SFB796 - Sub project C4
(sub project concluded on Dec 31 2012)

Suche


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

 

Project summary

We propose to develop and apply a new strategy to monitor gene expression at the protein level. For this purpose we will combine the minimal disturbing effects that peptide tags exhibit on the function and/or localisation of tagged proteins with the sensitive and convenient readout of Gfp and luciferase reporter proteins. We will construct sensitive Tet-controlled Gfp and luciferase expression systems in Salmonella Typhimurium and in a HeLa cell line. Virulence proteins from S. Typhimurium will be tagged with a 17-mer oligopeptide which specifically induces the Tet repressor, thereby leading to reporter protein expression. The S. Typhimurium mutants will be analyzed for their protein expression patterns during intracellular growth and in vivo in mouse infection models. We expect to determine tissue and cell type specific protein expression patterns for S. Typhimurium. The HeLa reporter cell line will be employed to detect microbial effectors that are located in the nucleus of the infected cell and to study the location of viral proteins for which nuclear shuttling is part of the infection mechanism.

 

Schematic presentation of an in vivo system to analyze the expression of virulence proteins in S. Typhimurium. The bacteria cell is depicted as an oval. The arrows indicate the respective gene. A dark green rectangle with an orange arrow shows a C-terminal fusion of TIP to a virulence gene (gene x). The Tet controlled promoter region is described as a grey rectangle and the Tet-Repressor as two blue ovals and circles. The expressed TIP fusion protein is shown as a green oval with an orange line. (A) In the absence of the TIP fusion protein the expression of the reporter gene is repressed by the Tet Repressor. (B) After expression of the TIP tagged virulence gene, Tet-Repressor is induced followed by expression of the reporter gene.

 

Project relevant publications

  • Schumacher, M. A., Allen, G.S., Diel, M., Seidel, G., Hillen, W. and Brennan, R. G. (2008). Structural basis for allosteric control of the transcription regulator CcpA by the phosphoprotein HPr-Ser46-P. Cell 118, 731-741.
  • Bertram, R. and Hillen, W. (2008). The application of Tet repressor in prokaryotic gene regulation and expression. Microbial Biotechnology 1, 2-16.
  • Gandotra, S., Schnappinger, D., Monteleone, M., Hillen, W. and Ehrt, S. (2007). In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential fort he bacteria to persist in mice. Nat Med 13, 1515-1520.
  • Knott, A., Drueppel, L., Beyer, T., Garke, K., Berens, C., Herrmann, M. and Hillen W.(2005). An optimized conditional suicide switch using doxycycline-dependent expression of human tBid. Cancer Biol Ther 4, 532-536.
  • Klotzsche, M., Berens, C. and Hillen, W.(2005). A peptide triggers allostery in Tet repressor by binding to a unique site. J Biol Chem 280, 24591-24599.