IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Study of bacterial pathogens using green fluorescent protein-biosensors: Characterization of lipid metabolism during Salmonella infections
Autor/es:
LORENZI L, DIACOVICH L, GRAMAJO H
Lugar:
Buenos Aires
Reunión:
Workshop; XV Giambiagi Winter School 2013 - Information processing in biological systems: from cells to equations, and back; 2013
Institución organizadora:
Departamento de Física. Fac. de Cs. Exactas y Naturales, UBA
Resumen:
Salmonella is a facultative intracellular pathogen capable of infecting a wide range of animals, causing diseases ranging from self-limiting enteritis to typhoid fever. The virulence of this pathogen relies on its ability to establish a replicative niche, named the Salmonella-containing vacuole (SCV), inside host cells. However, the microenvironment of the SCV and the bacteria and the metabolic pathways required during infection are largely undefined. The objective of the present application is to monitor the expression profile of genes involved in the biosynthesis or degradation of fatty acids during the infection process. For this, we have developed different biological probes called 'biosensors' whose expression is modulated by the environment and the physiological state of the bacterium. We constructed these probes by fusing regulatory regions of the genes under study to the gene codifying for the Green Fluorescent Protein (GFP). For this, we performed a bioinformatic analysis to determine the approximately 300 base pairs promoter regions of the genes, and cloned them into the pFPV25 plasmid, as transcriptional fusions to the gfpmuta3 gene. GFP absorbs light with an excitation maximum of 395 nm, and fluoresces with an emission maxima of 510 nm. Since this can occur in the absence of any cofactors, GFP is extremely useful as a marker for gene expression. Specifically, we selected genes whose products are involved in crucial steps of lipid biosynthesis or degradation (fabHDG, fadF, fadI, and fadAB). Wild type Salmonella strains were transformed with the generated plasmids and the expression of biosensors was analysed. The ability of these probes to be induced by a specific metabolic change was already tested in vitro, using different mediums. Then, we will test them during infection in macrophages. For this, we developed a methodology to rapidly lyse the infected cells and collect bacteria. Once isolated, bacteria are analysed by flow cytometry to quantify their fluorescence level and their population homogeneity. Altogether these studies can help us to better understand the host-pathogen interaction and to establish those molecular pathways, involved in lipid metabolism, essential for survival, replication and persistence of Salmonella.