Estudos comparativos do mecanismo de ação de peptídeos antimicrobianos
Arquivos
Data
2012
Autores
Martins, Marta Natividade Crizol 
Orientadores
Miranda, Antonio
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os peptideos antimicrobianos (PAMs) sao componentes importantes do sistema de defesa inato de plantas e animais contra micro-organismos, bacterias e fungos. A maioria dos PAMs sao cationicos e anfipaticos, caracteristicas que sao essenciais para a sua interacao nao especifica com a membrana de micro-organismos. Neste trabalho, comparamos as atividades antimicrobianas e hemoliticas, assim como o indice terapeutico (IT) dos peptideos gomesina, magainina II, protegrina I, taquiplesina I, polifemusina II e seus analogos lineares. A atividade antimicrobiana foi avaliada contra S. aureus, E. coli e C. albicans. As atividades hemoliticas dos peptideos foram avaliadas contra eritrocitos humanos. A microscopia optica foi empregada para estudar a interacao destes peptideos com vesiculas unilamelares gigantes (GUVs), compostas por misturas de um lipidio neutro (POPC) com um lipidio carregado negativamente (POPG). Os resultados mostraram que a gomesina e protegrina I tem a mesma potencia, enquanto que os outros peptideos foram menos ativos contra C. albicans. Protegrina I e taquiplesina I foram os mais potentes contra E.coli e protegrina I foi o mais ativo contra S. aureus (2 a 4 vezes). A maioria dos PAMs estudados apresentaram atividades hemoliticas significativas com excessao da magainina II, que nao foi hemolitica mesmo na concentracao de 100 μM. A magainina II apresentou o melhor IT contra todos os micro-organismos testados. Curiosamente, a gomesina, taquiplesina I, polifemusina II e seus analogos lineares e o analogo linear da protegrina I parecem atuar atraves do modelo de carpete, enquanto a magainina II e a protegrina I formam poros estaveis nas membranas das GUVs. Recentemente, estudos que envolvem a acao de PAMs contra celulas do cancer tem aumentado significatimente. No entanto os mecanismos pelos quais os PAMs regulam a morte celular em mamiferos nao estao bem esclarecidos. Diante do fato, no presente trabalho investigamos a atuacao destes peptideos contra as celulas eritroleucemicas humanas K562. Os resultados mostraram que a gomesina e protegrina I exibiram atividades citotoxicas nao mostradas pelos seus analogos lineares. Taquiplesina I e polifemusina II tambem induziram morte celular e seus analogos lineares nao perderam os efeitos. Atraves dos experimentos realizados foi possivel distinguir duas maneiras que os peptideos induzem morte celular, dependendo da concentracao empregada. Baixas concentracoes dos PAMs induzem mecanismos de morte celular de forma controlada, tais como apoptose, necrose secundaria e necrose/necroptose. Cada PAM testado promoveu morte celular controlada por um mecanismo intracelular diferente. Gomesina, taquiplesina I e [Trp0, Ser3,7,12,16]-taquiplesina I promoveram apoptose que foi caracterizada por anexina, sensibilidade a Z-VAD, e ativacao da caspase-3. Gomesina e protegrina I induziram morte celular que tambem foi dependente de mecanismos intracelulares de Ca2+. Necrostatina-1 tambem foi capaz de inibir a morte celular provocada pela gomesina, taquiplesina I e [Trp0, Ser4,8,13,17]-polifemusina II. Por outro lado o tratamento com concentracoes mais elevadas dos PAMs, acima da EC50, resultou principalmente em ruptura da membrana com diferentes acoes na membrana celular. Assim, em concentracoes de PAMs abaixo da EC50, a morte celular controlada pode ser induzida, mas em altas concentracoes ocorrem perturbacoes direta na membrana
Antimicrobial peptides (AMPs) are important components of the innate defense system of plants and animals against microorganisms, such as bacteria and fungi. Most AMPs are cationic and amphipathic, features which are essential for their non-specific interaction with the membrane of microorganisms. In this work, we compared the antimicrobial and hemolytic activities, as weIl as, the therapeutic index (TI) of gomesin, magainin II, protegrin I, tachyplesin I and polyphemusin II and its linear analogues. The antimicrobial activities were evaluated against S. aureus, E. coli and C. albicans. The hemolytic activities were evaluated against human erythrocytes. Optical microscopy was used to study the interaction of these peptides with giant unilamellar vesicles (GUVs) composed of mixtures of a neutral lipid (POPC) with a negatively charged (POPG). Our results showed that gomesin and protegrin I have the same potency while the other compounds were less active against C. albicans. Protegrin I and tachyplesin I were the most potent ones against E. coli and protegrin I was the most active against S. Aureus (2 to 4-fold). Most of AMPs studied presented significative hemolytic activities with the exception of magainin II that was not hemolytic even at 100 µM concentration. Magainin II presented the best TI against all the microorganisms tested. Intriguingly, as gomesin, tachyplesin I, polyphemusin