Síntese, caracterização, atividade biológica e sinérgica de peptídeos antimicrobianos análogos às bacteriocinas de Leuconostoc mesenteroides ta33a
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Data
2018-04-13
Autores
Vaz, Aline Buda dos Santos
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Universidade Estadual Paulista (Unesp)
Resumo
As leucocinas são peptídeos antimicrobianos, sintetizados no ribossomo de bactérias láticas do gênero Leuconostoc spp que são capazes de inibir microrganismos patogênicos e indicadores higiênico sanitários. Este trabalho tem como objetivo central, testar a atividade antimicrobiana de peptídeos derivados leucocinas em diferentes espécies de microrganismos de interesse na área de contaminação alimentar. Para atingir os objetivos, foram projetados peptídeos com base na estrutura secundária de Leucocinas A- e B-TA33a, os quais foram posteriormente sintetizados pelo método de síntese em fase sólida. As purificações dos peptídeos foram realizadas por cromatografia líquida de alta eficiência e a caracterização das moléculas por meio de espectrometria de massas. O análogo nativo LeuB, apresentou maior porcentagem de inibição de crescimento de Salmonella sorovar Typhimurium (71%) e Escherichia coli O157:H7 (69%). O análogo LeuA1 apresentou maior inibição contra as bactérias Listeria monocytogenes (79%) e Staphylococcus aureus (96%) e o análogo LeuAB2 para Pseudomonas aeruginosa (91%). Neste trabalho, evidencia-se ação bacteriostática para todos os peptídeos e ação parcialmente sinérgica entre os peptídeos LeuB e LeuA1, pelo ensaio de Cherckerboard (ΣCIF ≤ 0,75) contra Listeria monocytogenes. No ensaio de inibição enzimática o peptídeo LeuB, foi capaz de inibir a atividade da enzima DNA Girase e Topoisomerase IV a partir de 100 μg/mL e o LeuA1 inibiu a atividade de relaxamento da Topoisomerase IV em concentrações a partir de 100 μg.mL-1. Os peptídeos LeuB e LeuA1, atingiram 100% de vazamento de CF a concentração de 6,25 μg/ml, no primeiro minuto e para o peptídeo LeuAB2 aos 8 minutos houve uma porcentagem de vazamento em torno de 80%. Os peptídeos não apresentaram inibição da atividade da enzima topoisomerase I humana. No entanto, em concentrações de ≥ 200 μg/mL, foram capazes de inibir a enzima humana topoisomerase IIα. Mesmo assim, o percentual de hemólise dos peptídeos em hemácias humanas foi de no máximo 2%, resultados que indicam uma reduzida ação citotóxica em células eucarióticas. O análogo LeuA1 apresentou propriedade antioxidante de 406 μMol em equivalentes de Trolox o que corresponde a 24% de capacidade redutora, podendo inibir a deterioração oxidativa nos alimentos. Os espectros de dicroísmo demonstraram flexibilidade conformacional predominando estrutura de α-hélice em TFE 60% e SDS, para os peptídeos LeuB, LeuA1 e LeuAB2.
