The antimicrobial peptide Esc(1-21)-1c increases susceptibility of Pseudomonas aeruginosa to conventional antibiotics by decreasing the expression of the MexAB-OprM efflux pump

Carolina Canè; Bruno Casciaro; Angela Di Somma; Maria Rosa Loffredo; Elena Puglisi; Gennaro Battaglia; Marta Mellini; Floriana Cappiello; Giordano Rampioni; Livia Leoni; Angela Amoresano; Angela Duilio; Maria Luisa Mangoni

doi:10.3389/fchem.2023.1271153

The antimicrobial peptide Esc(1-21)-1c increases susceptibility of Pseudomonas aeruginosa to conventional antibiotics by decreasing the expression of the MexAB-OprM efflux pump

Front Chem. 2023 Oct 24:11:1271153. doi: 10.3389/fchem.2023.1271153. eCollection 2023.

Authors

Carolina Canè^#¹, Bruno Casciaro^#², Angela Di Somma^{1

3}, Maria Rosa Loffredo², Elena Puglisi², Gennaro Battaglia¹, Marta Mellini⁴, Floriana Cappiello², Giordano Rampioni^{4

5}, Livia Leoni⁴, Angela Amoresano^{1

6}, Angela Duilio^{1

6}, Maria Luisa Mangoni²

Affiliations

¹ Department of Chemical Sciences, University of Naples "Federico II", Naples, Italy.
² Laboratory Affiliated to Pasteur Italia-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
³ CEINGE Biotecnologie Avanzate, Naples, Italy.
⁴ Department of Science, University "Roma Tre", Rome, Italy.
⁵ IRCCS Fondazione Santa Lucia, Rome, Italy.
⁶ National Institute of Biostructure and Biosystems (INBB), Rome, Italy.

^# Contributed equally.

Abstract

Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa. Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice.

Keywords: Pseudomonas aeruginosa; antibiotics; antimicrobial peptide; efflux pumps; proteomic; tetracycline.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This research was partially supported by EU funding within the Next-Generation EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT, node 3, to MLM) and by Pasteur-Italia Fondazione Cenci Bolognetti- Anna Tramontano Grant 2020 (to MLM). This work was also supported by Project code PE00000003, Concession Decree No. 1550 of 11 October 2022 adopted by the Italian Ministry of University and Research, CUP D93C22000890001, Project title “ON Foods—Research and innovation network on food and nutrition Sustainability, Safety and Security—Working ON Foods” spoke 6 (AD).