Revealing the Interaction Mechanism between Mycobacterium tuberculosis GyrB and Novobiocin, SPR719 through Binding Thermodynamics and Dissociation Kinetics Analysis.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

POLQ inhibition attenuates the stemness and ferroptosis resistance in gastric cancer cells via downregulation of dihydroorotate dehydrogenase.

Gastric cancer (GC) contains subpopulations of cancer stem cells (CSCs), which are described as the main contributors in tumor initiation and metastasis. It is necessary to clarify the molecular mechanism underlying CSCs phenotype and develop novel biomarkers and therapeutic targets for gastric cancer. Here, we show that POLQ positively regulates stem cell-like characteristics of gastric cancer cells, knockdown of POLQ suppressed the stemness of GC cells in vitro and in vivo. Further mechanistic studies revealed that POLQ knockdown could downregulate the expression of dihydroorotate dehydrogenase (DHODH). DHODH overexpression rescued the reduced stemness resulted by POLQ knockdown. Furthermore, we found that POLQ expression correlated with resistance to ferroptosis, and POLQ inhibition renders gastric cancer cells more vulnerable to ferroptosis. Further investigation revealed that POLQ regulated DHODH expression via the transcription factors E2F4, thereby regulating ferroptosis resistance and stemness of gastric cancer cells. Given the importance of POLQ in stemness and ferroptosis resistance of GC, we further evaluated the therapeutic potential of POLQ inhibitor novobiocin, the results show that novobiocin attenuates the stemness of GC cells and increased ferroptosis sensitivity. Moreover, the combination of POLQ inhibitor and ferroptosis inducer synergistically suppressed MGC-803 xenograft tumor growth and diminished metastasis. Our results identify a POLQ-mediated stemness and ferroptosis defense mechanism and provide a new therapeutic strategy for gastric cancer.

Antimicrobial susceptibility of mastitis pathogens isolated from North American dairy cattle, 2011-2022.

A total of 10,890 bacterial isolates of Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Escherichia coli isolated as etiological agents from dairy cows with mastitis by 29 veterinary laboratories across North America between 2011 and 2022 were tested for in vitro antimicrobial susceptibility by broth microdilution to ampicillin, cefoperazone, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin-novobiocin and pirlimycin according to CLSI standards. Using available clinical breakpoints, antimicrobial resistance among S. dysgalactiae (n = 2406) was low for penicillin-novobiocin (0% resistance), ceftiofur (0.1%), erythromycin (3.2%) and pirlimycin (4.6%). Among S. uberis (n = 2398), resistance was low for ampicillin (0%) and ceftiofur (0.2%) and moderate for erythromycin (11.9%) and pirlimycin (18.4%). For S. aureus (n = 3194), resistance was low for penicillin-novobiocin (0%), ceftiofur (0.1%), oxacillin (0.2%), erythromycin (0.7%), cefoperazone (1.2%) and pirlimycin (2.8%). For E. coli (n = 2892), resistance was low for ceftiofur (2.8%) and cefoperazone (3.4%) and moderate for ampicillin (9.2%). Overall, the results indicate that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to antimicrobial drugs over the 12 years of the study, or among that of the proceeding survey from 2002-2010. The data support the conclusion that resistance to common antimicrobial drugs among mastitis pathogens, even to drugs that have been used in dairies for mastitis management for many years, continues to remain low.

Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics.

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon-controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

A Progressively Disassembled DNA Repair Inhibitors Nanosystem for the Treatment of BRCA Wild-Type Triple-Negative Breast Cancer.

