RESUMO
Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-gamma) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-gamma) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-gamma to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-gamma-activated armadillo MPhi did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-gamma-activated mouse MPhi produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MPhi to rDnIFN-gamma is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.
Assuntos
Tatus/metabolismo , Expressão Gênica , Interferon gama/metabolismo , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium leprae/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Tatus/genética , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , DNA Complementar/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/química , Interferon gama/genética , Macrófagos/citologia , Dados de Sequência Molecular , Nitritos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Proteínas Recombinantes , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.
Assuntos
Proteínas de Bactérias/metabolismo , Laminina/metabolismo , Mycobacterium leprae/química , Proteínas Ribossômicas/metabolismo , Animais , Tatus , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Histonas/metabolismo , Humanos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase , Ligação Proteica/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Células de Schwann/microbiologia , Células de Schwann/fisiologiaRESUMO
It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin