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:Phylogenomic characterization of the causative strain of one of the largest worldwide outbreaks of Legionnaires' disease occurred in Portugal in 2014
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 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.