individual

Abstract Previous studies have established that factor VII gene (F7) polymorphisms (5′F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype–phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with theIVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7Gallele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.

scholarly journals Identification of the Novel Lincosamide Resistance Genelnu(E) Truncated by ISEnfa5-cfr-ISEnfa5Insertion in Streptococcus suis:De NovoSynthesis and Confirmation of Functional Activity in Staphylococcus aureus

2013 ◽  
Vol 58 (3) ◽  
pp. 1785-1788 ◽  
Author(s):  
Qin Zhao ◽  
Sarah Wendlandt ◽  
Hui Li ◽  
Jun Li ◽  
Congming Wu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Promoter Region ◽  
De Novo ◽  
Shuttle Vector ◽  
Fold Increase ◽  
The Novel ◽  
Content Type ◽  
E Gene ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACTThe novel lincosamide resistance genelnu(E), truncated by insertion of an ISEnfa5-cfr-ISEnfa5segment, was identified inStreptococcus suis. The genelnu(E) encodes a 173-amino-acid protein with ≤69.4% identity to other lincosamide nucleotidyltransferases. Thelnu(E) gene and its promoter region werede novosynthesized, andStaphylococcus aureusRN4220 carrying a shuttle vector with the clonedlnu(E) gene showed a 16-fold increase in the lincomycin MIC. Mass spectrometry experiments demonstrated that Lnu(E) catalyzed the nucleotidylation of lincomycin.

scholarly journals DX-619, a Novel Des-Fluoro(6) Quinolone Manifesting Low Frequency of Selection of Resistant Staphylococcus aureus Mutants: Quinolone Resistance beyond Modification of Type II Topoisomerases

2005 ◽  
Vol 49 (12) ◽  
pp. 5051-5057 ◽  
Author(s):  
Jacob Strahilevitz ◽  
Que Chi Truong-Bolduc ◽  
David C. Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Low Frequency ◽  
Single Step ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dual Targeting ◽  
Selection Of ◽  
Stimulation Of

ABSTRACT DX-619, a novel des-fluoro(6) quinolone, was 16- to 32-fold, twofold, and four- to eightfold more potent than ciprofloxacin, gemifloxacin, and garenoxacin, respectively, against wild-type Staphylococcus aureus. DX-619 manifested equal fourfold increases in MIC against a common parC mutant and a common gyrA mutant and selected for mutants at up to two- to fourfold its MIC, consistent with dual-targeting properties. Of the four independent single-step mutants selected, two had new single mutations in parC (V87F and R17H), and two shared a new gyrA mutation (A26V), one with an additional deletion mutation in parE (Δ215-7). By allelic exchange, the ParC but not the GyrA or ParE mutation was shown to be fully responsible for the resistance phenotypes, suggesting an as yet undefined mechanism of resistance operating in conjunction with type II topoisomerase mutations contributed to resistance to DX-619. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that DX-619 had similar activity against topoisomerase IV and gyrase (50% stimulation of cleavage complexes concentration, 1.25 and 0.62 to 1.25 μg/ml, respectively). Susceptibility studies with DX-619 and an array of efflux pump substrates with and without reserpine, an inhibitor of efflux pumps, suggested that resistance in DX-619-selected mutants is affected by mechanisms other than mutations in topoisomerases or known reserpine-inhibitable pumps in S. aureus and thus are likely novel.

scholarly journals Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells

2015 ◽  
Vol 83 (11) ◽  
pp. 4362-4372 ◽  
Author(s):  
Q. C. Truong-Bolduc ◽  
G. R. Bolduc ◽  
H. Medeiros ◽  
J. M. Vyas ◽  
Y. Wang ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Unsaturated Fatty Acids ◽  
Efflux Pump ◽  
Fold Increase ◽  
Growth Patterns ◽  
Heart Cell ◽  
Content Type

ABSTRACTWe previously identified the protein Tet38 as a chromosomally encoded efflux pump ofStaphylococcus aureusthat confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator oftet38, named tetracycline regulator 21 (TetR21), that bound specifically to thetet38promoter and repressed pump expression. A ΔtetR21mutant showed a 5-fold increase intet38transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to thetetR21promoter and indirectly repressed the expression oftet38. To further assess the full role of Tet38 inS. aureusadaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We usedS. aureusRN6390, its Δtet38, ΔtetR21, and ΔmgrAmutants, and a Δtet38 ΔtetR21double mutant. After 2 h of contact, the Δtet38mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrAmutant. The growth patterns of RN6390 and the ΔmgrAand ΔtetR21mutants within A549 cells were similar, while no growth was observed for the Δtet38mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability ofS. aureusto internalize and replicate within epithelial cells.

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