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03 04, 2020;10 (1): 3978.

In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts.

Alexandre Crépin, Jean-François Jégou, Sonia André, Florine Ecale, Anastasia Croitoru, Anne Cantereau, Jean-Marc Berjeaud, Ali Ladram, Julien Verdon
Author Information
  1. Alexandre Crépin: Laboratoire Ecologie & Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France.
  2. Jean-François Jégou: Laboratoire Inflammation, Tissus Epithéliaux et Cytokines, UPRES EA4331, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France.
  3. Sonia André: Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, IBPS, BIOSIPE, F-75252, Paris, France.
  4. Florine Ecale: Laboratoire Ecologie & Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France.
  5. Anastasia Croitoru: Laboratoire d'Optique et Biosciences, INSERM U1182 - CNRS UMR7645, Ecole polytechnique, 91128, PALAISEAU, Cedex, France.
  6. Anne Cantereau: Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France.
  7. Jean-Marc Berjeaud: Laboratoire Ecologie & Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France.
  8. Ali Ladram: Sorbonne Université, CNRS, Institut de Biologie Paris-Seine, IBPS, BIOSIPE, F-75252, Paris, France.
  9. Julien Verdon: Laboratoire Ecologie & Biologie des Interactions, UMR CNRS 7267, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073, POITIERS, Cedex 9, France. julien.verdon@univ-poitiers.fr. ORCID
PMID: 32132569 DOI: 10.1038/s41598-020-60829-2

Abstract

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K]SHa, has been designed to improve those activities. SHa and [K]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K]SHa and exhibited low half maximal lethal concentrations (LC). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.

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MeSH Term

Acanthamoeba castellanii
Animals
Antimicrobial Cationic Peptides
Anura
Cell Line
Humans
Intracellular Space
Legionella pneumophila
Macrophages
Permeability
Skin

Chemicals

Antimicrobial Cationic Peptides
temporin
Journal Article Research Support, Non-U.S. Gov't
Article has an altmetric score of 1

Word Cloud

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