Síntese, caracterização e estudos da atividade biológica de peptídeos antimicrobianos derivados de Leucocinas TA33a
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Data
2017-09-06
Autores
Silva, Jesseleine Cristine Monteiro da [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Devido ao crescente aumento de doenças transmitidas por alimentos, a segurança microbiológica se torna uma questão de saúde pública pelas suas características de endemicidade, alta morbidade e pela dificuldade da adoção de medidas de controle desses microrganismos. Diante deste fato, o objetivo deste trabalho foi sintetizar e caracterizar os análogos peptídicos LeuB e LeuC-1 derivados de bacteriocinas naturais denominadas Leucocinas. Os peptídeos foram sintetizados manualmente pelo método de síntese em fase sólida, submetidos à desproteção total e clivagem, com liberação dos peptídeos brutos. Foram realizadas as análises comparativas usando HPLC e ESI-MS, e com os respectivos peptídeos puros foram feitos os ensaios antimicrobiano, enzimático, permeabilização, antioxidante, hemolítico e de espectroscopia de dicroísmo circular. Com isso, observou-se que o método de síntese dos análogos foi adequado e o processo de purificação possibilitou a obtenção dos peptídeos com alto grau de pureza. O peso molecular teórico dos peptídeos foi confirmado por espectrometria de massas. O LeuB apresentou uma maior capacidade em inibir o crescimento de Escherichia coli O157:H7 e em Salmonella sorovar Typhimurium, enquanto que LeuC-1 apresentou efeito de inibição de crescimento de Listeria monocytogenes e também de S. sorovar Typhimurium. É importante destacar que todas essas bactérias são de interesse na área de alimentos, já que são as causadoras da maioria dos casos de infecção alimentar. O ensaio de inibição enzimática com DNA girase e Topoisomerase IV mostrou que apenas o peptídeo LeuB possui capacidade de inibição destas enzimas bacterianas, sugerindo um possível mecanismo de ação deste derivado de Leucocina. Este peptídeo também apresentou a capacidade de permeabilizar miméticos de membrana bacteriana composto de POPC/POPG (75/25). Por sua vez, o peptídeo LeuC-1 não apresentou capacidade significativa de inibição da atividade das enzimas DNA girase e Topoisomerase IV e também não apresentou capacidade de permeabilização de miméticos de membrana. Porém, LeuC-1 apresentou uma boa atividade antioxidante, obtida pelo método de ABTS. Ambos os peptídeos apresentaram baixa toxicidade em eritrócitos, comprovadas pelos ensaios hemolíticos. Estruturalmente, os peptídeos LeuB e LeuC-1 tendem a se estruturar em α-hélice. Assim sendo, este trabalho possibilitou a obtenção de dois peptídeos com potencial de aplicação como conservantes alimentares a partir de mecanismos distintos de ação sem apresentar citotoxicidade para células vermelhas do sangue. LeuB possui uma suposta atuação como inibidor de topoisomerases bacterianas e capacidade de permeabilização de miméticos de membrana. LeuC-1 possivelmente atua em diferentes vias metabólicas da bactéria, porém ainda não foi possível elucidar o mecanismo alvo deste mimético peptídico.