Background: Olaparib, a poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor has demonstrated promising efficacy in patients with triple-negative breast cancer (TNBC) carrying breast cancer gene (BRCA) mutations. However, its impact on BRCA wild-type (BRCAwt) TNBC is limited. Hence, it is crucial to sensitize BRCAwt TNBC cells to olaparib for effective clinical practice. Novobiocin, a DNA polymerase theta (POLθ) inhibitor, exhibits sensitivity towards BRCA-mutated cancer cells that have acquired resistance to PARP inhibitors. Although both of these DNA repair inhibitors demonstrate therapeutic efficacy in BRCA-mutated cancers, their nanomedicine formulations' antitumor effects on wild-type cancer remain unclear. Furthermore, ensuring effective drug accumulation and release at the cancer site is essential for the clinical application of olaparib. Materials and Methods: Herein, we designed a progressively disassembled nanosystem of DNA repair inhibitors as a novel strategy to enhance the effectiveness of olaparib in BRCAwt TNBC. The nanosystem enabled synergistic delivery of two DNA repair inhibitors olaparib and novobiocin, within an ultrathin silica framework interconnected by disulfide bonds. Results: The designed nanosystem demonstrated remarkable capabilities, including long-term molecular storage and specific drug release triggered by the tumor microenvironment. Furthermore, the nanosystem exhibited potent inhibitory effects on cell viability, enhanced accumulation of DNA damage, and promotion of apoptosis in BRCAwt TNBC cells. Additionally, the nanosystem effectively accumulated within BRCAwt TNBC, leading to significant growth inhibition and displaying vascular regulatory abilities as assessed by magnetic resonance imaging (MRI). Conclusion: Our results provided the inaugural evidence showcasing the potential of a progressively disassembled nanosystem of DNA repair inhibitors, as a promising strategy for the treatment of BRCA wild-type triple-negative breast cancer.

Novobiocin blocks nucleic acid binding to Polθ and inhibits stimulation of its ATPase activity.

Polymerase theta (Polθ) acts in DNA replication and repair, and its inhibition is synthetic lethal in BRCA1 and BRCA2-deficient tumor cells. Novobiocin (NVB) is a first-in-class inhibitor of the Polθ ATPase activity, and it is currently being tested in clinical trials as an anti-cancer drug. Here, we investigated the molecular mechanism of NVB-mediated Polθ inhibition. Using hydrogen deuterium exchange-mass spectrometry (HX-MS), biophysical, biochemical, computational and cellular assays, we found NVB is a non-competitive inhibitor of ATP hydrolysis. NVB sugar group deletion resulted in decreased potency and reduced HX-MS interactions, supporting a specific NVB binding orientation. Collective results revealed that NVB binds to an allosteric site to block DNA binding, both in vitro and in cells. Comparisons of The Cancer Genome Atlas (TCGA) tumors and matched controls implied that POLQ upregulation in tumors stems from its role in replication stress responses to increased cell proliferation: this can now be tested in fifteen tumor types by NVB blocking ssDNA-stimulation of ATPase activity, required for Polθ function at replication forks and DNA damage sites. Structural and functional insights provided in this study suggest a path for developing NVB derivatives with improved potency for Polθ inhibition by targeting ssDNA binding with entropically constrained small molecules.

HSP90 C-terminal domain inhibition promotes VDAC1 oligomerization via decreasing K274 mono-ubiquitination in Hepatocellular Carcinoma.

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

Antibacterial potential of Thymus linearis essential oil collected from Wasturwan mountain: A combination of experimental and theoretical studies involving in silico molecular docking simulation of the major compounds against Novobiocin-resistant mutant of DNA Gyrase-B.

Antibiotic resistant bacteria are immune to most antibiotics and are therefore very difficult to treat and in most cases lead to death. As such there is a pressing need for alternative and more efficient antibacterial drugs which can target these drug-resistant strains as well. The objective of this research work was to investigate the antibacterial properties of Thymus linearis essential oil (EO) against multiple disease-causing bacterial pathogens. Additionally, the study aimed to examine the molecular docking and molecular dynamic (MD) simulations of the primary components of the EO with the essential bacterial proteins and enzymes. Gas chromatography-mass spectrometry was employed to analyse the chemical composition of Thymus linearis EO. The initial screening for antibacterial properties involved the use of disc diffusion and microdilution techniques. Molecular docking studies were conducted utilising Autodock Vina. The outcomes were subsequently visualised through BIOVIA Discovery Studio. MD simulations were conducted using iMODS, an internet-based platform designed for MD simulations. The essential oil (EO) was found to contain 26 components, with thymol, carvacrol, p-cymene, and γ-terpinene being the primary constituents. The study findings revealed that Thymus linearis EO demonstrated antibacterial effects that were dependent on both the dose and time. The results of molecular docking studies revealed that the primary constituents of the EO, namely thymol, carvacrol, and p-cymene, exhibited robust interactions with the active site of the bacterial DNA gyrase enzyme. This finding provides an explanation for the antibacterial mechanism of the EO. The results indicate that Thymus linearis EO possesses potent antibacterial properties against the MDR microorganisms. Molecular docking analyses revealed that the essential oil's primary components interact with the amino acid residues of the DNA-Gyrase B enzyme, resulting in a favourable docking score.

