Astaxanthin-Mediated Bacterial Lethality: Evidence from Oxidative Stress Contribution and Molecular Dynamics Simulation

The involvement of cellular oxidative stress in antibacterial therapy has remained a topical issue over the years. In this study, the contribution of oxidative stress to astaxanthin-mediated bacterial lethality was evaluated in silico and in vitro. For the in vitro analysis, the minimum inhibitory concentration (MIC) of astaxanthin was lower than that of novobiocin against Staphylococcus aureus but generally higher than those of the reference antibiotics against other test organisms. The level of superoxide anion of the tested organisms increased significantly following treatment with astaxanthin when compared with DMSO-treated cells. This increase compared favorably with those observed with the reference antibiotics and was consistent with a decrease in the concentration of glutathione (GSH) and corresponding significant increase in ADP/ATP ratio. These observations are suggestive of probable involvement of oxidative stress in antibacterial capability of astaxanthin and in agreement with the results of the in silico evaluations, where the free energy scores of astaxanthins’ complexes with topoisomerase IV ParC and ParE were higher than those of the reference antibiotics. These observations were consistent with the structural stability and compactness of the complexes as astaxanthin was observed to be more stable against topoisomerase IV ParC and ParE than DNA Gyrase A and B. Put together, findings from this study underscored the nature and mechanism of antibacterial action of astaxanthin that could suggest practical approaches in enhancing our current knowledge of antibacterial arsenal and aid in the novel development of alternative natural topo2A inhibitor.

Evaluation of the Inhibitory Effects of Coumermycin A1 on the Growth of Theileria and Babesia Parasites in vitro and in vivo

Coumermycin A1, a coumarin antibiotic, has anticancer, antibacterial, antiviral, and antimalarial activities. We aimed to evaluate the anti-thielerial and anti-babesial activity of coumermycin A1 in mice in vivo. Coumermycin A1 efficacy was determined by the transcription of DNA gyrase, a type II DNA topoisomerase using reverse transcriptase-polymerase chain reaction (RT-PCR) transcription. Coumermycin A1 significantly inhibited the development of preliminary parasitemia (1%). Theileria equi and the Babesia species B. bigemina, B. bovis, and B. caballi were observed with IC50 values of 80, 70, 57, and 65 nM, respectively. Their development was remarkably inhibited at observed concentrations of 10, 25, 50, and 100µM for the studied organisms T. equi, and the Babesia species B. caballi, B. bovis and B. bigemina, respectively. In the subsequent viability test, parasite re-growth was suppressed at 100µM for B. bigemina and B. bovis and at 50 µM for B. caballi and T. equi. Coumermycin A1 Treatment of B. bovis cultures with Coumermycin A1 completely suppressed the transcription of the DNA gyrase subunits B and A genes. In BALB/c mice, the development of Babesia microti was inhibited by 70.73% using 5 mg/kg of Coumermycin A1.

The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

Synthesis, spectral characterization, antimicrobial activity and docking studies against DNA Gyrase-A of new 4-chloro-3-nitrobenzene sulfonamide derivatives

A series of new 4-chloro-3-nitrobenzene sulfonamide derivatives 3(a-j) were synthesized from 4-chloro-3-nitrobenzene sulfonyl chloride by reacting various amino acid esters and amines 2(a-j) in high yields. The structures of all the synthesized compounds were characterized by the IR, NMR (1H & 13C), mass and elemental analyses. Further, all the synthesized compounds were tested for the antimicrobial activity and docking studies were carried out with DNA Gyrase-A. Most of the compounds showed good to moderate antimicrobial activities and binding affinity towards DNA Gyrase-A structure.

Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation

The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity.

In silico Screening of Approved Drugs to Describe Novel E. coli DNA Gyrase A Antagonists

The Green House Gas Emission (GHGs) from the carp culture ponds (n = 12) of West Godavari, Krishna, and Guntur districts of Andhra Pradesh and from the ponds (n = 4) of Moyna, East Medinipur district of West Bengal, India was assessed through carbon storage and carbon footprint analysis. The average inputs as Carbon Equivalent (CE) were 14407 ± 2651, and 9231 ± 1007 kg/ha in Andhra Pradesh, and West Bengal, respectively. The average carbon storage were 6216 ± 2291, and 5360 ± 1439 kg/ha, in Andhra Pradesh, and Moyna, West Bengal respectively. The emissions of CO2-e and CH4-e were 1.91 ± 0.42 kg CO2-e/kg fish and 0.122 ± 0.027 kg CH4-e/kg fish, respectively in Andhra Pradesh. The emissions of CO2-e and CH4-e were 0.006 to 2.07 (average 0.72) kg CO2-e /kg fish, and 0.0004 to 0.132 (average 0.046) kg CH4-e /kg fish production, respectively in Moyna, West Bengal.

Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

ABSTRACTDNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of “poisons”, namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

Diversity and Distribution of a Novel Genus of Hyperthermophilic Aquificae Viruses Encoding a Proof-reading Family-A DNA Polymerase

ABSTRACTDespite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional species, Thermocrinis Great Boiling Spring virus (TGBSV), Aquificae Joseph’s Coat Spring Virus (AJCSV), and Aquificae Conch Spring Virus (ACSV). Four near-complete UViGs, ranged from 37,256 bp to 41,208 bp and encoded 48 to 53 open reading frames. Despite low overall similarity between viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a polyprotein PolA-type polymerase with likely accessory functions, a DNA Pol III beta subunit (sliding clamp), a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsids, large and small terminases, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. TOSV and TGBSV had the highest coverage in their respective metagenomes and are predicted to infect Thermocrinis ruber and Thermocrinis jamiesonii, respectively, as multiple CRISPR spacers matching the viral genomes were identified within Thermocrinis ruber OC1/4T and Thermocrinis jamiesonii GBS1T. Based on the predicted, unusual bi-directional replication strategy, low sequence similarity to known viral genomes, and a unique position in gene-sharing networks, we propose a new putative genus, Pyrovirus, in the order Caudovirales.