Session: Session 2

Proteomics analysis of the mechanism of action of a King Penguin antimicrobial peptide

The identification and improvement/development of natural antimicrobial peptides is a promising avenue to mitigate antimicrobial resistance. In the early 2000s, two avian β-defensins, named spheniscins, have been fully characterized in the King penguin. The efficient, large antimicrobial activity of Speniscin-2 (Sphe-2) notably proved to be retained even at high salt concentration, supporting their potential against pathogens in salt-rich environments (e.g. during cystic fibrosis or ocular infections). To decipher the mechanism of action of Sphe-2, we investigated how it alters the proteome of E. coli.



We cultured E. coli with and without Sphe-2 for 0, 3, 5, 10 and 20 minutes (five replicates per condition). At the different timepoints, bacteria were collected and lysis was performed using Laemmli buffer and sonication. After extraction and electrophoresis (SDS-PAGE stacking gel), proteins were digested in gel with trypsin. Tryptic peptides were analyzed on a nanoUPLC system (nanoAcquityUPLC, Waters) Milford, MA, USA) coupled with a quadrupole-Orbitrap hybrid a Q- mass spectrometer (Q-Exactive HF-X, Thermo Scientific). MS data were processed using MaxQuant (v2.5.1.0) for label-free quantification in DDA mode.



We quantified 1803 proteins, including 1264 with no missing value (n=5/group). The abundance of up to 72 proteins was affected over time independently of Sphe-2, while Sphe-2 specifically affected the expression level of 278 proteins. Under control conditions, changes essentially concerned fuel metabolism while under Sphe-2 conditions also changed other biological processes such as carbon metabolism, nucleotide metabolism, genetic information processing or membrane transport. A few cases of proteins expressed only under control or only under Sphe-2 conditions also brought interesting information.



The results of this study show how large-scale proteomics data will enable us to better understand how penguin defensins are able to kill bacteria.