RESUMEN
BACKGROUND: Resistant infectious diseases caused by gram-negative bacteria are among the most serious worldwide health problems. Antimicrobial peptides (AMPs) have been explored as promising antibacterial, antibiofilm, and anti-infective candidates to address these health challenges. MAJOR CONCLUSIONS: Here we report the potent antibacterial effect of the peptide PaDBS1R6 on clinical bacterial isolates and identify an immunomodulatory peptide fragment incorporated within it. PaDBS1R6 was evaluated against Acinetobacter baumannii and Escherichia coli clinical isolates and had minimal inhibitory concentration (MIC) values from 8 to 32 µmol L-1. It had a rapid bactericidal effect, with eradication showing within 3 min of incubation, depending on the bacterial strain tested. In addition, PaDBS1R6 inhibited biofilm formation for A. baumannii and E. coli and was non-toxic toward healthy mammalian cells. These findings are explained by the preference of PaDBS1R6 for anionic membranes over neutral membranes, as assessed by surface plasmon resonance assays and molecular dynamics simulations. Considering its potent antibacterial activity, PaDBS1R6 was used as a template for sliding-window fr agmentation studies (window size = 10 residues). Among the sliding-window fragments, PaDBS1R6F8, PaDBS1R6F9, and PaDBS1R6F10 were ineffective against any of the bacterial strains tested. Additional biological assays were conducted, including nitric oxide (NO) modulation and wound scratch assays, and the R6F8 peptide fragment was found to be active in modulating NO levels, as well as having strong wound healing properties. GENERAL SIGNIFICANCE: This study proposes a new concept whereby peptides with different biological properties can be derived by the screening of fragments from within potent AMPs.
Asunto(s)
Acinetobacter baumannii , Antibacterianos , Biopelículas , Escherichia coli , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Acinetobacter baumannii/efectos de los fármacos , Humanos , Escherichia coli/efectos de los fármacos , Biopelículas/efectos de los fármacos , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/química , Simulación de Dinámica Molecular , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/químicaRESUMEN
Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials.
Asunto(s)
Bacteriemia/tratamiento farmacológico , Proteínas Citotóxicas Formadoras de Poros/química , Venenos de Avispas/química , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Venenos de Avispas/uso terapéutico , Venenos de Avispas/toxicidadRESUMEN
BACKGROUND: Brewers' spent grain (BSG) is a relevant, protein-rich by-product of the brewing process. Protein hydrolysates from different sources exert immune-regulatory actions activating toll-like receptors (TLRs), nuclear factor kappa B (NFκB), and mitogen-activated protein kinases (MAPKs). Effects of gastrointestinal digestion have been poorly studied. Here, we studied the immune-regulatory effect of BSG hydrolysates, and their in-vitro-digested products, on rat splenocytes, macrophages, and T lymphocytes RESULTS: In primary cultures of rat spleen cells, BSG hydrolysates induced interleukin 10 and tumor necrosis factor production in basal conditions. Under stimulation with lipopolysaccharide or concanavalin A, hydrolysates further induced interleukin 10 production. Tumor necrosis factor and interferon-γ were inhibited in lipopolysaccharide- and concanavalin-A-stimulated cells respectively. In vitro gastrointestinal digestion attenuated the observed effects. Splenic macrophages and T lymphocytes behaved in a similar fashion. In spleen cells from TLR2-/- and TLR4-/- mice, immune-regulatory effects were greatly reduced or abrogated. The study of signal transduction pathways indicated a major involvement of NFκB, and the contribution of MAPKs p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinases 1 and 2. CONCLUSION: BSG hydrolysates, like those obtained from other food sources, regulate the immune response, involving TLR2 and TLR4 and the activation of NFκB and MAPKs, an effect partly maintained after in vitro gastrointestinal digestion. Our data support the hypothesis of a shared, rather unspecific, mechanism of action of protein hydrolysates. © 2020 Society of Chemical Industry.
Asunto(s)
Citocinas/metabolismo , Grano Comestible/química , Factores Inmunológicos/metabolismo , Hidrolisados de Proteína/farmacología , Animales , Células Cultivadas , Digestión , Femenino , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteínas de Plantas/química , Ratas Wistar , Bazo/efectos de los fármacos , Bazo/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Food proteins contain specific amino acid sequences within their structures that may positively impact bodily functions and have multiple immunomodulatory effects. The functional properties of these specific sequences, also referred to as bioactive peptides, are revealed only after the degradation of native proteins during digestion processes. Currently, milk proteins have been the most explored source of bioactive peptides, which presents an interesting opportunity for the dairy industry. However, plant- and animal-derived proteins have also been shown to be important sources of bioactive peptides. This review summarizes the in vitro and in vivo evidence of the role of various food proteins as sources of immunomodulatory peptides and discusses the possible pathways involving these properties. © 2016 Society of Chemical Industry.