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Trypanosoma brucei peptidase inhibitors.Immunolocalization, secretion and potential use as targets for therapy
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As peptidases encontram-se envolvidas em diversas funções biológicas possuindo um papel importante na patogenicidade de várias infecções parasitárias. Em mamíferos, a actividade peptidica é controlada por inibidores endógenos, como as cistatinas e as serpinas. Os genes que codificam para inibidores homólogos às cistatinas e serpinas de mamíferos, encontram-se ausentes do genoma de tripanossomatídeos. A ecotina é uma proteína de Escherichia coli, capaz de inibir uma grande variedade de peptidases serínicas da família S1A, tais como a tripsina. Existem duas proteínas do tipo da ecotina em T. brucei, ISP1 e ISP2. A ausência de peptidases sensíveis à acção dos ISPs no genoma de Trypanosoma brucei, sugere que estes tenham como alvo as peptidases serínicas do hospedeiro. Linhas celulares de T. brucei deficientes em ISP1 (Δisp1), ISP2 (Δisp2) e em ambos os ISPs (Δisp1/2) foram produzidas com sucesso e a ausência dos inibidores comprovada com o uso de anticorpos monoclonais específicos contra o ISP1 e o ISP2, que reconhecem a proteina alvo em extractos de proteína total de parasitas selvagens mas não em extractos de parasitas mutantes. O efeito da ausência dos ISPs nas células foi avaliado in vitro e in vivo, verificando-se que a delecção individual de cada ISP não produz qualquer efeito nos parasitas que revelam um crescimento normal em cultura e padrões de infectividade em murganhos idênticos aos de parasitas selvagens. Em contraste, os parasitas Δisp1/2, embora possuam um crescimento normal in vitro com ausência de alterações morfológicas grosseiras, são caracterizados por um alteração na mobilidade e pela acumulação de micro-vesiculas na região da bolsa flagelar, consistente com um defeito no processo de endocitose/exocitose. Adicionalmente, por imunofluorescência foi possível localizar os ISPs no citoplasma e na perto da bolsa flagelar. Em conjunto, estes dados sugerem uma função intracelular, independente da actividade inibitória dos ISPs e provavelmente associada ao flagelo ou à bolsa flagelar. O papel biológico dos ISPs na interação parasita-hospedeiro foi também avaliada através da infecção de murganhos com os parasitas Δisp1/2.A monitorização da infecção demonstrou que a deleção de ambos os inibidores resulta em sobrevivência prolongada dos murganhos com cargas parasitárias reduzidas, sugerindo que os ISPs possuem um papel importante na sobrevivência do parasita. Em contraste com os resultados obtidos em Leishmania que atribuem diferentes funções a ISP1 e ISP2, este estudo sugere que existe uma redundância funcional dos mesmos em T. brucei, sendo necessária a presença de ambos para que o parasita estabeleça eficientemente a infecção no hospedeiro mamifero. Foram também realizados estudos de imuno-protecção em murganhos de modo a determinar o potencial dos ISPs como alvos terapêuticos. A imunização com ISP recombinante e o tratamento com anticorpos específicos contra ISP1 e ISP2 não produzem qualquer efeito protector ou neutralizante da infecção de murganhos, sugerindo que estes inibidores não constituem bons candidatos para o desenvolvimento de uma vacina ou terapia baseada em anticorpos.
