Histopathologically, Legionnaires' disease, caused by the Gram-negative bacterium Legionella pneumophila, is an acute fibrinopurulent pneumonia. Since the first documented outbreak of Legionnaires' disease in 1976, several autopsy series have been published (1). Samples from patients who died from L. pneumophila pneumonia exhibit a massive infiltration of neutrophils and macrophages into the alveoli and destruction of alveolar septa. Moreover, the alveolar epithelium shows sloughs, and inflammatory cells exhibit intense necrosis. L. pneumophila is present mainly in alveoli and tends to cluster inside macrophages. In late infection stages, bacteria disseminate to the patient's spleen, kidneys, bone marrow, and lymph nodes (1-4).Different models have been established to analyze specific aspects of infection. Besides human monocellular systems such as macrophages and epithelial cells, protozoa such as Acanthamoeba castellanii, Hartmannella vermiformis, and Dictyostelium discoideum were used to study the cellular and molecular pathogenicity of L. pneumophila (5-9). These studies revealed that L. pneumophila primarily enters phagocytes and resides within a unique membrane-bound compartment termed the Legionellacontaining vacuole (LCV). The establishment of this replication niche requires the translocation of about 300 effector proteins into the host cell via a functional Dot/Icm type IV secretion (10-12). Studying transcriptional responses of L. pneumophila-infected macrophages and D. discoideum vegetative cells also shed light on the cellular mechanisms of Legionnaires' disease (13-16). Moreover, proteomic approaches were shown to be powerful tools to characterize both sides of the host-pathogen interaction (17)(18)(19). Mammalian models such as guinea pigs, mice, rhesus monkeys, and marmosets were used to address immunological, pathological, and pharmacological questions (20)(21)(22). Despite providing enormous progress in the knowledge about mechanisms of L. pneumophila infections, each of the current infection models has intrinsic limitations. Cell culture assays lack the complex interaction networks between the specialized cell types and extracellular components in the human lung. Guinea pig infections require intraperitoneal or intratracheal inoculation techniques, and owing to a different genetic and immunological background, the adequacy and transferability to humans can be questioned.Given the different model-immanent limitations, numerous intra-and extracellular interactions of L. pneumophila factors with human lung tissue structures remain unknown. For example, early infection events appear to be underexplored, since histopathology studies were performed postmortem. Even conspicuous subcellular structures, such as the abundant outer membrane vesicles (OMVs) shed by L. pneumophila, have not yet been investigated in human lung tissue. OMVs contain large amounts of degradative enzymes and other virulence-related proteins, which

Summary Bacterial lipid homeostasis plays an important role for the adaptation to changing environments and under conditions of antimicrobial treatment. The tRNA-dependent aminoacylation of the phospholipid phosphatidylglycerol catalyzed by aminoacyl-phosphatidylglycerol synthases was shown to render various organisms less susceptible to antibacterial agents. Therefore, this type of enzyme might provide a new target to potentiate the efficacy of existing antimicrobials. This study makes use of the Pseudomonas aeruginosa alanyl-phosphatidylglycerol synthase to identify the minimal core domain of this transmembrane protein which is capable of alanyl-phosphatidylglycerol biosynthesis. Using this catalytic fragment we established a reliable activity assay which was used to study the enzymatic mechanism by analyzing an overall of 33 mutant proteins in vitro. Substrate recognition was analyzed by using aminoacylated microhelices as analogs of the natural tRNA substrate. The enzyme even tolerated mutated versions of this minimal substrate which indicates that neither the intact tRNA, nor the individual sequence of the acceptor stem is a determinant for substrate recognition. Furthermore, the analysis of derivatives of phosphatidylglycerol indicated that the polar headgroup of the phospholipid is specifically recognized by the enzyme, whereas modification of an individual fatty acid or even the deletion of a single fatty acid did not abolish A-PG synthesis.

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