*Legionella pneumophila* is a Gram-negative bacterium that is usually found in stagnant water where it replicates inside amoebae. Additionally, *L. pneumophila* can be infectious for humans if contaminated aerosols are inhaled followed by colonization of alveolar macrophages and epithelial cells. *L. pneumophila* remodels the phagosome into the replicative niche termed Legionella containing vacuole (LCV). This is accompanied by recruitment of mitochondria, ER-derived vesicles and ribosomes as well as changes in the lipid composition of the LCV membrane. After replication, the bacteria initiate lysis of the LCV and later of the host cell membrane. Among others, phospholipases are involved in this process. There are 19 phospholipases known for Legionella pneumophila, 15 of the PLA class, 3 PLCs and one PLD.
One goal of this master thesis is the analysis of the lipolytic activity of the phospholipase A PlaD, which has not been characterized extensively yet. To this end, recombinant PlaD is produced in *E. coli*, purified and used for activity assays. Coexpression of PlaD with its eukaryotic interaction partner FttB lead to induction of weak LPLA and phospholipase A activity.
Additionally, the release of *L. pneumophila* from the LCV into the cytoplasm shall be visualized. To this end, a fluorescence reporter that detects glucose-6-phosphate in the host cell cytoplasm will be established. This construct will be useful for the characterization of proteins that are involved in the exit from the LCV.
