Characterization of an antagonistic peptide produced by a Bacteroides Fragilis isolate obtained from a patient with intra-abdominal infection

Abstract

The indigenous microbiota of humans is extremely rich and diverse, with special emphasis on the intestinal microbiota. As a constituent of this microbiota, it is mentioned the genus Bacteroides, whose members are Gram negative rods, obligate anaerobes, amphibionts, associated to the etiopathogenesis of important infectious diseases, such as intra-abdominal infections. Bacteroides strains have the ability to synthesize antagonistic substances that play an ecological role, especially in densely colonized habitats, giving a competitive advantage to the producing samples. The objective of this study was to evaluate the synthesis capacity of antagonistic substances by 40 samples of Bacteroides and Parabacteroides isolated from patients with intra-abdominal infections. The expression of antagonism was evaluated by the overlay diffusion method, using, as well as the test samples, 36 reference samples of Gram negative and Gram-positive bacteria. Subsequently, a production strain (Bacteroides fragilis) was used for extraction, purification and partial characterization of the detected antagonistic substance. As indicator strains, Bacteroides ovatus and Bacteroides caccae were used. The production strain was submitted to protein extraction, and the activity of the precipitated intracellular extract was detected with (NH4) 2SO4 in concentrations of 30% (C30) and 50% (C50). C30 and C50 were inactivated by proteases and high temperatures and remained active after exposure to organic solvents and a wide pH range. Both fractions presented antagonistic activity of bacteriostatic nature. The C50 extract was subjected to ion exchange chromatography, and 50 fractions were recovered. Among them, fractions 1 to 4, referring to a single peak, that were not able to bind to the column, presented antagonistic activity. Fractions from the ion exchange chromatography were applied in gel filtration chromatography. Among them, fractions 2 and 3 were able to inhibit the developing sample. These fractions were submitted to reverse phase chromatography, and 50 fractions were collected. One of them, fraction 2C, remained active against the revealing sample. Mass spectrometry, from fraction 2C obtained from reverse phase chromatography, presented ions of approximately 1300.00 Da, which generated a more intense signal. The search performed by similarity between the sequenced peptides and proteins described in the BLASTP database, from fragmentation obtained in reverse phase chromatography, resulted in 100% identity between two peptides. One of the sequenced peptides showed 100% identity to a type VII secretion protein. The search performed by similarity between the sequenced peptides and proteins described in the ANTIMICROBIAL PEPTIDE DATABASE database, from fragmentation obtained with trypsin digestion, resulted in 42% identity with a Streptomyces microcine. Together, the results indicate the production of antagonistic substances by the B. fragilis sample under study. It is plausible to assume that they play a relevant role in interbacterial relationships, as a virulence factor, in a complex environment such as intra-abdominal infection.