Role ofampDHomologs in Overproduction of AmpC in Clinical Isolates ofPseudomonas aeruginosa

Abstract

AmpD indirectly regulates the production of AmpC β-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multipleampDgenes inPseudomonas aeruginosaandEscherichia coli. In the prototypeP. aeruginosastrain, PAO1, the threeampDgenes (ampD,ampDh2, andampDh3) contribute to a stepwise regulation ofampCβ-lactamase and help explain the partial versus full derepression ofampC. In the present study, the roles of the threeampDhomologs in nine clinicalP. aeruginosaisolates with either partial or full derepression ofampCwere evaluated. In eight of nine isolates, decreased RNA expression of theampDgenes was not associated with an increase inampCexpression. Sequence analyses revealed that every derepressed isolate carried mutations inampD, and in two fully derepressed strains, onlyampDwas mutated. Furthermore, everyampDh2gene was of the wild type, and in some fully derepressed isolates,ampDh3was also of the wild type. Mutations inampDandampDh3were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of theampDhomologs ofP. aeruginosacan explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes toampCregulation inP. aeruginosa.