title: Exposing a β-Lactamase "Twist": the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase.
abstract: Around the world, Burkholderia spp. are emerging as pathogens highly resistant to β-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A β-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A β-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant β-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI β-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a β-lactamase profoundly affects β-lactam resistance.
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA papp@va.gov robert.bonomo@va.gov.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Becka
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Magdalena A
authors.initials: MA
authors.lastname: Taracila
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Marisa L
authors.initials: ML
authors.lastname: Winkler
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Gatta
authors.affiliation: Department of Biology, North Park University, Chicago, Illinois, USA.
authors.forename: Drew A
authors.initials: DA
authors.lastname: Rholl
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine Emerging Pathogens Institute, Institute for Therapeutic Innovation, University of Florida, Gainesville, Florida, USA.
authors.forename: Herbert P
authors.initials: HP
authors.lastname: Schweizer
authors.affiliation: Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA papp@va.gov robert.bonomo@va.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016957:Burkholderia pseudomallei / Q000187:drug effects / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D002442:Ceftazidime / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics* / Q000648:ultrastructure
chemical_list: D000900:Anti-Bacterial Agents; D002442:Ceftazidime; D001618:beta-Lactamases