RESUMEN
Antimicrobial peptides (AMPs) are promising tools for developing new antibiotics. We described the design of IKR18, an AMP designed with the aid of computational tools. IKR18 showed antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). CD studies revealed that IKR18 assumes an alpha-helical structure in the membrane-mimetic environment. The action mechanism IKR18 involves damage to the bacteria membrane, as demonstrated by Sytox green uptake. Furthermore, IKR18 displayed synergic and additive effects in combination with antibiotics ciprofloxacin and vancomycin. The peptide showed anti-biofilm activity in concentration and efficiency compared with commercial antibiotics, involving the direct death of bacteria, as confirmed by scanning electron microscopy. The anti-infective activity of IKR18 was demonstrated in the Galleria mellonella model infected with S. aureus, MRSA, and Acinetobacter baumannii. The novel bioinspired peptide, IKR18, proved to be effective in the control of bacterial infection, opening opportunities for the development of further assays, including preclinical models.
Asunto(s)
Antiinfecciosos , Staphylococcus aureus Resistente a Meticilina , Mariposas Nocturnas , Animales , Péptidos Antimicrobianos , Staphylococcus aureus , Pruebas de Sensibilidad Microbiana , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Antibacterianos/farmacología , Antibacterianos/química , BacteriasRESUMEN
The Escherichia coli GhoT/GhoS system is a type V toxin-antitoxin system in which the antitoxin GhoS cleaves the GhoT mRNA, controlling its translation. GhoT is a small hydrophobic protein that damages bacterial membranes. OrtT is a GhoT-like toxin, but it apparently lacks a corresponding antitoxin and serves a different physiologic role. Using a profile hidden Markov model approach, a Salmonella enterica serovar Houten genome was screened to obtain homologs of GhoT/OrtT. We only found one protein (referred to here as OrtT-Sal) that shared more sequence identity with OrtT than GhoT. The chromosomal region around the coding sequence of OrtT-Sal suggests that it is an orphan toxin and can be under RpoH activation. To study OrtT-Sal, we chemically synthesized and expressed in E. coli the whole toxin and its N- and C-terminal regions (OrtT-Sal1-29 and OrtT-Sal29-57, respectively). Our findings have shown that the overproduction of the polypeptides resulted in severe growth inhibition and cell lysis. Using circular dichroism, we found that OrtT-Sal, OrtT-Sal1-29, and OrtT-Sal29-57 form an alpha-helical structure in the presence of SDS micelles or TFE. Finally, using carboxyfluorescein-loaded lipid vesicles, we determined that the polypeptides damage lipid membrane directly.