Leucocins are antimicrobial peptides, synthesized on the ribosome of lactic acid bacteria of the genus Leuconostoc that ar e capable of inhibiting pathogenic and / or deteriorating microorganisms. This work aims to test the antimicrobial activity of peptides derived from leucocins in different species of microorganisms of interest in food contamination scenario. Peptides were d esigned based on the secondary structure of Leucocin A - and B - TA33a, which were later synthesized by the solid - phase synthesis method. Peptide purifications were performed by high performance liquid chromatography and the characterization of the molecules was performed by mass spectrometry. The native analog LeuB showed a higher percentage of growth inhibition against Salmonella sorovar Typhimurium (71%) and Escherichia coli O157: H7 (69%). The LeuA1 analog showed the highest inhibition against the bacteria Listeria monocytogenes (79%) and Staphylococcus aureus (96%) while the LeuAB2 analogue presented highest inhibition against Pseudomonas aeruginosa (91%). In this work, the bacteriostatic action for all peptides and partially synergistic action between the LeuB and LeuA1 peptides were evidenced by the Cherckerboard test ( Σ CIF ≤ 0.75) against Listeria monocytogenes . In the enzymatic inhibition assay the LeuB peptide was able to inhibit the activity of the DNA Girase enzyme from 100 μg / ml and inhibit Topoisomerase IV from 100 μg / ml. LeuA1 inhibited the relaxation activit y of Topoisomerase IV at concentrations of 100 μg.mL - 1. LeuB and LeuA1 peptides reached 100% CF leaching at 6.25 μg / ml in the first minute and for LeuAB2 peptide at 8 minutes there was a leakage rate of around 80%. The peptides did not show inhibition of the activity of the enzyme topoisomerase I human. However, at concentrations of ≥ 200 μ g / mL, they were able to inhibit the human enzyme topoisomerase II α . Nevertheless, the percentage of hemolysis of the peptides in human red blood cells was lower than 2 %, indicating a reduced cytotoxic action in eukaryotic cells. The LeuA1 analog presented an antioxidant property of 406 μ Mol in Trolox equivalents, corresponding to 24% reducing capacity, which could supply oxidative deterioration in food. Dichroism spectr a demonstrated conformational flexibility predominating α - helix structure in 60% TFE and SDS for the LeuB, LeuA1 and LeuAB2 peptides
Leucocins are antimicrobial peptides, synthesized on the ribosome of lactic acid bacteria of the genus Leuconostoc that ar e capable of inhibiting pathogenic and / or deteriorating microorganisms. This work aims to test the antimicrobial activity of peptides derived from leucocins in different species of microorganisms of interest in food contamination scenario. Peptides were d esigned based on the secondary structure of Leucocin A - and B - TA33a, which were later synthesized by the solid - phase synthesis method. Peptide purifications were performed by high performance liquid chromatography and the characterization of the molecules was performed by mass spectrometry. The native analog LeuB showed a higher percentage of growth inhibition against Salmonella sorovar Typhimurium (71%) and Escherichia coli O157: H7 (69%). The LeuA1 analog showed the highest inhibition against the bacteria Listeria monocytogenes (79%) and Staphylococcus aureus (96%) while the LeuAB2 analogue presented highest inhibition against Pseudomonas aeruginosa (91%). In this work, the bacteriostatic action for all peptides and partially synergistic action between the LeuB and LeuA1 peptides were evidenced by the Cherckerboard test ( Σ CIF ≤ 0.75) against Listeria monocytogenes . In the enzymatic inhibition assay the LeuB peptide was able to inhibit the activity of the DNA Girase enzyme from 100 μg / ml and inhibit Topoisomerase IV from 100 μg / ml. LeuA1 inhibited the relaxation activit y of Topoisomerase IV at concentrations of 100 μg.mL - 1. LeuB and LeuA1 peptides reached 100% CF leaching at 6.25 μg / ml in the first minute and for LeuAB2 peptide at 8 minutes there was a leakage rate of around 80%. The peptides did not show inhibition of the activity of the enzyme topoisomerase I human. However, at concentrations of ≥ 200 μ g / mL, they were able to inhibit the human enzyme topoisomerase II α . Nevertheless, the percentage of hemolysis of the peptides in human red blood cells was lower than 2 %, indicating a reduced cytotoxic action in eukaryotic cells. The LeuA1 analog presented an antioxidant property of 406 μ Mol in Trolox equivalents, corresponding to 24% reducing capacity, which could supply oxidative deterioration in food. Dichroism spectr a demonstrated conformational flexibility predominating α - helix structure in 60% TFE and SDS for the LeuB, LeuA1 and LeuAB2 peptides
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Palavras-chave
Peptídeos catiônicos antimicrobianos, Antioxidantes, Alimentos-Conservação, Bactérias produtoras de ácido láctico, Bacteriocinas