Detection of predicted small RNA of Legionella pneumophila
Ontology highlight
ABSTRACT:
ORGANISM(S): Legionella pneumophila subsp. pneumophila str. Philadelphia 1
PROVIDER: GSE19196 | GEO | 2010/01/01
SECONDARY ACCESSION(S): PRJNA123749
REPOSITORIES: GEO
Similar Datasets
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.
2010-01-01 | GSE19200 | GEO
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 Ï70-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. The 70-mer microarray representing all annotated ORFs of L. pneumophila has been previously described (Charpentier et al., 2008). RNA was extracted from ssrS mutant strain and the wild-type strain grown to PE phase. gDNA was used as a reference channel on each slides. Labelling of samples, hybridization strategy and data acquisition were performed as described in the Methods section. Local background was removed from spot signal intensity and normalization was carried out by calculating the fraction over the total signal intensity in both channel as previously described (Faucher et al., 2006). Signal levels that were lower than background in experiments and controls were filtered out. A total of six cDNA to reference ratios were recorded for each time point. Statistical analysis between mutant and wild-type strain was performed using unpaired one-tailed studentâs t-test. Genes were considered as differentially expressed if they demonstrated a ratio to control value of ±2-fold with a p < 0.001.
2010-06-24 | E-GEOD-19200 | biostudies-arrayexpress
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 Ï70-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. A microarray was designed for the detection of 101 predicted sRNAs by using OligiWiz version 2.1.3 (Nielsen et al., 2003, Wernersson & Nielsen, 2005). The prokaryotic setting was used to design 50-mers probes located, when possible, at the 3â end of the predicted genes. Probes for 10 negative controls, representing 10 genes of the L. pneumophila Paris plasmid, were also designed. Probes (Illumina) were dissolved in 50% DMSO to a final concentration of 30 uM and printed in triplicate on UltraGAPS coated glass slides (Corning) using a PerkinElmer SpotArray microarray printer. Fifteen micrograms of total RNA was labelled during cDNA synthesis using Superscript II reverse transcriptase (Invitrogen) and amino-allyl dUTP (Sigma). Bacterial genomic DNA was used as the reference channel on each slide to allow comparison of each time point and different samples (Talaat et al., 2002). Five micrograms of genomic DNA (gDNA) was labelled with amino-allyl dUTP by using Klenow fragment and random primers (Invitrogen) at 37 °C for 18 h. DNA was subsequently coupled to the succinimidyl ester fluorescent dye AlexaFluor 546 (for cDNA) or Alexa Fluor 647 (for gDNA) (Invitrogen) following manufacturerâs protocols. Hybridization and data acquisition were performed as previously described (Hovel-Miner et al., 2009).Very low density array, like the sRNA microarray used here, cannot be normalised with common procedure like total intensity or lowess. Local background was removed from spot signal intensity and the noise signal was estimated by recording the average signal intensity of 10 negative controls printed on the chip. Normalisation of signal intensity was carried out by calculating the fold increase over the noise signal value. Correlation of replicates using this normalisation procedure was ⥠0.95.
2010-06-24 | E-GEOD-19196 | biostudies-arrayexpress
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
2010-06-24 | E-GEOD-19451 | biostudies-arrayexpress
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.
2011-01-01 | GSE23032 | GEO
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.
2011-01-01 | GSE23029 | GEO
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. To validate the use of SCOTS, we compare the expression patterns obtained by SCOTS to those obtained when a standard microarray protocol was used. RNA from bacteria in E phase was treated using SCOTS or a standard microarray protocol where the cDNA is labeled during the reverse transcription reaction as previously published. The resulting cDNA was hybridized to the microarray slides as described previously and data analysis was carried out the same way as for the SCOTS treated samples.
2011-01-01 | E-GEOD-23032 | biostudies-arrayexpress
Project description:These data were obtained using a recently-developed high throughput method to probe regional RNA accessibility by mimicking in vivo antisense hybridization. The method (INTERFACE) is an engineered RNA system (for use in E. coli) that exploits conserved bacterial mechanisms of translational stalling and Rho-dependent transcription termination mechanisms to quantify RNA hybridization (via an asRNA targeting an RNA region of interest) via a transcriptional elongation response. We first establish the high throughput system by evaluating the accessibility profile of a well-characterized gI intron (overexpressed). We then apply this method with bioinformatics and machine learning approaches to profile over 900 RNA-interaction interfaces in 71 validated, but largely mechanistically under-characterized, sRNAs of Escherichia coli, in the presence and absence of a global regulator, Hfq. Importantly, we showcase the utility of INTERFACE in detecting RNA regions whose hybridization is Hfq-sensitive, finding two thirds of tested sRNAs with Hfq dependency. Further, we identify in vivo hybridization patterns that hallmark functional regions in 16 well-characterized sRNAs and couple these insights to a computational target prediction algorithm to identify novel mRNA targets for six under-characterized and two well-characterized sRNAs.
2018-10-06 | GSE117939 | GEO
Project description:In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon and nitrogen limitation. RNAseq analysis identified 98 sRNAs, of which 69 were cis-encoded and located antisense to their target genes, and 30 were trans-encoded and linked to predicted target genes through complementarity analysis. The most prevalent functional roles of target genes were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Thirty-two percent of the sRNAs had been identified in a previous study of R. pomeroyi growth on organic sulfur compounds, and may be constitutively expressed, while 69% were not identified in previous studies. Eighty-six percent and were transcribed equally under both carbon and nutrient limitation, and may be involved in a general stress response; 14% were differentially regulated under carbon versus nitrogen stress, and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome rather than central metabolism encoded in the core genome.
2016-02-27 | E-MTAB-4468 | biostudies-arrayexpress