Stepwise Upregulation of thePseudomonas aeruginosaChromosomal Cephalosporinase Conferring High-Level β-Lactam Resistance Involves Three AmpD Homologues

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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 ofPseudomonas aeruginosainfections. AlthoughampDinactivation has been previously found to lead to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility, the regulation ofampCinP. aeruginosais far from well understood. We demonstrate thatampCexpression 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 stepwiseampCupregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal β-lactam resistance, as shown by analysis of the three singleampDmutants, the three doubleampDmutants, and the tripleampDmutant. 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 β-lactam resistance (ampDmutant), high-level hyperinducible expression with high-level β-lactam resistance (ampD ampDh3double 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.

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