Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

Abstract

AbstractThe impermeable outer membrane ofPseudomonas aeruginosais bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials againstPseudomonasinfections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTDis among the longest peptides produced from a “single-shot” synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTDwith circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTDbinds toP. aeruginosaexpressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms ofP. aeruginosaand leveraged to develop selective antimicrobial agents that bypass the outer membrane.