The relationship between the cellular location of Leishmania (Leishmania) amazonensis arginase and its role during murine macrophage infection / Relação entre a localização celular da enzima arginase de Leishmania (Leishmania) amazonensis e seu papel na infecção de macrófagos murinos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

In the mammal host, Leishmania parasites live inside macrophages escaping from their microbicidal mechanisms, such as the nitric oxide (NO) production. The macrophage NO production by inducible nitric oxide synthase (iNOS) requires L-arginine as substrate, the same amino acid required by arginase to generate ornithine and urea. So, arginase may play a dual role in Leishmania survival reducing the NO by competing with iNOS, and participating in the polyamines pathway, which is essential for the cells replication. Considering this, the aim of this study is to elucidate the role of L. (L.) amazonensis arginase during the parasite life cycle, mainly its function for the establishment and maintenance of the host cell infection, besides to elucidate the way that this enzyme plays its role. With this in mind, we obtained polyclonal anti-arginase sera using purified recombinant L. (L.) amazonensis arginase, these sera were used in immunolabelling assays of L. (L.) amazonensis promastigotes and macrophages infected with L. (L.) amazonensis amastigotes. These experiments determined that arginase is compartmentalized in the glycosomes of both promastigotes and amastigotes, during infection. Besides, we obtained several mutants with altered arginase expression, modified in terms of quantity and location, which permitted us to evaluate the importance of glycosome arginase compartmentalization. Among these mutants are: overexpressors of arginase, with and without glycosomal addressing signal; parasites with one arginase allele knocked out and the other one replaced by a sequence containing the ddFKBP-ARG fusion that would allow us to regulate arginase expression, working like a functional arginase knockout; and finally, we also obtained arginase null knockouts parasites. The mutants analyses lead us to important conclusions for the knowledge of the parasite physiology and its relationship with the host macrophage, revealing that the Leishmania arginase role appears to be more complex than previously thought, playing an important role in the regulation of other metabolic pathways, of the own parasite and of the host cell. In the other hand, we also determined that the ddFKBP system is functional in L. (L.) amazonensis, and then can be used for functional studies of other important parasite´s proteins.

ASSUNTO(S)

arginase arginase glycosome glicossomo infection infecção

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