Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum

Abstract

Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF.

Bacterial chitinases and chitin-binding proteins as virulence factors

“…Transcription of the virulence-related chitinase ChiB of Listeria monocytogenes and a chitinase from Enterococcus faecalis (efChi18A) has been shown to be upregulated in vitro in body fluids, such as blood and urine (Toledo-Arana et al, 2009;Vebø et al, 2009;Vebø et al, 2010). Similarly, chitinase upregulation has also been seen during growth of P. aeruginosa in artificial cystic fibrosis sputum (Fung et al, 2010), which is in agreement with the fact that the expression of a P. aeruginosa chitinase is enhanced in clinical isolates as compared with that in a laboratory strain (Salunkhe et al, 2005).…”

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

“…Artificial sputum medium (Fung et al, 2010) contained 20 mg ml À 1 pig stomach mucin (Sigma-Aldrich, St Louis, MO, USA), 1.4 mg ml À 1 DNA, (Salmon Sperm DNA, Sigma-Aldrich), 2.2 mg ml À 1 KCl, 5 mg ml À 1 NaCl, 7.225 Â amino acids (Sigma-Aldrich), 14.45 Â MEM-Non-Essential amino acids (SigmaAldrich) and 0.005% egg yolk emulsion (Dalynn Biologicals, Calgary, AB, Canada). The media was modified to include 3 mg ml À 1 ferritin as an alternate iron source to more appropriately reflect the conditions of the CF lung (Stites et al, 1998;Reid et al, 2004;Ghio et al, 2012;Hare et al, 2012).…”

Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY

“…Based on these studies one would expect there to be more overlap between the transcriptomes of the anr and rsmA mutants, which suggests that experimental conditions may be crucial when using transcriptome profiling to study RsmA regulation. However, the RsmA regulatory profiles do exhibit substantial overlap with the profiles from microaerobic growth conditions in biofilms and in artificial sputum media (Fung et al, 2010;Waite & Curtis, 2009), whereby the transcriptional regulator PtxS, and genes associated with phenazine, PQS production and early biofilm formation, are modified in common. It has been reported that a subtle environmental difference can exert significant variation in the control of target genes of the Gac/Rsm pathway (Ventre et al, 2006), and therefore a fine balance can exist between positive and negative regulation of target genes by RsmA.…”