Dataset Information

Stenotrophomonas maltophilia differential gene expression in synthetic cystic fibrosis sputum identifies shared and cystic fibrosis strain-specific expression responses to the sputum environment

ABSTRACT: We characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum media (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect different metabolic pathways used by S. maltophilia in sputum, and altered stress responses. The latter correlated with increased resistance to peroxide exposure after pre-growth in SCFM2. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates with the SCFM2 transcriptome of the acute infection model strain, S. maltophilia K279A, allowing us to identify CF isolate-specific signatures in differential gene expression that may be suggestive of adaptation to the CF lung. Each strain also possessed genes not shared by the other two and here we show that expression of some of the accessory genes in each strain are changed in response to SCFM2. Collectively, this work details the response of S. maltophilia to the CF lung environment, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.

ORGANISM(S): Stenotrophomonas maltophilia

PROVIDER: GSE125704 | GEO | 2019/01/30

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Cystic fibrosis (CF) is an inherited, multi-system disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a ubiquitous ion channel important for epithelial hydration. A direct consequence of this dysfunction is impaired mucociliary clearance, chronic airway infection and a persistent neutrophilic inflammatory response that results in progressive loss of lung function, development of respiratory failure and premature death. Partial restoration of CFTR function is now possible for most CF patients through mutation specific CFTR modulators. Ivacaftor monotherapy produces significant clinical improvement in CF patients with the G511D mutation. Dual therapy, combining ivacaftor with lumacaftor or tezacaftor, results in modest clinical improvements in patients homozygous for F508del. More recently, triple therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic improvements in lung function and quality of life in patients homozygous and heterozygous for F508del. Sputum proteomics is a powerful research technique capable of identifying important airway disease mechanisms by interrogating the proteome, an entire set of proteins within biological samples. It has confirmed the central role of neutrophilic immune dysregulation in CF and non-CF bronchiectasis, particularly involving the release of antimicrobial proteins and neutrophil-extracellular traps (NETs), and through impaired anti-inflammatory mechanisms. These processes produce distinct molecular signatures within the sputum proteome that become increasingly abnormal with chronic airway infection and progressive lung disease severity. In CF patients, airway and systemic inflammatory cytokines potentially related to these signatures reduce with the various forms of CFTR modulation. To date, no studies of ETI therapy in CF lung disease have assessed large-scale change in protein expression using untargeted proteomics. We hypothesised that ETI therapy would shift the sputum proteome toward health, potentially normalising airway biology in people with CF. The objectives of this study were to investigate changes in the CF sputum proteome with the introduction of ETI, correlate these with changes in clinical markers of disease severity, and make comparisons with the sputum proteome in healthy controls and in repeat samples from CF patients not suitable for ETI therapy. We also explored which molecular pathways associated with CF lung disease did not change with ETI.

Project description:Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This paves the way for the colonization by microbial pathogens and the concomitant establishment of chronic infections leading to lung tissue damage, reduced lung function, and to decreased life expectancy. Although microbial infections play a key role during disease progression, only a few studies investigated the pathophysiology of the microbial community in vivo so far. Moreover, no CF study so far applied metaproteomics, a powerful approach to unravel molecular mechanisms of microbial infection, mainly reasoned due to (I) the challenging processability of inhomogeneous, viscous, slimy sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. Consequently, we developed a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, and subsequent metaproteomics analyses with a focus on microbial pathogens overcoming the aforementioned challenges. Metaproteomics data were complemented and validated by 16S sequencing, metabolomic as well as microscopic analyses. In total, we processed 21 CF sputum samples and selected three for detailed metaproteome analysis. The number of bacterial proteins/protein groups increased from 199-425 to 392-868. Moreover, our data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera are so far underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies against mucoviscidosis.

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:Cystic Fibrosis lung disease progresses by a combination of accelerated airways inflammation and bacterial colonization and infection. Airways inflammation in CF is predominantly neutrophilic and complicates airway clearance therapies through cellular debris, excessive DNA, excessive and viscous mucous, and high concentrations of neutrophils,Il-8 and related cytokines liberated along the NFkB signaling pathway. We conducted a single site, randomized, double blind, placebo-controlled, proof-of-concept trial in which we evaluated the effects of 28 days of two dose levels (0.05 mg and 0.10 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease. Nasal epithelial cells from 23 subjects were analyzed for microarray analysis. CF patients 18 to 45 years old, any genotype combination, were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure: 1) the pharmacokinetics of digitoxin in serum in stable CF patients, 2) safety indices, including ECG changes and sputum microbiology, in stable CF patients, 3) the effect of digitoxin on gene expression in nasal epithelial cells of stable CF patients and 4) quality of life scores using the CFQ-R©. As expected, it took several weeks to achieve a detectable serum level of digitoxin in CF. Digitoxin showed a trend towards reduction in sputum free neutrophil elastase and neutrophil counts, but was not associated with a reduction in sputum Il-8. Digitoxin did not dampen sputum inflammation as measured by primary and secondary outcome measures over the 28 day study period. However the patient groups receiving placebo and 50 micro gram digitoxin daily fell into similar patterns which were distinctly different from the patient group taking 100 micro gram digitoxin. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway mediated reductions in Il-8, Il-6, lung epithelial inflammation, neutrophil recruitment and mucus hypersecretion. It is possible that a longer treatment period would be necessary to detect a meaningful, dose- dependent change in sputum indices of inflammation in CF. Importantly, there were no safety issues over the course of the study

Dataset Information

Stenotrophomonas maltophilia differential gene expression in synthetic cystic fibrosis sputum identifies shared and cystic fibrosis strain-specific expression responses to the sputum environment

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure