Protein phosphorylation-dependent virulence regulation in Listeria monocytogenes – LimoPhosProt

In many pathogens, protein phosphorylation represents one of several mechanisms that control their virulence. In this project, we will study the mechanisms by which proteins of the Listeria monocytogenes phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), a transport system for numerous sugars, control the activity of the virulence regulator PrfA. A series of mutants will be constructed affected in PTS componenets specific for glucose- and cellobiose, the two carbon sources most strongly inhibiting PrfA activity, and in the non-PTS glucose transporter GlcU. Alleles encoding non-phosphorylatable or phosphomimetic forms of these proteins will be ectopically inserted in the int-comK region of L. monocytogenes. The effect of the mutations on PrfA activity will be studied and those affecting PrfA function will also be tested for their influence on L. monocytogenes virulence. PTS proteins and PrfA will be purified and protein/protein interaction studies with various techniques (plasmon resonance spectroscopy, pull down and elution retardation experiments) will be carried out in order to demonstrate by in vitro studies which PTS components interact with PrfA in a phosphorylation-dependent manner. In a global approach, we will determine the Ser/Thr/Tyr phospho-proteome (P-proteome) of L. monocytogenes by using the most advanced techniques of mass spectrometry (LTQ Orbitrap) and software specifically developed for the analysis of P-proteome data (MSQuant). These experiments will be carried out with the wild-type EGD-e strain (grown under various conditions) and a few protein kinase/P-protein phosphatase mutants as well as a prfA* strain (Gly145Ser replacement; PrfA constitutively active) derived from it. Mutants based on identified P-proteins playing a role in major metabolic pathways or carrying out other important cellular functions will also be constructed. These proteins will be purified and biochemically characetized. The effect of phosphorylation on their activity will be tested in vivo and in vitro in order to better understand global regulatory networks of this pathogen. P-proteins suspected to play a role in Listeria virulence will be identified and corresponding deletion mutants and mutants, in which the phosphorylatable amino acids will be replaced with non-phosphorylatable or phosphomimetic ones, will be constructed. The P-proteome data will also be compared to recently obtained transcriptome results in order to identify P-proteins, the genes of which exhibit an altered expression during the infection process or under specific growth conditions. This approach became possible, because transcriptome data were obtained in the laboratory of partner 3 after growth of L. monocytogenes in the intestinal lumen of axenic mice and in human blood. Additional transcriptome studies and virulence assays will be carried out with selected mutants. We will determine the entrance and survival in human epithelial cell lines as well as the survival rate in mice, the lethal dose LD50 and measure the appearance of the pathogen in liver and spleen after infection by intraveinous inoculation. Oral infections will be carried out in order to study the effect of the mutations on early steps of infection.