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

Increasing death tolls accounted for 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 chromatography based assay was applied to quantify the unlabeled internalized full-length peptides and their proteolytic degradation products (metabolites) using UV absorbance 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.