Combinational Effect of Selected Medicinal Plants and Antibiotics Against Pathogenic Bacteria.

<b>Background and Objective:</b> The emergence of antibiotic resistance is a primary global health concern. As a result, there is an urgent need for new strategies to combat antibiotic-resistant bacteria. One of these essential strategies is the combination of medicinal plants and antibiotics as an alternative to using antibiotics alone which was the objective of this article. <b>Materials and Methods:</b> Nine plant materials were collected from different Egypt localities and then extracted by water. Water extracts were filtered and added with Mueller-Hinton agar during preparation. Nine test bacteria and 13 standard antibiotics were used in the disc diffusion sensitivity method. <b>Results:</b> The activity of Amikacin was increased when combined with most different plant extracts against <i>Escherichia coli</i> while antagonistic against <i>Pseudomonas aeruginosa</i>. Aztreonam, Ceftriaxone, Gentamicin and Nalidixic acid antibiotics showed antagonistic or indifferent effects when combined with most different plant extracts against <i>E. coli</i>, <i>Klebsiella pneumonia</i> and <i>P. aeruginosa</i>. The synergistic effect was achieved in Aztreonam when combined with all plant extracts, while Nalidixic acid showed antagonistic when combined with most plant extracts against <i>Proteus mirabilis</i>. The antagonistic effect was achieved in Aztreonam, Ceftriaxone and Nalidixic acid when combined with <i>Achillea fragrantissima</i>, <i>Artemisia monosperma</i> and <i>Leptadenia pyrotechnica</i>, also Aztreonam with <i>Lycium shawii</i> extract against <i>Salmonella typhimurium</i>. The <i>A. fragrantissima</i> and <i>A. monosperma</i> increase the activity of Novobiocin and Vancomycin against <i>Bacillus cereus</i> and Ampicillin and Cefazolin against <i>Staphylococcus aureus</i> but Novobiocin activity increased with most plant extracts against <i>S. aureus</i>. <b>Conclusion:</b> The combinations of antibiotics with the extracts of medicinal plants displayed varying degrees of effects, synergistic, antagonistic and indifferent according to antibiotic type, plant extract and test organism.

Synthesis of novobiocin derivatives and evaluation of their antigonococcal activity and pharmacokinetics.

Gonorrhea has become a serious problem because the number of infected people is increasing and the multi-drug resistance of the causative bacteria, Neisseria gonorrhoeae, is progressing. To develop novel drugs against resistant N. gonorrhoeae, we focused on the antibiotic novobiocin (1). This natural product has a different mechanism of action from existing drugs for gonorrhea, which may make it effective against resistant strains. Actually, it was applied to resistant N. gonorrhoeae, and moderate antibacterial activity was confirmed. Based on this result, we investigated the development of an antigonococcal drug with 1 as the lead compound. The pharmacophore is thought to be the noviose sugar moiety, especially around the 3'-position, so we derivatized this part in order to improve antibacterial activity. As a result, we found that 5 with an methylpyrrole ester structure have a very potent antibacterial activity. This derivative also showed excellent antigonococcal activity against resistant strains in vitro, however it has poor water solubility and pharmacokinetics because it is the acidic lipid-soluble compound. Therefore, we considered introduction of a basic substituent into the molecule would result in an amphoteric compound with improved water solubility, and we investigated further derivatization. As a result of synthesizing various derivatives, we found 47 containing imidazole with strong antigonococcal activity and greatly improved water solubility. This derivative has also improved metabolism and blood concentration in vivo, and is expected to be orally absorbed. Based on these results, we believe that 47 is a very promising anti-gonococcal lead compound and has great potential for further development.