II, their linear analogues and the linear analogue of protegrin I seem to act via the carpet model, while magainin II and protegrin I formed stable pores in the membranes of GUVs. Recently, studies involving the action of AMPs against cancer cells have increased significantly. However, the mechanisms by which AMPs regulate cell death in mammals are not well understood. Given the fact, in the stydy we investigated the role of these peptides against human erythroleukemia K562 cell line. Gomesin and protegrin I displayed cytotoxic properties that their linear analogues did not. Tachyplesin I and polyphemusin II also induced cell death, and their analogues did not lose this effect. We distinguished two ways in which AMPs induced cell death depending on the concentration range utilized. Lower concentrations of AMPs induced controlled cell death mechanisms such as apoptosis, secondary necrosis and necrosis/necroptosis. Each AMP tested promoted controlled cell death by a different intracellular mechanism. Gomesin, tachyplesin I and [Trp0 , Ser3,7,12,16]-tachyplesin I promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation. Gomesin and protegrin I induced cell death were also dependent on intracellular Ca2+ mechanisms. Necrostatin-1 was also able to inhibit gomesin, tachyplesin I and [Trp0, Ser4,8,13,17]-polyphemusin II mediated cell death. Conversely, treatment with higher concentrations of AMPs, above the EC50, primarily resulted in membrane disruption, although different actions on cell membranes were observed. Thus, at AMP concentrations below the EC50, controlled cell death could be induced but at higher concentrations, direct disruption of membranes occurred.
Antimicrobial peptides (AMPs) are important components of the innate defense system of plants and animals against microorganisms, such as bacteria and fungi. Most AMPs are cationic and amphipathic, features which are essential for their non-specific interaction with the membrane of microorganisms. In this work, we compared the antimicrobial and hemolytic activities, as weIl as, the therapeutic index (TI) of gomesin, magainin II, protegrin I, tachyplesin I and polyphemusin II and its linear analogues. The antimicrobial activities were evaluated against S. aureus, E. coli and C. albicans. The hemolytic activities were evaluated against human erythrocytes. Optical microscopy was used to study the interaction of these peptides with giant unilamellar vesicles (GUVs) composed of mixtures of a neutral lipid (POPC) with a negatively charged (POPG). Our results showed that gomesin and protegrin I have the same potency while the other compounds were less active against C. albicans. Protegrin I and tachyplesin I were the most potent ones against E. coli and protegrin I was the most active against S. Aureus (2 to 4-fold). Most of AMPs studied presented significative hemolytic activities with the exception of magainin II that was not hemolytic even at 100 µM concentration. Magainin II presented the best TI against all the microorganisms tested. Intriguingly, as gomesin, tachyplesin I, polyphemusin II, their linear analogues and the linear analogue of protegrin I seem to act via the carpet model, while magainin II and protegrin I formed stable pores in the membranes of GUVs. Recently, studies involving the action of AMPs against cancer cells have increased significantly. However, the mechanisms by which AMPs regulate cell death in mammals are not well understood. Given the fact, in the stydy we investigated the role of these peptides against human erythroleukemia K562 cell line. Gomesin and protegrin I displayed cytotoxic properties that their linear analogues did not. Tachyplesin I and polyphemusin II also induced cell death, and their analogues did not lose this effect. We distinguished two ways in which AMPs induced cell death depending on the concentration range utilized. Lower concentrations of AMPs induced controlled cell death mechanisms such as apoptosis, secondary necrosis and necrosis/necroptosis. Each AMP tested promoted controlled cell death by a different intracellular mechanism. Gomesin, tachyplesin I and [Trp0 , Ser3,7,12,16]-tachyplesin I promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation. Gomesin and protegrin I induced cell death were also dependent on intracellular Ca2+ mechanisms. Necrostatin-1 was also able to inhibit gomesin, tachyplesin I and [Trp0, Ser4,8,13,17]-polyphemusin II mediated cell death. Conversely, treatment with higher concentrations of AMPs, above the EC50, primarily resulted in membrane disruption, although different actions on cell membranes were observed. Thus, at AMP concentrations below the EC50, controlled cell death could be induced but at higher concentrations, direct disruption of membranes occurred.
Descrição
Em processo de proteção intelectual
Citação
MARTINS, Marta Natividade Crizol. Estudos comparativos do mecanismo de ação de peptídeos antimicrobianos. Tese (Doutorado em Ciências) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2012.