Study of gene expression using biosensors in the context of Salmonella infection: Lipid Metabolism

DIACOVICH L, LORENZI L, GRAMAJO H

Rosario

Congreso; IX Congreso Nacional de Microbiología General (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Salmonella is a facultative intracellular pathogen capable of infecting a wide range of animals, causing diseases ranging from self-limiting enteritis to typhoid fever. In last years problems related to Salmonella have increased significantly, both in terms of incidence and severity of cases of human salmonellosis. The emergence of multidrug-resistance Salmonella strains with resistance to fluoroquinolones and third generation cephalosporins is a serious development, which results in severe limitation for effective treatment of human infections. Importantly, non-typhoidal Salmonella serovars can cause invasive infections in immuno-compromised adults. This has emerged as a major public health problem in the context of HIV in sub-Saharan Africa. Salmonella uses two type III secretion systems to deliver effector proteins directly into the host cell to promote infection and disease. 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 this work is to develop different biological probes called ´biosensors´ whose expression is modulated by the environment and the physiological state of the bacterium. These probes are designed to monitor the expression profile of genes involved in the biosynthesis or degradation of fatty acids. Consequently the results will reflect the adaptive metabolism used by Salmonella to survive during the infection process. We constructed transcriptional fusions by fusing promoter regions, corresponding to the genes under study, to the gfpmut3a gene. Specifically, we selected genes whose products are involved in crucial step of lipid biosynthesis or degradation (accB, aceB, fabHDG, fabB, fadL, fadD, fadE, fadG, 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 first tested in vitro, using different mediums. Then the biosensors were analysed during infection in different cell lines: macrophages and epithelial cells. For this, we developed a methodology to rapidly lyse the infected cells and collect bacteria. Once isolated, bacteria were analysed by flow cytometry to quantify their fluorescence level and their population homogeneity. The data collected in previous steps will allow us to select one or two genes as interesting targets for the generation of mutants. These Salmonella mutants will be evaluated in the context of in vivo infection in mice. 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.