Project description:Legionella pneumophila wild-type and hfq mutant were grown to post-exponential. The wild-type strain is used as the control. Two strains: wild-type (control) and hfq mutant (test). Three biological replicates. Ch1 is gDNA, use as a reference channel (Talaat, 2002, NAR)
Project description:This SuperSeries is composed of the following subset Series: GSE19196: Detection of predicted small RNA of Legionella pneumophila GSE19200: Gene affected by deletion of 6S RNA in post-exponential phase Refer to individual Series
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.
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 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. The aim of this work was to study the gene expression profile of Legionella during infection of macrophages infected at a low multiplicity of infection (MOI). SCOTS is a method that allows amplification of small amounts of bacterial RNA from infected host cells, while discarding host cell transcripts and ribosomal RNA. Before infection, macrophages derived from the THP-1 monocyte cell line were pre-treated with antibodies against the L. pneumophila major outer membrane protein, which increases the efficiency of bacterial entry into host cells . After 2 hours of infection, the macrophages were washed and treated with gentamicin for 1 hour, to synchronize the infection and kill extracellular bacteria, and cells were washed 3 times and fresh medium was added. Samples for the first time point (T0) were collected after the gentamicin treatment. Samples were also collected after 6h (T6) and 18h (T18). Samples from all conditions, including growth in AYE broth to exponential (E) or post exponential (PE) phase, were treated with three consecutive rounds of SCOTS and the resulting cDNA was labeled and hybridized to the microarray slides. As a reference channel, labeled gDNA was also hybridized. For each condition studied, three independent biological replicates and two technical replicates were analyzed, resulting in six replicates for each condition. The data was background subtracted and normalized by calculating the contribution of each spot to the total intensity and the ratio to gDNA was recorded. A one tailed student's T test was used for statistical analysis and the ratio between test conditions (PE, T0, T6 and T18) and the control conditions (E phase or T0) was calculated.
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.
Project description:Comparaison of the transcriptional profile of mutant of the lpp1663 gene and the parent strain Legionella pneumophila strain Paris. lpp1663 codes for a protein with a ProQ domain (PFAM PF04352). A mutant of this gene was obtained by deleting the lpp1663 ORF and replacing it with a gene confering resistance to kanamycin. Transcriptional profiling was done on cultures grown to exponential phase (OD=0.8). Three independent biological replicates were analysed.
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:Comparaison of the transcriptional profile of mutant of the lpp0148 gene and the parent strain Legionella pneumophila strain Paris. lpp0148 codes for a protein with a ProQ domain (PFAM PF04352). A mutant of this gene was obtained by inserting a premature stop codon in the lpp0148 ORF. The lpp0148 mutant is constitutively competent for natural transformation as described here http://www.ncbi.nlm.nih.gov/pubmed/26526572. Transcriptional profiling was done on cultures grown to exponential phase (OD=0.8). Three independent biological replicates were analysed.