Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing
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ABSTRACT:
ORGANISM(S): Escherichia coli Staphylococcus aureus
PROVIDER: GSE141018 | GEO | 2020/05/25
REPOSITORIES: GEO
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Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of ϕNM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Following strains were grown in TSA broth: Staphylococcus aureus USA300 (reference) Staphylococcus aureus USA300 with deletion of ϕSa2usa (Query) Staphylococcus aureus USA300 with deletion of ϕSa3usa (Query) Staphylococcus aureus USA300 Prophage-free mutant (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa2mw (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa3usa (Query) strain: Staphylococcus aureus USA300 Prophage-free mutant lysogenized with both ϕSa2mw and ϕSa3usa (Query) RNA samples were harvested at early log, midlog and stationary phase.Samples were hybridized on aminosilane coated slides with 70-mer oligos.
2011-02-28 | E-GEOD-26623 | biostudies-arrayexpress
Project description:Conventional prokaryotic RNA labeling method usually requires large amounts of starting materials and tends to generate high background signals. Recently, two novel methods based on amplification systems were introduced. Here, we compared three alternative strategies: direct labeling method, ployadenylation-involved oligo-dT priming amplification method and random priming amplification method (hereafter referred to as DL, PAOD and RPA method in this article) for prokaryotic RNA labeling employing the expression profiling investigation in Escherichia coli (E. coli) heat shock model.
2009-07-25 | GSE17074 | GEO
Project description:Conventional prokaryotic RNA labeling method usually requires large amounts of starting materials and tends to generate high background signals. Recently, two novel methods based on amplification systems were introduced. Here, we compared three alternative strategies: direct labeling method, ployadenylation-involved oligo-dT priming amplification method and random priming amplification method (hereafter referred to as DL, PAOD and RPA method in this article) for prokaryotic RNA labeling employing the expression profiling investigation in Escherichia coli (E. coli) heat shock model. To identify an optimal RNA labeling method for prokaryotic microarray analysis, experiments were performed with starting RNA obtained from E.coli growing at control (37°C) or heat shock (43°C) condition. We employed 0.5 μg of total RNA for a single round of amplification using PAOD and RPA methods coupling to IVT to generate cDNA targets. In addition, an approach using DL method was also performed. Three types of cDNA mixtures containing Cy5-labeled (or Cy3-labeled) control DNA and Cy3-labeled (or Cy5-labeled) DNA targets were hybridized with E.coli microarrays in a dye-swap strategy. Replicate experiments were conducted from the same batch of total RNA to assess technique reproducibility of each approach.
2010-05-15 | E-GEOD-17074 | biostudies-arrayexpress
Project description:Compilation fo whole genome gene expression changes in Staphylococcus aureus USA300 LAC cultures grown in the presence of vehicle or the anti-gout drug benzbromarone. The drug was intially screened as effective against the agr quorum sensing system in Staphylococcus aureus AH1677. A microarray study using total RNA harvested from three cultures of Staphylococcus aureus USA300 LAC plus vehicle control and three cultures of Staphylococcus aureus USA300 LAC plus 12 uM benzbromarone.
2014-03-19 | E-GEOD-55980 | biostudies-arrayexpress
Project description:The study aims to compare gene expression patterns of Staphyloccoccus aureus USA300 vs USA300 sae knockout. Samples were hybridized on aminosilane coated slides with 70-mer oligos.
2011-04-08 | GSE26243 | GEO
Project description:S. aureus response to fakA deletion Gene expression profiles were generated by microarray analysis of S. aureus WT (USA300) or fakAKO
2014-08-01 | GSE57340 | GEO
Project description:Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the hostM-bM-^@M-^Ys inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 95 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.82). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.94, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues. To create a host gene expression-based classifier for S. aureus infection, mice from a variety of experimental conditions were utilized. Seven different strains of inbred mice (n=187 total) were challenged with four different S. aureus strains via intraperitoneal inoculation and sacrificed at various time points as described in Methods. The comparator group for model derivation included 50 A/J or C57BL/6J mice inoculated with E. coli (O18:K1:H7) as well as 54 uninfected mice. Next, the murine S. aureus classifier was externally validated in outbred CD-1 mice with S. aureus infection (Sanger 476 or USA300), E. coli infection (O18:K1:H7), or uninfected controls (10 animals per condition). Method: All experiments were performed on mice 6-8 weeks old. For the murine S. aureus predictor, seven inbred mouse strains (3 mice/strain: 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, C57BL/6J, C3H/HeJ, and NOD/LtJ) were IP inoculated with 107 CFU/g of S. aureus Sanger476, euthanized at 2h after injection, and bled. This was repeated using four different S. aureus strains (USA100, USA300, MW2, and Sanger476) in A/J mice (n=3 per S. aureus strain). For time series experiments, both A/J and C57BL/6J mouse strains were IP inoculated with S. aureus Sanger476 as above, and sacrificed at 2, 4, 6, and 12h after injection (n=5 per time point).
2013-01-11 | E-GEOD-33341 | biostudies-arrayexpress
Project description:In the community and clinical settings MRSA infections are more commonly caused by a USA300 strain vs a USA400 strain. The study aims to compare gene expression patterns of Staphyloccoccus aureus USA300(reference) vs USA400(query) and to determine why USA400 is being replaced by a USA300 strain.
2011-04-08 | GSE26255 | GEO
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) infections result in more than 200,000 hospitalizations and 10,000 deaths in the United States each year and remain an important medical challenge. To better understand the transcriptome of Staphylococcus aureus USA300 NRS384, a community-acquired MRSA strain, we have conducted an RNA-Seq experiment on WT samples.
2017-11-15 | GSE102279 | GEO
Project description:The study aims to compare gene expression patterns of Staphyloccoccus aureus USA300 wild type (reference) vs USA300 agr knockout(query). The agr quorum sensing system is an important regulator of density dependent gene expression. To determine the role of agr in USA300 virulence a agr mutant was constructed and differential gene expression compared to the parental wildtype.
2011-04-08 | GSE26254 | GEO