21 research outputs found
Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates
The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNAāprotein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time
Endonuclease II - a GIY-YIG enzyme of bacteriophage T4
Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding. It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them
Endonuclease II - a GIY-YIG enzyme of bacteriophage T4
Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding. It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them
Endonuclease II - a GIY-YIG enzyme of bacteriophage T4
Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding. It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them
Evaluation of In Vitro Activity of Double-Carbapenem Combinations against KPC-2-, OXA-48- and NDM-Producing Escherichia coli and Klebsiella pneumoniae
Double-carbapenem combinations have shown synergistic potential against carbapenemase-producing Enterobacterales, but data remain inconclusive. This study evaluated the activity of double-carbapenem combinations against 51 clinical KPC-2-, OXA-48-, NDM-1, and NDM-5-producing Escherichia coli and Klebsiella pneumoniae and against constructed E. coli strains harboring genes encoding KPC-2, OXA-48, or NDM-1 in an otherwise isogenic background. Two-drug combinations of ertapenem, meropenem, and doripenem were evaluated in 24 h time-lapse microscopy experiments with a subsequent spot assay and in static time-kill experiments. An enhanced effect in time-lapse microscopy experiments at 24 h and synergy in the spot assay was detected with one or more combinations against 4/14 KPC-2-, 17/17 OXA-48-, 2/17 NDM-, and 1/3 NDM-1+OXA-48-producing clinical isolates. Synergy rates were higher against meropenem- and doripenem-susceptible isolates and against OXA-48 producers. NDM production was associated with significantly lower synergy rates in E. coli. In time-kill experiments with constructed KPC-2-, OXA-48- and NDM-1-producing E. coli, 24 h synergy was not observed; however, synergy at earlier time points was found against the KPC-2- and OXA-48-producing constructs. Our findings indicate that the benefit of double-carbapenem combinations against carbapenemase-producing E. coli and K. pneumoniae is limited, especially against isolates that are resistant to the constituent antibiotics and produce NDM
A Novel Microfluidic Assay for Rapid Phenotypic Antibiotic Susceptibility Testing of Bacteria Detected in Clinical Blood Cultures
Background Appropriate antibiotic therapy is critical in the management of severe sepsis and septic shock to reduce mortality, morbidity and health costs. New methods for rapid antibiotic susceptibility testing are needed because of increasing resistance rates to standard treatment. Aims The purpose of this study was to evaluate the performance of a novel microfluidic method and the potential to directly apply this method on positive blood cultures. Methods Minimum inhibitory concentrations (MICs) of ciprofloxacin, ceftazidime, tigecycline and/or vancomycin for Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus were determined using a linear antibiotic concentration gradient in a microfluidic assay. Bacterial growth along the antibiotic gradient was monitored using automated time-lapse photomicrography and growth inhibition was quantified by measuring greyscale intensity changes in the images. In addition to pure culture MICs, vancomycin MICs were determined for S. aureus from spiked and clinical blood cultures following a short centrifugation step. The MICs were compared with those obtained with the Etest and for S. aureus and vancomycin also with macrodilution. Results The MICs obtained with the microfluidic assay showed good agreement internally as well as with the Etest and macrodilution assays, although some minor differences were noted between the methods. The time to possible readout was within the range of 2 to 5 h. Conclusions The examined microfluidic assay has the potential to provide rapid and accurate MICs using samples from positive clinical blood cultures and will now be tested using other bacterial species and antibiotics
Activity of polymyxin B combinations against genetically well-characterised Klebsiella pneumoniae producing NDM-1 and OXA-48-like carbapenemases
Background: Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae ( K. pneumoniae). Methods: Twenty clinical K. pneumoniae strains producing NDM-1 (n = 8), OXA-48-like (n = 10), or both NDM-1 and OXA-48-like (n = 2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influ-encing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. Results: Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocy-cline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P = 0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P = 0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. Conclusion: Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype as-sociations were found. Such information might accelerate the search for promising combinations and guide individualised treatment
Combination of polymyxin B and minocycline against multidrug-resistant Klebsiella pneumoniae : interaction quantified by pharmacokinetic/pharmacodynamic modelling from in vitro data
Lack of effective treatment for multidrug-resistant Klebsiella pneumoniae (MDR-Kp) necessitates finding and optimising combination therapies of old antibiotics. The aims of this study were to quantify the combined effect of polymyxin B and minocycline by building an in silico semi-mechanistic pharmacokinetic/pharmacodynamic (PKPD) model and to predict bacterial kinetics when exposed to the drugs alone and in combination at clinically achievable unbound drug concentration-time profiles. A clinical K. pneumoniae strain resistant to polymyxin B [minimum inhibitory concentration (MIC) = 16 mg/L] and minocycline (MIC = 16 mg/L) was selected for extensive in vitro static time-kill experiments. The strain was exposed to concentrations of 0.0625-48 ? MIC, with seven samples taken per experiment for viable counts during 0-28 h. These observations allowed the development of the PKPD model. The final PKPD model included drug-induced adaptive resistance for both drugs. Both the minocycline-induced bacterial killing and resistance onset rate constants were increased when polymyxin B was co-administered, whereas polymyxin B parameters were unaffected. Predictions at clinically used dosages from the developed PKPD model showed no or limited antibacterial effect with monotherapy, whilst combination therapy kept bacteria below the starting inoculum for 20 h at high dosages [polymyxin B 2.5 mg/kg + 1.5 mg/kg every 12 h (q12h); minocycline 400 mg + 200 mg q12h, loading + maintenance doses]. This study suggests that polymyxin B and minocycline in combination may be of clinical benefit in the treatment of infections by MDR-Kp and for isolates that are non-susceptible to either drug alone. (C) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/
Synergy of polymyxin B and minocycline against KPC-3- and OXA-48-producing Klebsiella pneumoniae in dynamic time-kill experiments: high agreement with in silico predictions
Objectives. Combination therapy is often used for carbapenem-resistant Gram-negative bacteria. We previously demonstrated synergy of polymyxin B and minocycline against carbapenem-resistant Klebsiella pneumoniae in static time-kill experiments and developed an in silico pharmacokinetic-pharmacodynamic (PKPD) model. The present study assessed the activity of this antibiotic combination in dynamic experiments.Ā Methods. Two clinical K. pneumoniae isolates producing KPC-3 and OXA-48 (polymyxin B MICs 0.5 mg/L and 8 mg/L, and minocycline MICs 1 mg/L and 8 mg/L, respectively) were included. Activities of the single drugs and the combination were assessed in 72-h dynamic time-kill experiments mimicking patient pharmacokinetics. Population analysis was performed every 12 h using plates containing antibiotics at 4 and 8 x MIC. Whole-genome sequencing was applied to reveal resistance genes and mutations. Results. The combination showed synergistic and bactericidal effects against the KPC-3-producing strain from 12 h onwards. Subpopulations with decreased susceptibility to polymyxin B were frequently detected after single-drug exposures but not with the combination. Against the OXA-48-producing strain, synergy was observed between 4 and 8 h and was followed by regrowth. Subpopulations with decreased susceptibility to polymyxin B and minocycline were detected throughout experiments. For both strains, the observed antibacterial activities showed high agreement with the in silico predictions.Ā Conclusion. Polymyxin B and minocycline in combination showed synergistic effects mainly against the KPC-3-producing K. pneumoniae. The high agreement between the experimental results and in silico predictions supports the use of PKPD models based on static time-kill data to predict the activity of antibiotic combinations at dynamic drug concentrations