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:[1] Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum [2] Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment
