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Caracterização bioquímica e propriedades biológicas de diferentes metaloproteinases de tripanossomatídeos (Leishmania spp., Trypanosoma cruzi e Trypanosoma brucei brucei)
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Leishmania spp., Trypanosoma cruzi e Trypanosoma brucei são os agentes etiológicos da leishmaniose, doença de Chagas e doença do sono, respetivamente. Estas doenças caracterizam-se por serem maioritariamente prevalentes em regiões tropicais e subtropicais onde impera a pobreza. Não obstante os esforços no sentido de as controlar/erradicar, atualmente as Doenças Tropicais Negligenciadas são endémicas em 149 países. Desta realidade surge a necessidade de melhor compreensão das suas interações com o hospedeiro vertebrado. Neste sentido, pretendeu-se caracterizar bioquímicamente e biologicamente as metaloproteases, enzimas comuns a tripanossomatídeos envolvidas nos processos de virulência deste trio de protozoários de importância clínica. Traçou-se o perfil enzimático, em géis de colagénio, gelatina e caseína, de extratos de formas promastigotas de L. amazonensis, L. guyanensis, L. infantum, L. shawi; de formas epimastigotas e tripomastigotas das estirpes Y, Bolívia, e QMM5 de T. cruzi e de tripomastigotas sanguíneos de T. b. brucei. As concentrações mínimas inibitórias de metaloproteases foram estimadas na presença da 1,10-fenantrolina, um inibidor de metaloproteases. Para avaliar a atividade metaloproteolítica de cada extrato foi otimizada uma metodologia que permitiu quantificar o nível de atividade enzimática na presença e na ausência da 1,10-fenantrolina. O estudo das propriedades biológicas de cada extrato consistiu na avaliação da interação das metaloproteinases dos diferentes tripanosomatídeos com eritrócitos humanaos (Eh) e com possível atividade hemolítica mediada pelo sistema complemento. Todos os extratos foram enzimaticamente ativos nos diferentes substratos e cada parasita evidenciou um perfil zimográfico distinto. Nos extratos de Leishmania spp. foi observada a expressão de enzimas com massa molecular entre os 50 e 80 kDa, possivelmente a glicoproteína de 63 kDa (gp63). Em extratos de T. cruzi e T. b. brucei foi detetado, respetivamente, a expressão de enzimas de aproximadamente 40 e 20 kDa. O decréscimo da atividade enzimática total dos extratos na presença de concentrações crescentes de 1,10-fenentrolina confirmou a natureza metaloproteica das enzimas. O perfil de atividade das metaloproteases de L. shawi mostrou-se distinto do evidenciado pelas restantes espécies e cada estirpe de T. cruzi exibiu um padrão metaloproteolítico diferente. Das metaloproteases estudadas, a de T. b. brucei apresentou a menor velocidade de reação enzimática.
Os resultados obtidos nos ensaios de sensibilização de Eh por extratos de Leishmania, T.cruzi e T. b. brucei permitiram dar início à criação de um modelo biológico de infeção tendo ficado estabelecido que em Leishmania spp. ocorre a sensibilização de Eh pelas metaloproteases que, por sua vez, são lisados pelo sistema complemento. Dada a importância das funções biológicas destas enzimas, o conhecimento do perfil de expressão nos diferentes tripanossomatídeos e dos mecanismos biológicos de infeção pode constituir uma oportunidade para a identificação de novos alvos terapêuticos e, consequentemente, para o desenvolvimento de novas estratégias profiláticas e terapêuticas.
Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei are the etiologic agents of leishmaniasis, Chagas sickness and sleeping disease, respectively. These diseases are prevalent in tropical and subtropical regions between poor populations. Despite the efforts to control/eradicate, currently neglected tropical diseases are endemic in 149 countries. From this reality arises the need for better understanding the interactions established by the parasites with the respective vertebrate host. Taken into account the above considerations, we aimed to characterize biochemically and biologically metalloproteases, a set of enzymes common to Leishmania and Trypanosome that are involved in several aspects of infectious process. The enzymatic profile of L. amazonensis, L. guyanensis, L. infantum, L. shawi promastigotes, of T. cruzi (strains Y, Bolívia, QMM5) epimastigotes and trypomastigotes and, of T. b. brucei trypomastigotes were evaluated in collagen, gelatin and casein gels. Minimum inhibitory concentrations (MIC) were estimated in four species of Leishmania and in three T. cruzi strains in the presence of 1,10-phenanthroline, a specific inhibitor of metalloproteinase. To evaluate the proteolytic activity of each extract, an enzymatic fluorescent assay was optimized. This methodology allows to estimate the enzyme expression by measuring its activity in the presence and absence of 1,10-phenanthroline. Furthermore, the interaction of metalloproteinases of each trypanosomatid with human erythrocytes (Eh) and their presumable lysis mediated by complement system was also assessed. All extracts were enzymatically active in different substrates and each parasite evidenced a specific zymographic profile. An enzyme with a molecular mass of 50-80 kDa, possibly the 63 kDa glycoprotein, was detected in Leishmania species. Bands of approximately 40 and 20 kDa with enzymatic activity were observed in extracts of T. cruzi and T. b. brucei, respectively. The declining of extracts total enzymatic activity in the presence of increasing concentrations of 1,10-phenatroline confirmed the metalloproteolytic nature of enzymes. Furthermore, metalloproteolytic profile of L. shawi was different from other Leishmania species and each strain of T. cruzi exhibited a characteristic metalloproteolytic pattern. From all the studied parasites, T. b. brucei presented the lowest enzymatic reaction rate. Concerning the assays with Eh pre-treated with parasite extracts it was possible to establish a biological model of infection revealing that Leishmania spp. metalloproteases effectively sensitize Eh which are then lysed by complement system. Given the biological importance of metalloproteases, clarifying their role in the establishment of infection and in the survival of tripanosomatids may constitute an opportunity to identify new targets and, consequently, for the design and development of new therapeutic and prophylactic strategies.
Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei are the etiologic agents of leishmaniasis, Chagas sickness and sleeping disease, respectively. These diseases are prevalent in tropical and subtropical regions between poor populations. Despite the efforts to control/eradicate, currently neglected tropical diseases are endemic in 149 countries. From this reality arises the need for better understanding the interactions established by the parasites with the respective vertebrate host. Taken into account the above considerations, we aimed to characterize biochemically and biologically metalloproteases, a set of enzymes common to Leishmania and Trypanosome that are involved in several aspects of infectious process. The enzymatic profile of L. amazonensis, L. guyanensis, L. infantum, L. shawi promastigotes, of T. cruzi (strains Y, Bolívia, QMM5) epimastigotes and trypomastigotes and, of T. b. brucei trypomastigotes were evaluated in collagen, gelatin and casein gels. Minimum inhibitory concentrations (MIC) were estimated in four species of Leishmania and in three T. cruzi strains in the presence of 1,10-phenanthroline, a specific inhibitor of metalloproteinase. To evaluate the proteolytic activity of each extract, an enzymatic fluorescent assay was optimized. This methodology allows to estimate the enzyme expression by measuring its activity in the presence and absence of 1,10-phenanthroline. Furthermore, the interaction of metalloproteinases of each trypanosomatid with human erythrocytes (Eh) and their presumable lysis mediated by complement system was also assessed. All extracts were enzymatically active in different substrates and each parasite evidenced a specific zymographic profile. An enzyme with a molecular mass of 50-80 kDa, possibly the 63 kDa glycoprotein, was detected in Leishmania species. Bands of approximately 40 and 20 kDa with enzymatic activity were observed in extracts of T. cruzi and T. b. brucei, respectively. The declining of extracts total enzymatic activity in the presence of increasing concentrations of 1,10-phenatroline confirmed the metalloproteolytic nature of enzymes. Furthermore, metalloproteolytic profile of L. shawi was different from other Leishmania species and each strain of T. cruzi exhibited a characteristic metalloproteolytic pattern. From all the studied parasites, T. b. brucei presented the lowest enzymatic reaction rate. Concerning the assays with Eh pre-treated with parasite extracts it was possible to establish a biological model of infection revealing that Leishmania spp. metalloproteases effectively sensitize Eh which are then lysed by complement system. Given the biological importance of metalloproteases, clarifying their role in the establishment of infection and in the survival of tripanosomatids may constitute an opportunity to identify new targets and, consequently, for the design and development of new therapeutic and prophylactic strategies.
Descrição
Palavras-chave
Microbiologia médica Tripanossomas Metaloproteases Sistema complemento Leshmaniose Enzimas Tripanossomatídeos Metaloproteases
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical