title: Stepwise upregulation of the Pseudomonas aeruginosa chromosomal cephalosporinase conferring high-level beta-lactam resistance involves three AmpD homologues.
abstract: Development of resistance to the antipseudomonal penicillins and cephalosporins mediated by hyperproduction of the chromosomal cephalosporinase AmpC is a major threat to the successful treatment of Pseudomonas aeruginosa infections. Although ampD inactivation has been previously found to lead to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility, the regulation of ampC in P. aeruginosa is far from well understood. We demonstrate that ampC expression is coordinately repressed by three AmpD homologues, including the previously described protein AmpD plus two additional proteins, designated AmpDh2 and AmpDh3. The three AmpD homologues are responsible for a stepwise ampC upregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal beta-lactam resistance, as shown by analysis of the three single ampD mutants, the three double ampD mutants, and the triple ampD mutant. This is achieved by a three-step escalating mechanism rendering four relevant expression states: basal-level inducible expression (wild type), moderate-level hyperinducible expression with increased antipseudomonal beta-lactam resistance (ampD mutant), high-level hyperinducible expression with high-level beta-lactam resistance (ampD ampDh3 double mutant), and very high-level (more than 1,000-fold compared to the wild type) derepressed expression (triple mutant). Although one-step inducible-derepressed expression models are frequent in natural resistance mechanisms, this is the first characterized example in which expression of a resistance gene can be sequentially amplified through multiple steps of derepression.
authors.affiliation: Servicio de Microbiología, Hospital Son Dureta, C. Andrea Doria No. 55, 07014 Palma de Mallorca, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Juan
authors.forename: Bartolomé
authors.initials: B
authors.lastname: Moyá
authors.forename: José L
authors.initials: JL
authors.lastname: Pérez
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002510:Cephalosporinase / Q000235:genetics / Q000378:metabolism*; D002876:Chromosomes, Bacterial / Q000201:enzymology*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017124:Conserved Sequence; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009876:Operon; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation; D018440:beta-Lactam Resistance
chemical_list: D001426:Bacterial Proteins; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase; D002510:Cephalosporinase