Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance

Abstract

Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.

Prediction of Fluoroquinolone Susceptibility Directly from Whole-Genome Sequence Data by Using Liquid Chromatography-Tandem Mass Spectrometry To Identify Mutant Genotypes

“…Fluorescent dye accumulation assays revealed that the shape of the dye accumulation curve for representative rule 15 clinical isolates KP7 and KP8 ( Fig. 1A and B) was like that seen previously upon micF overexpression in Ecl8, i.e., causing reduced OmpF (OmpK35) porin production (26). Porin reduction retards the entry of the dye but does not prevent the accumulation of the dye over time.…”

“…Porin reduction retards the entry of the dye but does not prevent the accumulation of the dye over time. This phenotype is in stark contrast to efflux-mediated reduced envelope permeability, which gives a persistent level of reduced dye accumulation, as seen in the Ecl8 ΔramR mutant previously (26) and, for example, in clinical isolate KP21, which has a ramR mutation and overproduces AcrAB-TolC (Table 2 and Fig. 1C).…”

“…During growth in the same medium used to perform proteomic analyses of our clinical isolates (nutrient broth), the OmpF/OmpC ratio seen for Ecl8 was approximately 1:1 (26). For these 10 test clinical isolates, the OmpF/OmpC ratio was in a range of 0.25:1 to 0.40:1, or it was zero due to the apparent loss of OmpF (Table 1).…”

“…To confirm whether the identified regulatory SNPs activated efflux pump production in these clinical isolates, whole-cell LC-MS/MS proteomics was performed. Clinical isolates carrying Thr141Ile and 194K insertion mutations in RamR (6/10 clinical isolates carrying one or both mutations) did not hyperproduce AcrA and AcrB (Table 1), as would be expected from a true RamR loss-of-function mutation (26), so these variants were considered functionally of the wild type and to have arisen by random genetic drift. Two out of 10 isolates carried OqxR mutations, and both isolates were confirmed to hyperproduce OqxB (Table 1).…”

Mode of Action of the Monobactam LYS228 and Mechanisms Decreasing In Vitro Susceptibility in Escherichia coli and Klebsiella pneumoniae

“…Mutations in the repressor gene ramR can upregulate expression of the positive activator RamA, which in turn upregulates expression of the acrAB-tolC and oqxAB-tolC efflux pump genes along with other regulatory changes; ramR mutations have also been identified in clinical isolates (22,(28)(29)(30)(31)(32). RamAmediated upregulation of AcrAB-TolC and/or OqxAB-TolC has also been reported to decrease susceptibility to a range of ␤-lactams, including ATM (33). Furthermore, NB29002-JBA0001 was also less susceptible to TGC and CHL, known substrates of AcrAB-TolC that are structurally unrelated to LYS228.…”