Purification of NADP-dependent glutamate dehydrogenase from Pseudomonas aeruginosa and immunochemical characterization of its in vivo inactivation

1984 ◽  
Vol 801 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Rob A.M.M. Smits ◽  
Frank R. Pieper ◽  
Chris van der Drift
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Glutamate Dehydrogenase ◽  
Immunochemical Characterization

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections

2020 ◽  
Vol 76 (1) ◽  
pp. 91-100
Author(s):  
Jorge Arca-Suárez ◽  
Cristina Lasarte-Monterrubio ◽  
Bruno-Kotska Rodiño-Janeiro ◽  
Gabriel Cabot ◽  
Juan Carlos Vázquez-Ucha ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Genomic Analysis ◽  
Resistance Mechanisms ◽  
Comparative Genomic ◽  
Development Of Resistance ◽  
Structural Impact ◽  
Genetic Context

Abstract Background The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Objectives Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Methods Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. Results The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Conclusions Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

1973 ◽  
Vol 19 (10) ◽  
pp. 1225-1233 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Sodium Dodecyl ◽  
Current Theory ◽  
Outer Cell ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
A Subunit

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.

scholarly journals Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 945-954 ◽  
Author(s):  
Páraic Ó Cuív ◽  
Paul Clarke ◽  
Michael O'Connell
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Outer Membrane Receptor ◽  
Significant Similarity ◽  
Hydroxamate Siderophores ◽  
Insertional Inactivation ◽  
Titration Assay

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

scholarly journals Adaptation-Based Resistance to Siderophore-Conjugated Antibacterial Agents by Pseudomonas aeruginosa

2013 ◽  
Vol 57 (9) ◽  
pp. 4197-4207 ◽  
Author(s):  
Andrew P. Tomaras ◽  
Jared L. Crandon ◽  
Craig J. McPherson ◽  
Mary Anne Banevicius ◽  
Steven M. Finegan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Agents ◽  
Iron Acquisition ◽  
Content Type ◽  
Multiple Strains ◽  
Resistance Rates ◽  
Assay Conditions

ABSTRACTMultidrug resistance in Gram-negative bacteria has become so threatening to human health that new antibacterial platforms are desperately needed to combat these deadly infections. The concept of siderophore conjugation, which facilitates compound uptake across the outer membrane by hijacking bacterial iron acquisition systems, has received significant attention in recent years. While standardin vitroMIC and resistance frequency methods demonstrate that these compounds are potent, broad-spectrum antibacterial agents whose activity should not be threatened by unacceptably high spontaneous resistance rates, recapitulation of these results in animal models can prove unreliable, partially because of the differences in iron availability in these different methods. Here, we describe the characterization of MB-1, a novel siderophore-conjugated monobactam that demonstrates excellentin vitroactivity againstPseudomonas aeruginosawhen tested using standard assay conditions. Unfortunately, thein vitrofindings did not correlate with thein vivoresults we obtained, as multiple strains were not effectively treated by MB-1 despite having low MICs. To address this, we also describe the development of newin vitroassays that were predictive of efficacy in mouse models, and we provide evidence that competition with native siderophores could contribute to the recalcitrance of someP. aeruginosaisolatesin vivo.

Control of ammoniagenesis in the kidney of water- and air-breathing osteoglossids: characterization of glutamate dehydrogenase

1978 ◽  
Vol 56 (4) ◽  
pp. 845-851 ◽  
Author(s):  
K. B. Storey ◽  
H. E. Guderley ◽  
M. Guppy ◽  
P. W. Hochachka
Keyword(s):  
Glutamate Dehydrogenase ◽  
Product Inhibition ◽  
Energy Status ◽  
Ph Optimum ◽  
Arapaima Gigas ◽  
Marked Preference ◽  
Regulatory Properties ◽  
Role Activity

Glutamate dehydrogenases (EC 1.4.1.2) from the kidney of Osteoglossum bicirrhosum (called aruana) and Arapaima gigas were kinetically characterized. The two enzymes exhibited several common characteristics including Vmax activity ratio, pH optimum, affinity for cofactors, a marked preference for NAD(H) over NADP(H), and a very low affinity for NH4+. A variety of regulatory metabolites affected both enzymes. GTP and GDP were inhibitory while ADP, ATP, AMP, and leucine activated the enzymes. Both enzymes displayed potent product inhibition which was partially reversed by low levels of ADP. Arapaima kidney glutamate dehydrogenase was tightly regulated by the adenylate and guanylate nucleotides, inhibition by GTP and GDP and deinhibition by ADP and AMP being much stronger for this enzyme than for the aruana enzyme. Aruana glutamate dehydrogenase, however, was more responsive to NAD–NADH control. The enzyme was more sensitive to NAD(H) product inhibition and this inhibition was poorly reversed by ADP. From these data, it was concluded that both fish kidney glutamate dehydrogenases could function in glutamate oxidation in vivo. However, the Arapaima enzyme appeared most clearly adapted to a catabolic role, activity being more tightly linked to the energy status of the mitochondrion. Conversely, the aruana enzyme displayed regulatory properties allowing it the potential to function in NADH oxidation during periods of hypoxic stress.

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