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Science (New York, N.Y.) Apr 2024DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules,...
DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.
Topics: Cryoelectron Microscopy; DNA; DNA Gyrase; DNA, Superhelical; Escherichia coli; Escherichia coli Proteins; Protein Domains
PubMed: 38603484
DOI: 10.1126/science.adl5899 -
Microbiology and Immunology Jun 2024We have previously isolated a gram-negative microaerophilic strain, PAGU2000 from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study...
We have previously isolated a gram-negative microaerophilic strain, PAGU2000 from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000 shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000 has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000 and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000 was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000 = GTC 16811 = LMG 33095). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis.
Topics: Helicobacter; RNA, Ribosomal, 16S; Humans; Phylogeny; DNA, Bacterial; Helicobacter Infections; Base Composition; Japan; Sequence Analysis, DNA; Bacterial Typing Techniques; DNA Gyrase
PubMed: 38599769
DOI: 10.1111/1348-0421.13127 -
Bioorganic Chemistry Jun 2024The identification of novel 4-hydroxy-2-quinolone-3-carboxamide antibacterials with improved properties is of great value for the control of antibiotic resistance. In...
The identification of novel 4-hydroxy-2-quinolone-3-carboxamide antibacterials with improved properties is of great value for the control of antibiotic resistance. In this study, a series of N-heteroaryl-substituted 4-hydroxy-2-quinolone-3-carboxamides were developed using the bioisosteric replacement strategy. As a result of our research, we discovered the two most potent GyrB inhibitors (WBX7 and WBX18), with IC values of 0.816 µM and 0.137 µM, respectively. Additional antibacterial activity screening indicated that WBX18 possesses the best antibacterial activity against MRSA, VISA, and VRE strains, with MIC values rangingbetween0.5and 2 µg/mL, which was 2 to over 32 times more potent than that of vancomycin. In vitro safety and metabolic stability, as well as in vivo pharmacokinetics assessments revealed that WBX18 is non-toxic to HUVEC and HepG2, metabolically stable in plasma and liver microsomes (mouse), and displays favorable in vivo pharmacokinetic properties. Finally, docking studies combined with molecular dynamic simulation showed that WBX18 could stably fit in the active site cavity of GyrB.
Topics: Anti-Bacterial Agents; Humans; DNA Gyrase; Topoisomerase II Inhibitors; Microbial Sensitivity Tests; Structure-Activity Relationship; Animals; Molecular Structure; Dose-Response Relationship, Drug; Mice; Hep G2 Cells; Molecular Docking Simulation; Microsomes, Liver
PubMed: 38581967
DOI: 10.1016/j.bioorg.2024.107314 -
Journal of Infection and Chemotherapy :... Apr 2024Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of...
INTRODUCTION
Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni.
METHODS
The structure-activity relationship of the examined drugs was investigated by measuring IC and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase.
RESULT
WQ-3334 exhibited the lowest IC against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases.
CONCLUSION
WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.
PubMed: 38580055
DOI: 10.1016/j.jiac.2024.04.002 -
Cell Reports Apr 2024In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a...
In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. In vitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25 μg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.
Topics: Topoisomerase II Inhibitors; DNA Gyrase; Anti-Bacterial Agents; Escherichia coli; Transcription, Genetic; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests
PubMed: 38578824
DOI: 10.1016/j.celrep.2024.114053 -
Antimicrobial Agents and Chemotherapy May 2024) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an...
) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in . The reference strain of (ATCC19977) and 60 clinical isolates, including 41 subsp. and 19 . subsp. , were investigated. The drug sensitivity of against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the and genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the and genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against . The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in .
Topics: Levofloxacin; Anti-Bacterial Agents; Microbial Sensitivity Tests; Mycobacterium abscessus; Reserpine; Bacterial Proteins; Carbonyl Cyanide m-Chlorophenyl Hydrazone; DNA Gyrase; Membrane Transport Proteins; Drug Resistance, Bacterial; Humans; Verapamil
PubMed: 38572960
DOI: 10.1128/aac.01348-23 -
Chemistry & Biodiversity Jun 2024In order to develop novel antimicrobial agents, we prepared quinoline bearing pyrimidine analogues 2-7, 8 a-d and 9 a-d and their structures were elucidated by...
In order to develop novel antimicrobial agents, we prepared quinoline bearing pyrimidine analogues 2-7, 8 a-d and 9 a-d and their structures were elucidated by spectroscopic techniques. Furthermore, our second aim was to predict the interactions between the active compounds and enzymes (DNA gyrase and DHFR). In this work, fourteen pyrimido[4,5-b]quinoline derivatives were prepared and assessed for their antimicrobial potential by estimating zone of inhibition. All the screened candidates displayed antibacterial potential with zone of inhibition range of 9-24 mm compared with ampicillin (20-25 mm) as a reference drug. Moreover, the target derivatives 2 (ZI=16), 9 c (ZI=17 mm) and 9 d (ZI=16 mm) recorded higher antifungal activity against C. albicans to that exhibited by the antifungal drug amphotericin B (ZI=15 mm). Finally, the most potent pyrimidoquinoline compounds (2, 3, 8 c, 8 d, 9 c and 9 d) were docked inside DHFR and DNA gyrase active sites and they recorded excellent fitting within the active regions of DNA gyrase and DHFR. These outcomes revealed us that compounds (2, 3, 8 c, 8 d, 9 c and 9 d) could be lead compounds to discover novel antibacterial candidates.
Topics: Quinolines; DNA Gyrase; Microbial Sensitivity Tests; Molecular Docking Simulation; Tetrahydrofolate Dehydrogenase; Candida albicans; Anti-Bacterial Agents; Structure-Activity Relationship; Antifungal Agents; Pyrimidines; Molecular Structure; Topoisomerase II Inhibitors; Dose-Response Relationship, Drug
PubMed: 38570192
DOI: 10.1002/cbdv.202400200 -
ACS Infectious Diseases Apr 2024Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone... (Review)
Review
Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.
Topics: DNA Topoisomerase IV; Fluoroquinolones; DNA Gyrase; Topoisomerase II Inhibitors; Anti-Bacterial Agents; DNA; Mycobacterium tuberculosis
PubMed: 38564341
DOI: 10.1021/acsinfecdis.4c00128 -
Advances in Pharmacological and... 2024is one of the traditional medicinal plants used in Ethiopia to treat different ailments, such as cancer and diseases related to the respiratory system. This study aimed...
is one of the traditional medicinal plants used in Ethiopia to treat different ailments, such as cancer and diseases related to the respiratory system. This study aimed to isolate the phytochemical components of the root of and evaluate their and biological activities. Oleic acid (1), palmitic acid (2), sterols (3 and 4), boehmenan (5), and carolignans E (6 and 7) were isolated by silica gel column chromatography and preparative thin layer chromatography and characterized by NMR spectroscopy. Compounds 5-7 were isolated from the plant for the first time. At 5 mg/mL, the inhibition zone of evaluated compounds ranged from 8.80 to 11.10 mm against all selected bacteria. The MIC of the MeOH and -hexane: EtOAc (1 : 1) extracts was greater than or equal to 50 mg/mL against all selected bacteria. At 62.5 g/mL, the % DPPH radical scavenging activity of tested compounds ranged from 30.3% to 92.1% with an IC value of 19.4 to 2.1 g/mL. The results of molecular docking studies indicated that the docking scores of compounds 3-7 ranged from -6.4 to -7.9 kcal/mol against DNA gyrase B, -8.3 to -9.0 kcal/mol against the quinolone signal A, -7.1 to -8.5 kcal/mol against pyruvate kinase M2, and -7.9 to -8.5 kcal/mol against human topoisomerase . The results of the antibacterial activity of compounds , , and supported the antibacterial test results. Compound had a better docking score against human topoisomerase than the other test samples demonstrating its potential as an anticancer agent. Therefore, compounds 3-7 could be considered as a lead for developing antibacterial and anticancer drugs. Moreover, the presence of these active phytochemicals supports the traditional use of this plant against cancer and bacteria.
PubMed: 38562543
DOI: 10.1155/2024/3152929 -
Nature Communications Mar 2024In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is...
In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to "unroll" disrupting the H-NS bridges and releasing H-NS.
Topics: Chromatin; DNA-Binding Proteins; Bacterial Proteins; Bacteria; DNA-Directed RNA Polymerases; DNA; Gene Silencing; Gene Expression Regulation, Bacterial; DNA, Bacterial; Transcription, Genetic
PubMed: 38555352
DOI: 10.1038/s41467-024-47114-w