Mutational Activation of the AmgRS Two-Component System in Aminoglycoside-Resistant Pseudomonas aeruginosa

TheamgRS operon encodes a presumed membrane stress-responsive two-component system linked to intrinsic aminoglycoside resistance inPseudomonas aeruginosa . Genome sequencing of a lab isolate showing modest pan-aminoglycoside resistance, strain K2979, revealed a number of mutations, including a substitution inamgS that produced an R182C change in the AmgS sensor kinase product of this gene. Introduction of this mutation into an otherwise wild-type strain recapitulated the resistance phenotype, while correcting the mutation in the resistant mutant abrogated the resistant phenotype, confirming that theamgS mutation is responsible for the aminoglycoside resistance of strain K2979. TheamgS R182 mutation promoted an AmgR-dependent, 2- to 3-fold increase in expression of the AmgRS target geneshtpX and PA5528, mirroring the impact of aminoglycoside exposure of wild-type cells onhtpX and PA5528 expression. This suggests thatamgS R182 is a gain-of-function mutation that activates AmgS and the AmgRS two-component system in promoting modest resistance to aminoglycosides. Screening of several pan-aminoglycoside-resistant clinical isolates ofP. aeruginosa revealed three that showed elevatedhtpX and PA5528 expression and harbored single amino acid-altering mutations inamgS (V121G or D106N) and no mutations inamgR . Introduction of theamgS V121G mutation into wild-typeP. aeruginosa generated a resistance phenotype reminiscent of theamgS R182 mutant and produced a 2- to 3-fold increase inhtpX and PA5528 expression, confirming that it, too, is a gain-of-function aminoglycoside resistance-promoting mutation. These results highlight the contribution ofamgS mutations and activation of the AmgRS two-component system to acquired aminoglycoside resistance in lab and clinical isolates ofP. aeruginosa .