Disparate properties of Burkholderia multivorans and Pseudomonas aeruginosa regarding outer membrane chemical permeabilization to the hydrophobic substances novobiocin and triclosan.

Burkholderia multivorans causes opportunistic pulmonary infections and is intrinsically resistant to many antibacterial compounds including the hydrophobic biocide triclosan. Chemical permeabilization of the Pseudomonas aeruginosa outer membrane affects sensitization to hydrophobic substances. The purpose of the present study was to determine if B. multivorans is similarly susceptive suggesting that outer membrane impermeability properties underlie triclosan resistance. Antibiograms and conventional macrobroth dilution bioassays were employed to establish baseline susceptibility levels to hydrophobic antibacterial compounds. Outer membrane permeabilizers compound 48/80, polymyxin B, polymyxin B-nonapeptide, and ethylenediaminetetraacetic acid were used in attempts to sensitize disparate B. multivorans isolates to the hydrophobic agents novobiocin and triclosan, and to potentiate partitioning of the hydrophobic fluorescent probe 1-N-phenylnapthylamine (NPN). The lipophilic agent resistance profiles for all B. multivorans strains were essentially the same as that of P. aeruginosa except that they were resistant to polymyxin B. Moreover, they resisted sensitization to hydrophobic compounds and remained inaccessible to NPN when treated with outer membrane permeabilizers. These data support the notion that while both phylogenetically-related organisms exhibit general intrinsic resistance properties to hydrophobic substances, the outer membrane of B. multivorans either resists permeabilization by chemical modification or sensitization is mitigated by a supplemental mechanism not present in P. aeruginosa.

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae.

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

CYP broth: a tool for Yersinia pestis isolation in ancient culture collections and field samples.

We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

Targeting novel sites in DNA gyrase for development of anti-microbials.

Antimicrobial resistance in bacteria poses major challenges in selection of the therapeutic regime for managing the infectious disease. There is currently an upsurge in the appearance of multiple drug resistance in bacterial pathogens and a decline in the discovery of novel antibiotics. DNA gyrase is an attractive target used for antibiotic discovery due to its vital role in bacterial DNA replication and segregation in addition to its absence in mammalian organisms. Despite the presence of successful antibiotics targeting this enzyme, there is a need to bypass the resistance against this validated drug target. Hence, drug development in DNA gyrase is a highly active research area. In addition to the conventional binding sites for the novobiocin and fluoroquinolone antibiotics, several novel sites are being exploited for drug discovery. The binding sites for novel bacterial type II topoisomerase inhibitor (NBTI), simocyclinone, YacG, Thiophene and CcdB are structurally and biochemically validated active sites, which inhibit the supercoiling activity of topoisomerases. The novel chemical moieties with varied scaffolds have been identified to target DNA gyrase. Amongst them, the NBTI constitutes the most advanced DNA gyrase inhibitor which are in phase III trial of drug development. The present review aims to classify the novel binding sites other than the conventional novobiocin and quinolone binding pocket to bypass the resistance due to mutations in the DNA gyrase enzyme. These sites can be exploited for the identification of new scaffolds for the development of novel antibacterial compounds.

Whole-genome metagenomic analysis of the oral microbiota in patients with obstructive sleep apnea.

