title: Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.
abstract: Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.
authors.affiliation: Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.
authors.forename: Linnell B
authors.initials: LB
authors.lastname: Randall
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Dobos
authors.affiliation: Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA.
authors.forename: Krisztina M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bonomo
authors.affiliation: Department of Molecular Genetics and Microbiology, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA hschweizer@ufl.edu.
authors.forename: Herbert P
authors.initials: HP
authors.lastname: Schweizer
mesh_terms: D016957:Burkholderia pseudomallei / Q000187:drug effects* / Q000201:enzymology* / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000502:physiology; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D017830:Octoxynol; D010455:Peptides / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D010455:Peptides; D011092:Polyethylene Glycols; C017339:globomycin; D017830:Octoxynol; C010615:Nonidet P-40; D001618:beta-Lactamases