Due to the increase of foodborne diseases, microbiological safety becomes a public health issue due to its characteristics of endemicity, high morbidity and the difficulty of adopting control measures of these microorganisms. In view of this fact, the objective of this work was to synthesize and characterize LeuB and LeuC-1 peptidics analogues derived from natural bacteriocins called Leucocins. The peptides were synthesized manually by the solid phase synthesis method, subjected to total deprotection and cleavage, with release of the crude peptides. Comparative analyzes were performed using HPLC and ESI-MS, and with the respective pure peptides the antimicrobial, enzymatic, permeabilization, antioxidant, hemolytic and circular dichroism spectroscopy. With this, it was observed that the method of synthesis of the analogs was adequate and the purification process allowed to obtain the peptides with high purity. The theoretical molecular weight of the peptides was confirmed by mass spectrometry. LeuB showed a greater capacity to inhibit the growth of Escherichia coli O157: H7 and Salmonella serovar Typhimurium, whereas LeuC-1 presented inhibition effect of growth of Listeria monocytogenes and also of S. serovar Typhimurium. It is important to highlight that all these bacteria are interesting in the area of food, since they are the cause of most cases of food infection. The enzyme inhibition assay with DNA gyrase and Topoisomerase IV showed that only the LeuB peptide has the ability to inhibit these bacterial enzymes, suggesting a possible mechanism of action of this Leucocin derivative. This peptide also showed the ability to permeabilize bacterial membrane mimetics composed of POPC/POPG (75/25). On the other hand, the LeuC- 1 peptide did not present significant capacity to inhibit the activity of the enzymes DNA gyrase and Topoisomerase IV and also did not present permeabilization capacity of membrane mimetics. However, LeuC-1 presented a good antioxidant activity, obtained by the ABTS method. Both peptides had low erythrocyte toxicity, as demonstrated by hemolytic assays. Structurally, the LeuB and LeuC-1 peptides tend to be α-helix structured. Therefore, this work enabled two peptides with application potential as food preservatives to be obtained from distinct mechanisms of action without presenting red blood cell cytotoxicity. LeuB has a supposed action as inhibitor of bacterial topoisomerases and permeabilization capacity of membrane mimetics. LeuC-1 possibly acts on different metabolic pathways of the bacterium, but it has not yet been possible to elucidate the target mechanism of this peptidic mimetic.
Due to the increase of foodborne diseases, microbiological safety becomes a public health issue due to its characteristics of endemicity, high morbidity and the difficulty of adopting control measures of these microorganisms. In view of this fact, the objective of this work was to synthesize and characterize LeuB and LeuC-1 peptidics analogues derived from natural bacteriocins called Leucocins. The peptides were synthesized manually by the solid phase synthesis method, subjected to total deprotection and cleavage, with release of the crude peptides. Comparative analyzes were performed using HPLC and ESI-MS, and with the respective pure peptides the antimicrobial, enzymatic, permeabilization, antioxidant, hemolytic and circular dichroism spectroscopy. With this, it was observed that the method of synthesis of the analogs was adequate and the purification process allowed to obtain the peptides with high purity. The theoretical molecular weight of the peptides was confirmed by mass spectrometry. LeuB showed a greater capacity to inhibit the growth of Escherichia coli O157: H7 and Salmonella serovar Typhimurium, whereas LeuC-1 presented inhibition effect of growth of Listeria monocytogenes and also of S. serovar Typhimurium. It is important to highlight that all these bacteria are interesting in the area of food, since they are the cause of most cases of food infection. The enzyme inhibition assay with DNA gyrase and Topoisomerase IV showed that only the LeuB peptide has the ability to inhibit these bacterial enzymes, suggesting a possible mechanism of action of this Leucocin derivative. This peptide also showed the ability to permeabilize bacterial membrane mimetics composed of POPC/POPG (75/25). On the other hand, the LeuC- 1 peptide did not present significant capacity to inhibit the activity of the enzymes DNA gyrase and Topoisomerase IV and also did not present permeabilization capacity of membrane mimetics. However, LeuC-1 presented a good antioxidant activity, obtained by the ABTS method. Both peptides had low erythrocyte toxicity, as demonstrated by hemolytic assays. Structurally, the LeuB and LeuC-1 peptides tend to be α-helix structured. Therefore, this work enabled two peptides with application potential as food preservatives to be obtained from distinct mechanisms of action without presenting red blood cell cytotoxicity. LeuB has a supposed action as inhibitor of bacterial topoisomerases and permeabilization capacity of membrane mimetics. LeuC-1 possibly acts on different metabolic pathways of the bacterium, but it has not yet been possible to elucidate the target mechanism of this peptidic mimetic.
Descrição
Palavras-chave
Peptídeos catiônicos antimicrobianos, Bacteriocinas, Alimentos-Contaminação, Alimentos-Conservação, Bactérias produtoras de ácido láctico, Antimicrobial cationic peptides, Bacteriocins, Food-Contamination, Food-Preservation, Bacteria producing lactic acid