Christiane Georgi

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C4 In vivo expression analysis of Salmonella Typhimurium virulence proteins in macrophages and mouse infection models

Principal investigator
Wolfgang Hillen

Mentor
Frederik Börnke

PhD exam: 25.02.2013

In vivo expression analysis of Salmonella Typhimurium virulence proteins in macrophages and mouse infection models

So far there is only little known about the in vivo protein expression pattern of the human pathogen Salmonella enterica serovar Typhimurium (Gerlach and Hensel, 2007). To investigate the expression profiles of Salmonella virulence factors during an infection we want to use a reporter system based on the 17-mer oligo peptide TIP (transcription inducing peptide) which specifically induces Tet repressor controlled transcription (Klotzsche et al., 2005; Klotzsche et al., 2007). By chromosomally fusing the DNA sequence of TIP to genes encoding Salmonella virulence proteins their expression during an infection in macrophages and mice can be determined according to the expression of a reporter gene which is under the control of the Tet repressor (Figure 1). Therefore we will first have to construct sensitive Salmonella Typhimurium reporter strains using the gfp reporter gene for single cell detection and luc for quantification of protein expression. To validate the constructed reporter system TIP will be fused to Salmonella virulence proteins known to be highly induced during intracellular growth. By infecting a macrophage cell line and mice with the Salmonella reporter strains the expression of the respective fusion proteins can be quantitatively determined by luciferase activity. Furthermore, the intracellular GFP fluorescence can be determined to analyse tissue and cell type specific virulence protein expression. Afterwards the reporter system will be applied to analyse the so far unknown expression profiles of several Salmonella virulence factors and to identify novel proteins contributing to the pathogenicity of Salmonella.
This work should provide an insight into the proteomic situation of Salmonella Typhimurium during an infection and thereby contribute to a better understanding of Salmonella pathogenicity.

Figure: Schematic illustration of the Salmonella Typhimurium reporter strains. At different sites in the genome are located: i) the gene of interest (green) encoding a Salmonella virulence factor tagged with the sequence of the oligo peptide TIP (orange), ii) the tetR gene (blue) and iii) the reporter gene (yellow) under the control of the Tet operator (gray). A If the virulence factor is not expressed there is no induction of the TetR dimer (blue ovals) by TIP. As a result the reporter gene transcription is inhibited. B If expression of the fusion protein (green oval with orange tag) occurs in the course of infection, TIP binds into the binding pockets of TetR and specifically induces the dissociation of the Tet repressor from the operator sequence so that the resulting reporter gene activity can be quantified.

 

Publications

Georgi, C., Bürger, J., Hillen, W. and Berens C. (2012). Promoter Strength Driving TetR Determines the Regulatory Properties of Tet-Controlled Expression Systems. PLoS One 7(7), e41620.

 

Presentations

July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Establishing a novel, sensitive TetR-controlled reporter system for the in vivo expression analysis of Salmonella Typhimurium virulence proteins
Talk
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Creating a sensitive TetR controlled reporter system for the in vivo expression analysis of Salmonella Typhimurium virulence proteins
Poster
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Creating a sensitive TetR controlled reporter system for the in vivo expression analysis of Salmonella Typhimurium virulence proteins
Talk and Poster
     
September 2010 2nd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Creating a sensitive TetR-controlled reporter system
Talk
     
September 2009 First Annual Retreat, Erlangen School of Molecular Communication, Schloss Atzelsberg, Atzelsberg, Germany
Creating a sensitive TetR-controlled reporter system
Talk