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.
Altering the gut microbiota can negatively affect human health. Efforts may be sustained to predict the intended or unintended effects of molecules not naturally produced or expected to be present within the organism on the gut microbiota. Here, culture-dependent and DNA-based approaches were combined to UHPLC-MS/MS analyses in order to investigate the reciprocal interactions between a constructed Human Gut Microbiota Model (HGMM) and molecules including antibiotics, drugs, and xenobiotics. Our HGMM was composed of strains from the five phyla commonly described in human gut microbiota and belonging to Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria. Relevantly, the bacterial diversity was conserved in our constructed human gut model through subcultures. Uneven richness distribution was revealed and the sensitivity of the HGMM was mainly affected by antibiotic exposure rather than by drugs or xenobiotics. Interestingly, the constructed model and the individual cultured strains respond with the same sensitivity to the different molecules. UHPLC-MS/MS analyses revealed the disappearance of some native molecules in the supernatants of the HGMM as well as in those of the individual strains. These results suggest that biotransformation of molecules occurred in the presence of our gut microbiota model and the coupled approaches performed on the individual cultures may emphasize new bacterial strains active in these metabolic processes. From this study, the new HGMM appears as a simple, fast, stable, and inexpensive model for screening the reciprocal interactions between the intestinal microbiota and molecules of interest.
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