Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

Abstract

The discovery of therapies that modulate Pseudomonas aeruginosa virulence or that can eradicate chronic P. aeruginosa lung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding of P. aeruginosa behavior in vivo. We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances of P. aeruginosa transcripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiology in vitro and in vivo. The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety of P. aeruginosa strains as well as RNA serial sputum samples from four P. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis of P. aeruginosa grown in vitro identified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates. P. aeruginosa transcript profiles in RNA from CF sputum indicated alginate production in vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory. P. aeruginosa gene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grown P. aeruginosa showed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CF P. aeruginosa lung infections. Several factors complicate the comparison of in vitro studies on Pseudomonas aeruginosa with infections in the cystic fibrosis (CF) lung. First, many different P. aeruginosa strains cause CF lung infections; thus, reliance on commonly used laboratory strains might limit our understanding of P. aeruginosa in CF (1-3). Second, laboratory or cell culture studies rarely incorporate coinfecting species and lack components of the lung environment, such as immune response factors, that shape P. aeruginosa phenotypes (4, 5). We know little about how these environmental stimuli influence P. aeruginosa (6-8). To complicate matters further, a single CF sputum sample contains mixtures of P. aeruginosa genotypes and phenotypes. A recent study has suggested that a mixture of strains influenced traits such as drug response in ways that were difficult to predict from the study of single strains (9). Clinical isolates of P. aeruginosa, even from the same respiratory sample, can have striking differences in phenotypes, including colony morphology, quorum-sensing regulation, and motili...

Exogenous alginate protectsStaphylococcus aureusfrom killing byPseudomonas aeruginosa

“…To determine how exogenous alginate impacts P. aeruginosa gene expression more generally, we used the Nanostring PAV2 codeset (49) to compare P. aeruginosa gene expression in the presence and absence of exogenous alginate and S. aureus . Nansotring is a digital multiplexed technology for direct quantification of RNA transcripts.…”

“…Nansotring is a digital multiplexed technology for direct quantification of RNA transcripts. The PAV2 codeset used in this study contains probes for 75 transcripts associated with P. aeruginosa genes known or suspected to be expressed in the CF airway (49). P. aeruginosa PAO1 and P. aeruginosa PA14 were cultured to mid-log phase and then sub-cultured into fresh media +/− 1% alginate.…”

“…We chose to perform our Nanostring experiment with CFBRPA32 because there is publicly available Nanostring data (49) for several other mucoid clinical isolates we examined (CFRL8, CI2224, CI228, and FRD1). We re-analyzed this data to determine whether we could identify a shared expression pattern between mucoid killers (Fig.…”

“…Total RNA was prepared as described for q-RT-PCR. The PAV2 codeset (49) was incubated with total RNA as described by the manufacturer’s protocol.…”

Pseudomonas aeruginosa lasRmutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin

“…Compared to other Anr regulated genes, expression of mhr and the Anr-regulated ccoNOPQ-2 high affinity terminal oxidase followed similar patterns across a large data compendium and the loss of Mhr did not cause a further fitness defect in a strain lacking high affinity cytochrome c oxidase activity, suggesting that Mhr and high affinity cytochrome c oxidases work together directly or indirectly. Both proteomic and transcriptomic studies provided evidence for the coordinated induction of Mhr and high affinity cytochrome c oxidases (40) in synthetic cystic fibrosis medium under microoxic conditions compared to normoxic conditions, and in CF sputum (40, 41).…”