Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila in comparison to uninfected cells was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co-regulated genes, an enrichment of preferentially up- or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host-pathogen interactions and for further studies on the Dictyostelium response to Legionella infection. The bacterial strain used in this study was L. Pneumophila Philadelphia I JR32. The strain was grown on buffered charcoal yeast extract agar (BCYE) at 37M-BM-0C with 5% CO2 atmosphere for 3 days. The D. discoideum wild-type strain AX2 was grown at 23M-BM-0C in 75 cm2 cell-culture flasks with 10 ml HL5 medium. For infection, Dictyostelium cells were harvested, resuspended in a 1:1 solution of HL5 medium and Soerensen buffer. Fifteen millilitres of a 1M-CM-^W10e6 cells/ml suspension were seeded into a 75 cme2 cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. After different time intervals of incubation (1, 3, 6, 24, and 48 h) the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells. Usually two or three parallel cultures for the experiment and the control were inoculated per infection. The percentage of infected cells was determined by in situ hybridization with Legionella-specific 16S rRNA probes. The average from three independent determinations was 34, 42, 42, 57 and 82% of infected Dictyostelium cells after 1, 3, 6, 24 and 48 h, respectively.

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Differential expression of Dictyostelium discoideum AX2 upon infection with wt L. pneumophila JR32 vs. uninfected 48h p.i. (timecourse experiment)

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