Banca de DEFESA: CLEIDIANE DOS SANTOS ORSSATTO

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : CLEIDIANE DOS SANTOS ORSSATTO
DATA : 20/12/2021
HORA: 14:00
LOCAL: Sala Virtual Remota
TÍTULO:

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PALAVRAS-CHAVES:

Leptodactylus. Ocellatin-KN. In silico. In vitro. Antimicrobial profile. Immunomodulation.

PÁGINAS: 103
GRANDE ÁREA: Ciências Biológicas
ÁREA: Microbiologia
SUBÁREA: Biologia e Fisiologia dos Microorganismos
ESPECIALIDADE: Bacterologia
RESUMO:

The growing development of antimicrobial resistance, and the decrease in the supply of anti-infective drugs, arouse the need to look for therapeutic alternatives that are effective and safe. Therefore, as a strategy, antimicrobial peptides have numerous advantages, from less likely to induce microbial resistance, potent and fast microbicidal action at micromolar concentrations, synergistic capacity, in addition to immunomodulating properties. Among those, the vast amount of peptides from the skin of anurans stand out, such as Ocellatin-KN, constructed from Ocellatin-K1, isolated from the secretion of the skin of Leptodactylus knudseni. In this frame, the objective of this work was to evaluate in vitro the antimicrobial profile of OKN against ATCC strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and ATCC strain of the yeast Candida albicans, in addition to the evaluation of the cytotoxic and immunomodulatory characteristics related to the production of ROS, IL-6 and OKN phagocytosis stimulation on murine peritoneal macrophages. The physical-chemical and structural characteristics of OKN were determined in silico, using bioinformatics tools. The antimicrobial and biofilm inhibition (MIC and MBIC) potential of OKN was performed by the broth microdilution method, at concentrations from 500 to 3.9 μg/mL. With the same concentrations as related, the cytotoxicity of the peptide was tested against mouse peritoneal macrophages by the MTT method, with incubations of 24 and 48 hours. An assay for the production of ROS was carried out based on a fluorescence reaction, with concentrations of OKN from 500 to 31.2 μg/mL. The supernatant (from the cytotoxicity assay) produced in 6 hours was used to measure Interleukin 6 using the immunoenzymatic method, with concentrations of OKN from 62.5 to 7.8 μg/mL. The phagocytosis assay was conducted using opsonized zymosan particles using OKN concentrations of 125, 62 and 31 μg/ml. The results were analyzed using the GraphPad Prism software version 6.0, performing the analysis of variance (One-Way ANOVA) with the application of Tukey's post-test. It was observed that OKN has mild activity against the bacteria tested, with a MIC of 125 μg/mL for the two gram-negative strains (E. coli and P. aeruginosa) and 15.6 μg/mL for S. aureus; and no inhibitory effect on C. albicans. Biofilm formation of E. coli and P. aeruginosa was discretely inhibited at the highest concentrations (500 and 250 μg/mL) and noticeably inhibited for S. aureus and C. albicans at all concentrations (500 to 3.9 μg/mL). On murine peritoneal macrophages, OKN was cytotoxic at concentrations equal to and greater than 125 μg/mL, and did not stimulate the production of ROS even at the highest concentrations tested, however, an increase in the production of the cytokine IL-6 was observed in the concentration of 62.5 μg/mL, in addition to increasing the rate of phagocytosis by macrophages at all concentrations evaluated. These findings suggest that OKN has mild antimicrobial activity, antibiofilm potential and likely immunostimulating activity on murine macrophages

MEMBROS DA BANCA:
Presidente - 2148421 - CHRISTIAN COLLINS KUEHN
Externo à Instituição - JORGE JAVIER ALFONSO RUIZ DIAZ - FIOCRUZ
Externo à Instituição - SULAMITA DA SILVA SETUBAL - FIOCRUZ