RESUMEN
By integrating the microarray expression data and a global E. faecalis transcriptional network we identified a sub-network activated by zinc and copper. Our analyses indicated that the transcriptional response of the bacterium to copper and zinc exposure involved the activation of two modules, module I that contains genes implicated in zinc homeostasis, including the Zur transcriptional repressor, and module II containing a set of genes associated with general stress response and basal metabolism. Bacterial exposure to zinc and copper led to the repression of the zinc uptake systems of module I. Upon deletion of Zur, exposure to different zinc and copper conditions induced complementary homeostatic mechanisms (ATPase efflux proteins) to control the intracellular concentrations of zinc. The transcriptional activation of zinc homeostasis genes by zinc and copper reveals a functional interplay between these two metals, in which exposure to copper also impacts on the zinc homeostasis. Finally, we present a new zinc homeostasis model in E. faecalis, positioning this bacterium as one of the most complete systems biology model in metals described to date.
Asunto(s)
Proteínas Bacterianas/genética , Cobre/metabolismo , Enterococcus faecalis/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/genética , Zinc/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Homeostasis , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Alineación de SecuenciaRESUMEN
Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.