Extração, purificação de substâncias antagonistas produzidas por Shigella sonnei, síntese de peptídios ativos contra Shigella spp. e Escherichia coli e caracterização parcial das moléculas

Abstract

Shigella, a member of the Enterobacteriaceae family, is a bacterial genus that includes four species, S. boydii, S. dysenteriae and, of special importance in Brazil, S. flexneri and S. sonnei. The enteropathogen Shigella is the agent of shigellosis, a severe form of diarrheal disease associated with high levels of morbidity and mortality, which predominantly affects children under the age of five living in less developed countries. Like several bacterial groups, Shigella is capable of synthesizing substances of a protein nature with antagonistic action that confer competitive advantages to the producing microorganism. The general objective of the work was extract and purify antagonist substances produced by Shigella sonnei isolated from a fecal specimen of a child with shigellosis, based on these molecules, synthesize and characterize the peptides active against Shigella spp. and Escherichia coli. For the study, two samples of S. sonnei, producer and developer of the antagonist action were used. The intracellular extracts of samples producing antagonist substances were obtained by culture, lysis and protein precipitation with ammonium sulfate at concentrations of 15 and 30% w/v (EI-15 and EI-30). The antagonist action was determined and the extracts were submitted to protein purification by ion exchange, molecular exclusion and reversed phase chromatography, followed by mass spectrometry analysis and production of synthetic peptides. The 31 synthetic peptides were evaluated for antagonistic action, of which 8 were active against the revealing sample. Active peptides were challenged against a bacterial panel divided between Shigella spp. and E.coli, samples sensitive to the producing sample. Physicochemical analysis was also performed to characterize the substances. Based on assays for antagonistic action, a synthetic peptide, 8/J8, was named the main peptide. Followed by peptides 5/J5 and 10/J10 which also showed encouraging results. The data arouses biotechnological interest, since the World Health Organization advocates the urgent discovery of active compounds against the enteropathogen.