Peptidases are involved in several biological functions playing an important role in the pathogenicity of many parasitic infections. In mammals, one way in which the activity of these peptidases is controlledisby interaction with natural inhibitors, such as cystatins and serpins. In trypanosomatids, the genes encoding forendogenousinhibitors homologous to mammalian cystatins and serpins, are absent. Ecotin is an Escherichia coliprotein capable of inhibiting a wide range of serine peptidases from S1A family such as trypsin. Ecotin orthologues can be found in a restricted range of bacterial pathogens and also in trypanosomatid parasitic protozoa.In Trypanosomabruceitwo proteins of 19.7 kDa and 17.8 kDa were identified as ecotin-like inhibitors and given the generic name of Inhibitors of Serine Peptidases (ISP). The absence of peptidases predicted to be sensitive to the action of ISPs in T. bruceisuggests that these inhibitors are targeting the host’s serine peptidases, although their exact biological role was unknown. T. bruceimutant cell linesdeficientin ISP1 (Δisp1),ISP2 (Δisp2) and both ISPs (Δisp1/2) weresuccessfully generatedby targeted gene disruption. This was confirmed using monoclonal antibodies generated specifically against ISP1 and ISP2 that recognize the target protein in wild type and re-expressor cell lysates but not in the respective mutant cell line. Theeffect of ISP deletion in the parasiteswasdeterminedboth in culture andin micein vivo. The deletion of ISPgenes individually was shown to have no effect on the parasites in vitroor in vivo. However, different results were obtained for the simultaneous deletion of ISP1and ISP2. Although Δisp1/2parasites have normal growth and morphology in culture, they exhibit a motility defect and are characterized by the accumulation of small vesicles outside the flagellar pocket consistent with an endocytosis/exocytosis defect. Furthermore, immunofluorescence shows that both ISPs are localized in the cytosol and in small punctuated structures near the flagellar pocket region. These data suggests that ISPs have an intracellular function independent of their inhibitory activityand probably associated with the flagellar pocket.The biological role of ISP1 and ISP2 in the host-parasite interaction was also evaluated by infecting mice with Δisp1/2parasites. Parasitemia monitoring shows that the deletion of both ISPsresults in prolonged host survival with reduced or undetectable parasite loads, suggesting an important role for these inhibitors in the parasite’s survival. In contrast with recent findings of ISPs functionin Leishmania, that assigns different biological roles to ISP1 and ISP2, the present study reveals some functional redundancy of ISPs in T. brucei,with both inhibitors being required for the parasite to infect the mammalian host efficiently. Finally, immuno-protection studies were made, to assess the potential of ISPs as targets for therapy. Immunization with recombinant ISPs and treatment with antibodies specific against ISP1 and ISP2 had no protective or neutralizing effecton T. bruceiinfected mice, revealing that these inhibitors are poor candidates for an anti-disease vaccine or for antibody-based therapy.
Peptidases are involved in several biological functions playing an important role in the pathogenicity of many parasitic infections. In mammals, one way in which the activity of these peptidases is controlledisby interaction with natural inhibitors, such as cystatins and serpins. In trypanosomatids, the genes encoding forendogenousinhibitors homologous to mammalian cystatins and serpins, are absent. Ecotin is an Escherichia coliprotein capable of inhibiting a wide range of serine peptidases from S1A family such as trypsin. Ecotin orthologues can be found in a restricted range of bacterial pathogens and also in trypanosomatid parasitic protozoa.In Trypanosomabruceitwo proteins of 19.7 kDa and 17.8 kDa were identified as ecotin-like inhibitors and given the generic name of Inhibitors of Serine Peptidases (ISP). The absence of peptidases predicted to be sensitive to the action of ISPs in T. bruceisuggests that these inhibitors are targeting the host’s serine peptidases, although their exact biological role was unknown. T. bruceimutant cell linesdeficientin ISP1 (Δisp1),ISP2 (Δisp2) and both ISPs (Δisp1/2) weresuccessfully generatedby targeted gene disruption. This was confirmed using monoclonal antibodies generated specifically against ISP1 and ISP2 that recognize the target protein in wild type and re-expressor cell lysates but not in the respective mutant cell line. Theeffect of ISP deletion in the parasiteswasdeterminedboth in culture andin micein vivo. The deletion of ISPgenes individually was shown to have no effect on the parasites in vitroor in vivo. However, different results were obtained for the simultaneous deletion of ISP1and ISP2. Although Δisp1/2parasites have normal growth and morphology in culture, they exhibit a motility defect and are characterized by the accumulation of small vesicles outside the flagellar pocket consistent with an endocytosis/exocytosis defect. Furthermore, immunofluorescence shows that both ISPs are localized in the cytosol and in small punctuated structures near the flagellar pocket region. These data suggests that ISPs have an intracellular function independent of their inhibitory activityand probably associated with the flagellar pocket.The biological role of ISP1 and ISP2 in the host-parasite interaction was also evaluated by infecting mice with Δisp1/2parasites. Parasitemia monitoring shows that the deletion of both ISPsresults in prolonged host survival with reduced or undetectable parasite loads, suggesting an important role for these inhibitors in the parasite’s survival. In contrast with recent findings of ISPs functionin Leishmania, that assigns different biological roles to ISP1 and ISP2, the present study reveals some functional redundancy of ISPs in T. brucei,with both inhibitors being required for the parasite to infect the mammalian host efficiently. Finally, immuno-protection studies were made, to assess the potential of ISPs as targets for therapy. Immunization with recombinant ISPs and treatment with antibodies specific against ISP1 and ISP2 had no protective or neutralizing effecton T. bruceiinfected mice, revealing that these inhibitors are poor candidates for an anti-disease vaccine or for antibody-based therapy.
Descrição
Palavras-chave
Biologia molecular Trypanosoma brucei Infeções Endocitose Parasitas Hospedeiro Anticorpos monoclonais
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical