Project description:The social amoeba Dictyostelium discoideum is vulnerable to infection by the pathogen that causes Legionnaire's Disease, Legionella pneumophila. Dictystelium cells lacking the dual-specificity phospahatase DupA are at least partially resistant to infection, and strikingly the expression profile of uninfected dupA null cells mirrors the profile of infected wild-type cells to a considerable extent. This suggests that DupA has a key role in regulating host defence systems.
Project description:Legionella pneumophila is a water-borne pathogen, and thus survival in the aquatic environment is central to its transmission to humans. Hence, identifying genes required for its survival in water could help prevent Legionnaires’ disease outbreaks. In the present study, we investigate for the first time the role of the sigma factor RpoS in promoting the survival in water, where L. pneumophila experiences total nutrient deprivation. The rpoS mutant showed a significant survival defect compared to the wild-type strain in defined water medium (DFM). Then, we analyzed the transcriptome of the rpoS mutant during exposure to water using whole genome microarray analysis. We found that RpoS negatively affects the expression of several genes, including genes required for replication, cell division, translation and transcription, suggesting that the mutant fails to shutdown major metabolic programs.
Project description:Legionella pneumophila cells were harvested during exponential growth (RP) and stationary growth (TP). VBNC cells were also anylzed. Protein subfractions were studied.
Project description:Legionella pneumophila is a gram-negative opportunistic human pathogen that infects and multiplies in a broad range of phagocytic protozoan and mammalian phagocytes. Based on the observation that small regulatory RNAs (sRNAs) play an important role in controlling virulence-related genes in several pathogenic bacteria, we attempted to test the hypothesis that sRNAs play a similar role in L. pneumophila. We used computational prediction followed by experimental verification to identify and characterize sRNAs encoded in the L. pneumophila genome. A 50-mer probe microarray was constructed to test the expression of predicted sRNAs in bacteria grown under a variety of conditions. This strategy successfully identified 22 expressed RNAs, out of which six were confirmed by northern blot and RACE. One of the identified sRNAs is highly expressed when the bacteria enter post exponential phase and computational prediction of its secondary structure reveals a striking similarity to the structure of 6S RNA, a widely distributed prokaryotic sRNA, known to regulate the activity of σ70-containing RNAP. A 70-mer probe microarray was used to identify genes affected by L. pneumophila 6S RNA in stationary phase. The 6S RNA encoded by the ssrS gene positively regulates expression of genes encoding type IVB secretion system effectors, stress response genes such as groES and recA as well as many genes with unknown or hypothetical functions. Deletion of 6S RNA significantly reduced L. pneumophila intracellular multiplication in both protist and mammalian host cells, but had no detectable effect on growth in rich media.
