Correlating uptake and activity of proline-rich antimicrobial peptides in Escherichia coli.

Abstract

Increasing death tolls accountedfor by antimicrobial drug resistance demand novel antibiotic lead compounds. Among different promising candidate classes, proline-rich antimicrobial peptides (PrAMPs) are very favorable due to their intracellular mechanism, i.e., binding to the 70S ribosome and DnaK, after active uptake relying on bacterial transporters like SbmA and MdtM. Studies on peptide internalization as the first step of their complex mode of action rely typically on fluorophore or radioactive labeling and quantification using microscopy, flow cytometry, or radioactivity. Here, a liquid chromatographybased assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UVabsorbance and mass spectrometry. Knockout mutants lacking transporter proteins showed reduced PrAMP uptakes, explaining their reduced susceptibility against PrAMPs. Interestingly, major metabolites produced by bacterial proteases still bound to the 70S ribosome provide evidence that degradation by cytosolic proteases as a possible resistance mechanism is not very efficient. Graphical abstract The uptake of unlabeled proline-rich antimicrobial peptides (PrAMPs) is analyzed in Escherichia coli BW25113 wild-type and transporter knockout mutants ΔsbmA and BS2 (ΔsbmA yjiL::Tn10) by reversed-phase chromatography and quantified by UV detection or mass spectrometry with multi-reaction monitoring (scheme right). Internalized peptide amounts correlated to minimal inhibitory concentrations and bacterial transport activities based on the present transporter proteins (scheme left).