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Antimicrobial Agents and Chemotherapy Apr 2022Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of...
Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans. Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase. However, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in M. ulcerans remains unknown. This study investigated the detailed molecular mechanism of M. ulcerans DNA gyrase and the contribution of FQ resistance using recombinant proteins from the M. ulcerans subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC of FQs against Ala91Val and Asp95Gly mutants of M. ulcerans shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type (WT) enzyme. Similarly, the quinolone concentrations required to induce 25% of the maximum DNA cleavage (CC) was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of the WT/mutant M. ulcerans DNA gyrase and FQ side chains were assessed by molecular docking studies. This was the first elaborative study demonstrating the contribution of mutations in M. ulcerans DNA GyrA subunit to FQ resistance .
Topics: DNA Gyrase; Drug Resistance, Bacterial; Fluoroquinolones; Microbial Sensitivity Tests; Molecular Docking Simulation; Mutation; Mycobacterium ulcerans; Quinolones
PubMed: 35041504
DOI: 10.1128/AAC.01902-21 -
ACS Omega May 2023In pursuit of new antitubercular agents, we here report the antimycobacterial (HRv) and DNA gyrase inhibitory potential of daidzein and khellin natural products (NPs)....
In pursuit of new antitubercular agents, we here report the antimycobacterial (HRv) and DNA gyrase inhibitory potential of daidzein and khellin natural products (NPs). We procured a total of 16 NPs based on their pharmacophoric similarities with known antimycobacterial compounds. The HRv strain of was found to be susceptible to only two out of the 16 NPs procured; specifically, daidzein and khellin each exhibited an MIC of 25 μg/mL. Moreover, daidzein and khellin inhibited the DNA gyrase enzyme with IC values of 0.042 and 0.822 μg/mL, respectively, compared to ciprofloxacin with an IC value of 0.018 μg/mL. Daidzein and khellin were found to have lower toxicity toward the vero cell line, with IC values of 160.81 and 300.23 μg/mL, respectively. Further, molecular docking study and MD simulation of daidzein indicated that it remained stable inside the cavity of DNA GyrB domain for 100 ns.
PubMed: 37179626
DOI: 10.1021/acsomega.3c00684 -
Virus Evolution 2022Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription,...
Type II DNA topoisomerases of the family A (Topo IIAs) are present in all Bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation, and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologs and have no orthologs in Archaea. Interestingly, eukaryotic Topo IIAs have close homologs in viruses of the phylum , an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses. Topo IIAs are also encoded by some bacterioviruses of the class (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses on a dataset combining viral and cellular Topo IIA homologs. Topo IIAs encoded by Bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by and , respectively. Importantly, remained well separated from eukaryotes after removing both Bacteria and from the data set, indicating that the separation of and eukaryotes is probably not due to long-branch attraction artifact. The topologies of our trees suggest that the eukaryotic Topo IIA was probably acquired from an ancestral member of the of the class , before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.
PubMed: 36533149
DOI: 10.1093/ve/veac097 -
Antimicrobial Agents and Chemotherapy Apr 2022Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class...
Described here is a series of spiropyrimidinetrione (SPT) compounds with activity against Mycobacterium tuberculosis through inhibition of DNA gyrase. The SPT class operates via a novel mode of inhibition, which involves Mg-independent stabilization of the DNA cleavage complex with DNA gyrase and is thereby not cross-resistant with other DNA gyrase-inhibiting antibacterials, including fluoroquinolones. Compound 22 from the series was profiled broadly and showed cidality as well as intracellular activity against M. tuberculosis in macrophages. Evidence for the DNA gyrase mode of action was supported by inhibition of the target in a DNA supercoiling assay and elicitation of an SOS response seen in a reporter strain of M. tuberculosis. Pharmacokinetic properties of 22 supported evaluation of efficacy in an acute model of M. tuberculosis infection, where modest reduction in CFU numbers was seen. This work offers promise for deriving a novel drug class of tuberculosis agent without preexisting clinical resistance.
Topics: DNA Gyrase; Fluoroquinolones; Humans; Mycobacterium tuberculosis; Topoisomerase II Inhibitors; Tuberculosis
PubMed: 35266826
DOI: 10.1128/aac.02192-21 -
The Journal of Biological Chemistry May 2023DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection....
DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and EC. We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5× slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.
Topics: Adenosine Triphosphate; DNA; DNA Gyrase; DNA, Superhelical; Escherichia coli; Mycobacterium tuberculosis; Molecular Dynamics Simulation
PubMed: 36775125
DOI: 10.1016/j.jbc.2023.103003 -
Scientific Reports Jul 2019Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in...
Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.
Topics: Cell Culture Techniques; DNA Gyrase; DNA Replication; DNA, Bacterial; Leprosy; Mycobacterium leprae
PubMed: 31346236
DOI: 10.1038/s41598-019-47364-5 -
Molecules (Basel, Switzerland) Mar 2024There is an urgent need to discover and develop novel antibacterial agents. Accordingly, we synthesised 2-(piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione (PNT), which...
There is an urgent need to discover and develop novel antibacterial agents. Accordingly, we synthesised 2-(piperazin-1-yl)naphtho[2,3-d]thiazole-4,9-dione (PNT), which exhibits antimicrobial activity. The aim of this study was to characterise PNT as an effective antimicrobial agent. Fluorescence microscopy was used to measure PNT's uptake into microbial cells (strains of , , and methicillin-resistant (MRSA)), transmission electron microscopy (TEM) was used to investigate the influence of PNT on the configuration of microbial cells, and a DNA gyrase supercoiling assay was used to investigate whether PNT inhibits DNA gyrase. PNT was taken up by more than 50% of microbial cells within 30 min. Using TEM, hollowed-out bacterial cytoplasms were observed in the specimen treated with PNT, although there was no disintegration of the bacterial membrane. In the DNA gyrase supercoiling assay, a dose-dependent reduction in fluorescence intensity was observed as the concentration of PNT increased. This suggests that PNT is taken up by microbial cells, resulting in cell disruption, and it reveals that one of the mechanisms underlying the antimicrobial activity of PNT is the inhibition of DNA gyrase.
Topics: Staphylococcus; Thiazoles; Methicillin-Resistant Staphylococcus aureus; DNA Gyrase; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 38542913
DOI: 10.3390/molecules29061277 -
Molecules (Basel, Switzerland) Feb 2021Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the... (Review)
Review
Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the treatment of bacterial infections. Inhibition of gyrase activity is achieved by competitive inhibitors that interfere with ATP- or DNA-binding, or by gyrase poisons that stabilize cleavage complexes of gyrase covalently bound to the DNA, leading to double-strand breaks and cell death. Many of the current inhibitors suffer from severe side effects, while others rapidly lose their antibiotic activity due to resistance mutations, generating an unmet medical need for novel, improved gyrase inhibitors. DNA supercoiling by gyrase is associated with a series of nucleotide- and DNA-induced conformational changes, yet the full potential of interfering with these conformational changes as a strategy to identify novel, improved gyrase inhibitors has not been explored so far. This review highlights recent insights into the mechanism of DNA supercoiling by gyrase and illustrates the implications for the identification and development of conformation-sensitive and allosteric inhibitors.
Topics: Bacteria; DNA Gyrase; Models, Molecular; Topoisomerase II Inhibitors
PubMed: 33669078
DOI: 10.3390/molecules26051234 -
BioRxiv : the Preprint Server For... Oct 2023Fluoroquinolones (FQs) are potent antibiotics of clinical significance, known for their unique mechanism of action as gyrase poisons, which stabilize gyrase-DNA cleavage...
Fluoroquinolones (FQs) are potent antibiotics of clinical significance, known for their unique mechanism of action as gyrase poisons, which stabilize gyrase-DNA cleavage complexes and convert gyrase into a DNA-damaging machinery. Unfortunately, FQ resistance has emerged, and these antibiotics can cause severe side effects. Therefore, discovering novel gyrase poisons with different chemical scaffolds is essential. The challenge lies in efficiently identifying them from compound libraries containing thousands or millions of drug-like compounds, as high-throughput screening (HTS) assays are currently unavailable. Here we report a novel fluorescence-based, T5 exonuclease-amplified DNA cleavage assay for gyrase poison discovery. This assay capitalizes on recent findings showing that multiple gyrase molecules can simultaneously bind to a plasmid DNA molecule, forming multiple gyrase-DNA cleavage complexes on the same plasmid. These gyrase-DNA cleavage complexes, stabilized by a gyrase poison, can be captured using sarkosyl. Proteinase K digestion results in producing small DNA fragments. T5 exonuclease, selectively digesting linear and nicked DNA, can fully digest the fragmented linear DNA molecules and, thus, "amplify" the decrease in fluorescence signal of the DNA cleavage products after SYBR Green staining. This fluorescence-based, T5 exonuclease-amplified DNA cleavage HTS assay is validated using a 50-compound library, making it suitable for screening large compound libraries.
PubMed: 37904923
DOI: 10.1101/2023.10.16.562555 -
The Journal of Biological Chemistry Aug 2021DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate...
DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.
Topics: Catalytic Domain; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Bacterial; DNA, Superhelical; Models, Molecular; Mutagenesis, Site-Directed; Pneumococcal Infections; Protein Structural Elements; Streptococcus pneumoniae
PubMed: 34303706
DOI: 10.1016/j.jbc.2021.101000