PURPOSE: The oral microbiota is closely associated with systemic health, but few studies have investigated the oral microbiota in patients with obstructive sleep apnea (OSA). This study aimed to identify the variation of oral microbiota among patients with severe OSA, and the change of oral microbiota after treatment with continuous positive airway pressure (CPAP). METHODS: Participants were enrolled in the study from November 2020 to August 2021. Sleep parameters using full nocturnal polysomnography (PSG) were collected on healthy controls, patients with severe OSA, and patients with severe OSA after CPAP treatment for 3 months. Oral samples were also collected by rubbing disposable medical sterile swabs on the buccal mucosa. Routine blood tests and biochemical indicators were measured using the fully automated biochemical analyzer. Oral microbial composition of oral samples were determined using whole-genome metagenomic analysis in all participants. Correlations were analyzed between the oral microbiota and blood lipids. RESULTS: Study enrollment included 14 participants, 7 healthy controls and 7 patients with severe OSA. At the species level, the relative abundances of Prevotella, Alloprevotella, Bacteroides, Veillonella_tobetsuensis, Candidatus saccharimonas, and Leptotrichia in the groups with severe OSA were significantly lower than those in the healthy controls (P both < 0.05). The abundances of Capnocytophaga, Veillonella, Bacillus_anthracis, Eikenella, and Kingella were significantly higher whereas the abundances of Gordonia and Streptococcus were significantly lower in the group with severe OSA compared to the severe OSA-CPAP group (P < 0.05 for both). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), 4 pathways changed in the group with severe OSA compared with healthy controls (P both < 0.05). Pathways related to Novobiocin biosynthesis, 2-Oxocarboxylic acid metabolism, and Histidine metabolism were enriched in the patients with severe OSA. Nine pathways showed significant differences with regard to the relative abundances of phenylalanine metabolism; alanine, aspartate, and glutamate metabolism; one carbon pool by folate; monobactam biosynthesis; 2-oxocarboxylic acid metabolism; arginine biosynthesis and vitamin B6 metabolism; novobiocin biosynthesis; and arginine and proline metabolism, which were significantly higher in the group with severe OSA compared to the severe OSA-CPAP group (P both < 0.05). The Spearman correlation analysis between blood lipid parameters and oral microbiota components showed that negative correlations were observed between total cholesterol and Streptomyces (r = - 0.893, P = 0.007), and high-density lipoprotein cholesterol (HDL-C) and Gordonia (r = - 0.821, P = 0.023); positive correlations were observed between HDL-C and Candidatus saccharimonas (r = 0.929, P = 0.003), and low-density lipoprotein cholesterol (LDL-C) and Capnocytophaga (r = 0.893, P = 0.007). CONCLUSION: There was an apparent discrepancy of the oral microbiota and metabolic pathways between the group with severe OSA and controls, and CPAP significantly changed oral microbial abundance and metabolic pathways in patients with severe OSA. Correlation analysis showed that these oral bacteria were strongly correlated with the blood lipids level.

CycA-Dependent Glycine Assimilation Is Connected to Novobiocin Susceptibility in Escherichia coli.

Escherichia coli serine hydroxymethyltransferase (GlyA) converts serine to glycine, and glyA mutants are auxotrophic for glycine. CycA is a transporter that mediates glycine uptake. Deleting glyA in E. coli strain W3110 led to activation of CysB, which was related to novobiocin (NOV) susceptibility. Moreover, deleting glyA resulted in increased sensitivity to NOV, and this could be reversed by high concentrations of glycine. Reverse mutants of ΔglyA were selected and one of them had a mutation in yrdC, the gene encoding threonylcarbamoyl-AMP synthase. Subsequent proteome analysis showed that deleting glyA led to increased expression of TcyP and TdcB, making this bacterium dependent on CycA for glycine assimilation. Furthermore, deleting cycA in a ΔglyA background caused a severe growth defect on Luria-Bertani medium, which could be complemented by high concentrations of exogenous glycine. Mutation of yrdC led to decreased expression of TdcB but increased expression of ThrA/B/C and LtaE, which favored the conversion of threonine to glycine and thus avoided the dependence on CycA. Correspondingly, deleting of tcyP, tdcB, or gshA could reverse the NOV-sensitive phenotype of ΔglyA mutants. Overexpression of cycA resulted in increased sensitivity to NOV, whereas deleting this gene caused NOV resistance. Moreover, overexpression of cycA led to increased accumulation of NOV upon drug treatment. Therefore, inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which enhanced the accumulation of NOV and then made the bacterium more sensitive to this drug. These findings broaden our understanding of glycine metabolism and mechanisms of NOV susceptibility. IMPORTANCE Novobiocin (NOV) has been used in clinical practice as an ATPase inhibitor for decades. However, because it has been withdrawn from the market, pharmaceutical companies are searching for other ATPase inhibitors. Thus, probing the mechanisms of susceptibility to NOV will be beneficial to those efforts. In this study, we showed that inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which accompanied the accumulation of NOV and thereby increased the sensitivity to this drug. To date, this is the first report demonstrating the linkage between glycine assimilation and NOV susceptibility, and it is also the first report showing that YrdC is able to modulate the metabolic flux of threonine.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Methanobacterium alkalithermotolerans sp. nov., a novel alkaliphilic and hydrogen-utilizing methanogen isolated from an alkaline geothermal spring (La Crouen, New Caledonia).

