Project description:Transcriptional profiling of L. pneumophila JR32 (wild-type) and rpoS- (LM1376) strains grown to stat phase compared to cultures grown to log phase Keywords: growth phase
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 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. The WT and rpoS mutant were grown to exponential phase in rich AYE broth, washed three times in DFM (NaCl 0.5 g/L, KH2PO40.2 g/L, KCl 0.5 g/L, pH=6.9) and resuspended in DFM at an OD600 of 0.1, After 24h exposure, RNA was extracted.
Project description:Legionella pneumophila Philadelphia-1 strain was grown to stationary phase in AYE broth and starved in freshwater for 2 hours and RNA was harvested with or without sublethal heat shock via immersion in a 55 degree C hot water bath for 5 minutes
Project description:Legionella pneumophila (Lp) is a waterborne bacterium able to infect human alveolar macrophages, causing a severe pneumonia known as Legionnaires’ disease. In water, Lp grows inside ciliates and ameoba. Lp is able to survive for several months in water, while searching for host cells. In Lp, the sigma factor RpoS is important for survival in water. Several small regulatory RNAs (sRNA) are known to regulate the expression of RpoS in other bacteria. A previous transcriptomic study showed that RpoS positively regulates the sRNA Lpr10 in Lp. Microarray analysis was performed to investigate the genes regulated by Lpr10.
Project description:Legionella pneumophila (Lp) is an opportunistic pathogen and its survival in water is critical for human infection. Therefore, identifying the genes of Lp that are required for survival in water may help devise strategies to prevent Legionella outbreaks. In this study, we exposed Lp in rich medium and in an artificial freshwater medium (Fraquil) for 2, 6 and 24 hours to uncover the global transcriptomic changes of Lp in water. The repression of major metabolic pathways, such as division, transcription and translation, suggests that Lp enters a dormant state in water. The induction of the flagellar associated genes (flg, fli and mot), enhance entry genes (enh) and some Icm/Dot effectors suggests that Lp may be waiting to establish intracellular replication in suitable host. Moreover, many genes involved in resistance to antibiotic and oxidative stress were induced, suggesting that Lp may be more tolerant to environmental stresses in water. Indeed, Lp exposed to water is more resistant to erythromycin, gentamycin and kanamycin than those cultured in rich medium. Apart from this, the gene bdhA involved in the degradation of the intracellular energy storage compound poly-hydroxybutyrate is highly expressed in water. Further characterization shows that bdhA is positively regulated by RpoS during short-term exposure to water. The deletion mutant of bdhA had a survival defect in water at 37°C, demonstrating that this gene is important for maintaining the long-term survivorship of Lp in water. Other identified genes highly induced upon exposure to water could also be necessary for Lp to survive in water. Legionella pneumophila Philadelphia-1 strain JR32 was grown in AYE broth at 25°C shaking to OD600 of 1 in triplicate. Samples were taken for analysis; this is the control. Then the cultures were washed three times in Fraquil and resuspended in Fraquil to an OD600 of 1 and transfered to vessels of bioreactior (Biostat Q-plus). Samples were taken after 2h, 6h and 24h.
Project description:Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co-incubation with Legionella hackeliae and L. pneumophila DeltadotA. One hundred and thirty-one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. 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. 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 strains used in this study were L. pneumophila Philadelphia I JR32, L. pneumophila Philadelphia I JR32 LELA 3118 (dotA3118:Tn903 DLL LacZ) and L. hackeliae (ATCC 35250). The Legionella strains were 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 square-cm cell culture flask and the amoebae were inoculated with 10e7 bacteria/ml. Three different pairs of infection were compared: 1. AX2 infected with L. pneumophila JR32 versus uninfected cells; 19 microarrays of seven independent infections; 2. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. pneumophila JR32 delta DotA; 4 microarrays of two independent infections; 3. AX2 infected with L. pneumophila JR32 versus AX2 infected with L. hackeliae; 4 microarrays of two independent infections. 24h post infection the RNA was isolated from 1.5M-CM-^W10e7 Dictyostelium cells and microarray analysis was performed as described (Farbrother et al., 2006).
Project description:Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoan, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known virulence determinant used by L. pneumophila to infect host cells is a Type IVb translocation system named Icm/Dot, which is used to modify the host cell functions to the benefit of the bacteria. To date the Icm/Dot systeme is known to translocate more than 100 effectors. While the transcriptional response of Legionella to the intracellular environement of A. castelannii as already been investigated, much less is known of how Legionella reacts transcriptionnally inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth and during infection of human cultured macrophages by using microarray and a RNA amplification procedure called SCOTS to allow for the study of conditions of low bacterial loads. Among the genes induced intracellularly are those involved in amino acid synthesis pathway leading to L-arginine, L-histidne and L-proline as well as many transport system involved in amino acid and iron uptake. The Icm/Dot systems is not differentially expressed inside cells compare to the E phase control but the effectors are strongly induced. The intracellular transcriptome was further used to identify putative new Icm/Dot effectors and translocation was show to occur for 3 of them. This study provides a comprehensive view of how L. pneumophila react to the human macrophages intracellular environment.