Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide’s antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a ‘pair of swing-doors’ hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1’s bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested

C. elegans Germline-Deficient Mutants Respond to Pathogen Infection Using Shared and Distinct Mechanisms

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to Gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including Gram positive and Gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16

Cigarette smoking, nicotine dependence and anxiety disorders : a systematic review of population-based, epidemiological studies

Background Multiple studies have demonstrated that rates of smoking and nicotine dependence are increased in individuals with anxiety disorders. However, significant variability exists in the epidemiological literature exploring this relationship, including study design (cross-sectional versus prospective), the population assessed (random sample versus clinical population) and diagnostic instrument utilized.Methods We undertook a systematic review of population-based observational studies that utilized recognized structured clinical diagnostic criteria (Diagnostic and Statistical Manual of Mental Disorders (DSM) or International Classification of Diseases (ICD)) for anxiety disorder diagnosis to investigate the relationship between cigarette smoking, nicotine dependence and anxiety disorders.Results In total, 47 studies met the predefined inclusion criteria, with 12 studies providing prospective information and 5 studies providing quasiprospective information. The available evidence suggests that some baseline anxiety disorders are a risk factor for initiation of smoking and nicotine dependence, although the evidence is heterogeneous and many studies did not control for the effect of comorbid substance use disorders. The identified evidence however appeared to more consistently support cigarette smoking and nicotine dependence as being a risk factor for development of some anxiety disorders (for example, panic disorder, generalized anxiety disorder), although these findings were not replicated in all studies. A number of inconsistencies in the literature were identified.Conclusions Although many studies have demonstrated increased rates of smoking and nicotine dependence in individuals with anxiety disorders, there is a limited and heterogeneous literature that has prospectively examined this relationship in population studies using validated diagnostic criteria. The most consistent evidence supports smoking and nicotine dependence as increasing the risk of panic disorder and generalized anxiety disorder. The literature assessing anxiety disorders increasing smoking and nicotine dependence is inconsistent. Potential issues with the current literature are discussed and directions for future research are suggested

WSES Jerusalem guidelines for diagnosis and treatment of acute appendicitis

Acute appendicitis (AA) is among the most common cause of acute abdominal pain. Diagnosis of AA is challenging; a variable combination of clinical signs and symptoms has been used together with laboratory findings in several scoring systems proposed for suggesting the probability of AA and the possible subsequent management pathway. The role of imaging in the diagnosis of AA is still debated, with variable use of US, CT and MRI in different settings worldwide. Up to date, comprehensive clinical guidelines for diagnosis and management of AA have never been issued. In July 2015, during the 3rd World Congress of the WSES, held in Jerusalem (Israel), a panel of experts including an Organizational Committee and Scientific Committee and Scientific Secretariat, participated to a Consensus Conference where eight panelists presented a number of statements developed for each of the eight main questions about diagnosis and management of AA. The statements were then voted, eventually modified and finally approved by the participants to The Consensus Conference and lately by the board of co-authors. The current paper is reporting the definitive Guidelines Statements on each of the following topics: 1) Diagnostic efficiency of clinical scoring systems, 2) Role of Imaging, 3) Non-operative treatment for uncomplicated appendicitis, 4) Timing of appendectomy and in-hospital delay, 5) Surgical treatment 6) Scoring systems for intra-operative grading of appendicitis and their clinical usefulness 7) Non-surgical treatment for complicated appendicitis: abscess or phlegmon 8) Pre-operative and post-operative antibiotics.Peer reviewe

Molecular Evolution Perspectives on Intraspecific Lateral DNA Transfer of Topoisomerase and Gyrase Loci in Streptococcus pneumoniae, with Implications for Fluoroquinolone Resistance Development and Spread

Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread

Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in Staphylococcus aureus

Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA

Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, ΔH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme