Transcription profiling by array of Pseudomonas aeruginosa wild-type and phhR mutants grown in a defined synthetic cystic fibrosis sputum medium with and without aromatic amino acids
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ABSTRACT: Arrays comparing Pseudomonas aeruginosa growth in a defined synthetic cystic fibrosis sputum medium with and without aromatic amino acids. Additional arrays comparing wild-type Pseudomonas aeruginosa and phhR mutant P. aeruginosa in defined synthetic cystic fibrosis sputum medium.
Project description:Effect of anaerobic growth condition on gene expression profile of Pseudomonas aeruginosa PA14 grown in cystic fibrosis sputum with 100 mM nitrate added
Project description:Pseudomonas aeruginosa chronically colonizes the lungs of individuals with CF, where it reaches high cell densities and produces a battery of virulence factors. Upon infection, a single strain of P. aeruginosa can colonize an individualâs lungs throughout his or her lifetime. To understand the evolution of P. aeruginosa during chronic lung infection, we conducted both genotypic and phenotypic analyses on clinical isogenic strains obtained from the lungs of three different individuals with CF. These strains were isolated over a period of approximately ten years and possess phenotypes that are commonly observed in isolates from the CF lung, such as the antibiotic resistant dwarf and mucoid phenotypes. Microarray analyses were carried out on isolates grown in a chemically defined medium that mimics the nutritional environment of the CF lung, synthetic CF sputum medium (SCFM). 17 clinically isolated P. aeruginosa strains from three individuals with CF (5 strains from individual P1, 7 strains from individual P2, 5 strains from individual P3). Two reference strains PAO1 and PA14. All experiments were biologically duplicated.
Project description:One of the hallmarks of Pseudomonas aeruginosa cystic fibrosis (CF) infection is very high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by HCD quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection is largely unknown. Hence, understanding the nutrient factors contributing to HCD in the CF lung will yield new insights into the 'metabolic pathogenicity' and potential treatment of CF infections caused by P. aeruginosa. Herein, we performed microarray studies of P. aeruginosa directly isolated from the CF lung to demonstrate its metabolic capability and virulence in vivo. Our in vivo microarray data, confirmed by real-time reverse-transcription-PCR, indicated P. aeruginosa expressed several genes for virulence, drug-resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The data also indicates deregulation of several pathways, suggesting in vivo evolution by deregulation of a large portion of the transcriptome during chronic CF infection. To our knowledge, this is the first in vivo transcriptome of P. aeruginosa in a natural CF infection, and it indicates several important aspects of pathogenesis, drug-resistance, and nutrient-utilization never before observed in vivo. Experiment Overall Design: The purpose of the experiment was to observe which genes are upregulated in P. aeruginosa during chronic CF lung infection as compared to PAO1. All in vitro studies were grown in 1x M9 minimal media supplemented with 20 mM citrate and grown to mid-log phase prior to RNA isolation. The in vivo RNA was isolated directly from CF sputum samples after TRIzol treatment. Each in vitro sample (both for PAO1 and the CF sputum pool isolate) were processed individually and in triplicate. Two in vivo isolations from sputum were conducted from the same patient but two different sputum samples. After isolation of total RNA, samples were processed for microarrays (i.e. cDNA synthesis, fragmentation, labeling, etc) as recommended by Affymetrix, and processed on the GeneChip as recommended by Affymetrix.
Project description:The aim of this study is to evaluate the evolutionary robustness of the quorum sensing inhibitor (QSI) furanone C-30 for the treatment of P. aeruginosa biofilm infections. We repeatedly exposed P. aeruginosa biofilms to furanone C-30 (with or without tobramycin) in the synthetic cystic fibrosis sputum medium (SCFM2) and characterized the genotype and phenotype of the evolved lineages. P. aeruginosa biofilms were grown in SCFM2 for 24 h after which the treatment in fresh SCFM2 was added to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. After each cycle the number of CFU was determined and an aliquot of the culture was stored at -80°C in Microbank vials to allow further tests on the evolved strains. A sample from the treated biofilm was used to prepare a new overnight culture, in order to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. Whole-genome sequencing was performed on the wild type P. aeruginosa PAO1 and on the exposed lineages after 5, 10 and 16 cycles.
Project description:Pseudomonas aeruginosa was repeatedly and intermittently exposed to tobramycin. Bacteria were grown in synthetic cystic fibrosis medium in wells of a 96-well microtiter plate. After 24 hours, more medium with or without tobramycin was added. After another 24 hours of incubation, a subsample of the well content was used to inoculate fresh synthetic cystic fibrosis medium in a 96-well microtiter plate. This was repeated for a total of 15 cycles. Evolved lineages were then DNA-sequenced to screen for genome changes.
Project description:<p>While bacterial metabolism is known to impact antibiotic efficacy and virulence, the metabolic capacities of individual microbes in cystic fibrosis lung infections are difficult to disentangle from sputum samples. Here, we show that untargeted metabolomic profiling of supernatants of multiple strains of<em> Pseudomonas aeruginosa</em> and <em>Staphylococcus aureus </em>grown in monoculture in synthetic cystic fibrosis media (SCFM) reveal distinct species-specific metabolic signatures with limited strain-to-strain variability. The majority of metabolites significantly consumed by <em>S. aureus </em>were also consumed by <em>P. aeruginosa</em>, indicating that <em>P. aeruginosa</em> has the flexibility to metabolically outcompete<em> S. aureus </em>in coculture even in the absence of other pathogen-pathogen interactions. Finally, metabolites that were uniquely produced by one species or the other were identified. Specifically, the virulence factor precursor anthranilic acid as well as the quinoline 2,4-Quinolinediol (DHQ) were robustly produced across all tested strains of <em>P. aeruginosa</em>. Through the direct comparison of the extracellular metabolism of <em>P. aeruginosa</em> and <em>S. aureus</em> in a physiologically relevant environment, this work provides insight towards the potential metabolic interactions in vivo and supports the development of species-specific diagnostic markers of infection.</p>
Project description:The opportunistic pathogen Pseudomonas aeruginosa is among the main colonizers of the lungs of cystic fibrosis (CF) patients. We have isolated and sequenced several P. aeruginosa isolates from the sputum of CF patients and used phenotypic, genomic and proteomic analyses to compare these CF derived strains with each other and with the model strain PAO1.
Project description:Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method enabling improved identification of PA proteome with CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. We detected 1528 PA proteins (encoded by 1458 core genes and 70 accessory genes) that were expressed in CF airways, of which 1178 proteins were commonly identified with the ex vivo-grown PA populations. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.
Project description:This study was conducted to learn the transcriptional effects of deleting different components of the nitrogen-related phosphotransfer system (Nitro-PTS or PTS-Ntr) in Pseudomonas aeruginosa under infection-similar culture conditions using synthetic cystic fibrosis sputum medium (SCFM2). This system regulates a number of different genes and processes in different bacterial species, but few of its targets have been identified in P. aeruginosa. Because PtsP is required for PtsN phosphorylation, the ptsP mutant provides an opportunity to assess the impact of unphosphorylated PtsN (versus the ptsP ptsN double deletion). We also tested the transcriptional impact of adding 7.5 mM supplementary citrate to this medium. The different deletions showed distinct sets of differentially regulated genes that help yield insight into how the different components of the Nitro-PTS differentially influence expression. The results also aided in identifying previously unrecognized citrate transport proteins.