An anaerobic, hydrogenotrophic methane-producing archaeon was isolated from an alkaline thermal spring (42 °C, pH 9.0) in New Caledonia. This methanogen, designated strain CANT, is alkaliphilic, thermotolerant, with Gram-positive staining non-motile cells. Strain CANT grows autotrophically using hydrogen exclusively as an energy source and carbon dioxide as the sole carbon source (without the requirement of yeast extract or other organic compounds). It grows at 20-45 °C (optimum, 45 °C) and pH 7.3-9.7 (optimum, pH 9.0). NaCl is not required for growth (optimum 0 %) but is tolerated up to 1.5 %. It resists novobiocin, streptomycin and vancomycin but is inhibited by ampicillin and penicillin, among other antibiotics. The genome consists of a circular chromosome (2.2 Mb) containing 2126 predicted protein-encoding genes with a G+C content of 36.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain CANT is a member of the genus Methanobacterium, most closely related to the alkaliphilic Methanobacterium alcaliphilum WeN4T with 98.5 % 16S rRNA gene sequence identity. The genomes of strain CANT and M. alcaliphilum DSM 3459, sequenced in this study, share 71.6 % average nucleotide identity and 14.0 % digital DNA-DNA hybridization. Therefore, phylogenetic and physiological results indicate that strain CANT represents a novel species, for which the name Methanobacterium alkalithermotolerans sp. nov. is proposed, and strain CANT (=DSM 102889T= JCM 31304T) is assigned as the type strain.

Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump.

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters. IMPORTANCE RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a cationic antibiotic-sensitizing peptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.

Genetic and Chemical Screening Reveals Targets and Compounds to Potentiate Gram-Positive Antibiotics against Gram-Negative Bacteria.

Gram-negative bacteria are intrinsically resistant to a plethora of antibiotics that effectively inhibit the growth of Gram-positive bacteria. The intrinsic resistance of Gram-negative bacteria to classes of antibiotics, including rifamycins, aminocoumarins, macrolides, glycopeptides, and oxazolidinones, has largely been attributed to their lack of accumulation within cells due to poor permeability across the outer membrane, susceptibility to efflux pumps, or a combination of these factors. Due to the difficulty in discovering antibiotics that can bypass these barriers, finding targets and compounds that increase the activity of these ineffective antibiotics against Gram-negative bacteria has the potential to expand the antibiotic spectrum. In this study, we investigated the genetic determinants for resistance to rifampicin, novobiocin, erythromycin, vancomycin, and linezolid to determine potential targets of antibiotic-potentiating compounds. We subsequently performed a high-throughput screen of ∼50,000 diverse, synthetic compounds to uncover molecules that potentiate the activity of at least one of the five Gram-positive-targeting antibiotics. This led to the discovery of two membrane active compounds capable of potentiating linezolid and an inhibitor of lipid A biosynthesis capable of potentiating rifampicin and vancomycin. Furthermore, we characterized the ability of known inhibitors of lipid A biosynthesis to potentiate the activity of rifampicin against Gram-negative pathogens.