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

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.

Aggregation and Its Influence on the Bioactivities of a Novel Antimicrobial Peptide, Temporin-PF, and Its Analogues

“…The antimicrobial killing effect is generally recognized to permeabilize the cell membrane, resulting in the disruption of the structure [ 27 ]. Additionally, they also demonstrated potential in other aspects, including antifungal [ 28 ], anticancer [ 28 ], antiparasite [ 29 ], anti-inflammatory [ 30 ], and antiviral effects [ 31 ]. The native temporins show considerable drawbacks to the clinic application, such as significant hemolysis and cytotoxicity [ 32 ].…”

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus)

“…The similar killing of MRSA cells inside HaCaT keratinocytes was also observed for an analog of temporin-CEb (formerly temporin-1CEb) conjugated with dalargin, a Leu-enkephalin analog [ 44 ]. We have recently confirmed the intracellular activity of temporin-SHa, but this time against a bacterial pathogen, the Gram-negative Legionella pneumophila responsible for Legionnaire’s disease [ 31 ]. Temporin-SHa killed L. pneumophila bacteria within both amoebae and macrophages.…”

“…These analogs were able to affect a wider range of clinically relevant Gram-negative bacteria while retaining the non-cytotoxic character of the parent peptide [ 29 ], or to target Candida species [ 30 ]. Among temporins-SH, temporin-SHa is undoubtedly the most potent and broad-spectrum AMP with antibacterial, antifungal, antiparasitic and antiviral activities [ 14 , 22 , 31 ]. In addition, following covalent immobilization onto gold surfaces, this peptide retains its antimicrobial activity [ 32 ].…”

Design, Engineering and Discovery of Novel α-Helical and β-Boomerang Antimicrobial Peptides against Drug Resistant Bacteria

“…TA and TB were found to be active against Gram positive bacteria, including clinical isolates, while being only weakly hemolytic [79]. In addition, TA and TB also displayed growth inhibition of viruses, protoza, and parasitic activity in in vitro assays [93][94][95][96]. In vitro wound healing activity of TA and TB has also been reported [97,98].…”