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

Alexandre Crépin,Jean-François Jégou,Ali Ladram,Julien Verdon,Sonia André,Anastasia Croitoru,Anne Cantereau,Jean-Marc Berjeaud,Florine Ecale

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 [K3]SHa, has been designed to improve those activities. SHa and [K3]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 [K3]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 [K3]SHa and exhibited low half maximal lethal concentrations (LC50). 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.

Highlights

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus
To assess the activity of SHa and [K3]SHa against bacteria that belong to the Legionella genus, several species of human health concern were grown in the presence of different amount of peptides and minimal inhibitory concentrations (MICs) were determined after a growth period of 96 h at 37 °C (Table 1)
Biological activities of both parent peptides and analogs are widely described throughout the literature and include (i) direct broad-spectrum activity against bacterial, fungal or parasitic pathogens[9,10,16,17,21,22], (ii) anti-Herpes simplex virus type 1 activity[23,24], (iii) stimulation of human cells migration such as keratinocytes, monocytes and neutrophils[27,33], (iv) endotoxin neutralization[34,35], (v) chemoattraction of human phagocytes[33]; (vi) antibacterial/antiadhesive effect when grafted on surface[36,37], (vii) antibiofilm properties[38,39] and (viii) killing of intracellular microorganisms[10,17,27]

Summary

Introduction

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. Temporins SHa and SHd were reported to have potent activity against the human protozoan parasite Leishmania[10,16,17], the etiological agent of leishmaniasis, a vector-borne tropical disease leading to morbidity and mortality[25] Their antiparasitic activity was assessed against several Leishmania species responsible for visceral or cutaneous leishmaniases (L. infantum, L. major, L. tropica, L. amazonensis and L. braziliensis), and against other trypanosomatids that cause major diseases in humans: Trypanosoma brucei gambiense (sleeping sickness) and T. cruzi (Chagas disease). [K3]temporin-SHa, which was obtained by increasing the net positive charge of SHa from +2 to +3, has been described as a potent antiparasitic peptide active against all these trypanosomatids, including antimony-resistant L. infantum parasites[17] This analog showed broad antimicrobial spectrum, targeting several Gram-negative bacteria and Candida species[17]. Both peptides were demonstrated to trigger apoptotis-like death in Leishmania infantum in addition to their primary membranolytic mechanism[17]

Methods

Results

Conclusion