dPseudomonas aeruginosa chronically infects the lungs of more than 80% of adult patients with cystic fibrosis (CF) and is a major contributor to the progression of disease pathology. P. aeruginosa requires iron for growth and has multiple iron uptake systems that have been studied in bacteria grown in laboratory culture. The purpose of this research was to determine which of these are active during infection in CF. RNA was extracted from 149 sputum samples obtained from 23 CF patients. Reverse transcription-quantitative real-time PCR (RT-qPCR) was used to measure the expression of P. aeruginosa genes encoding transport systems for the siderophores pyoverdine and pyochelin, for heme, and for ferrous ions. Expression of P. aeruginosa genes could be quantified in 89% of the sputum samples. Expression of genes associated with siderophore-mediated iron uptake was detected in most samples but was at low levels in some samples, indicating that other iron uptake mechanisms are active. Expression of genes encoding heme transport systems was also detected in most samples, indicating that heme uptake occurs during infection in CF. feoB expression was detected in all sputum samples, implying an important role for ferrous ion uptake by P. aeruginosa in CF. Our data show that multiple P. aeruginosa iron uptake mechanisms are active in chronic CF infection and that RT-qPCR of RNA extracted from sputum provides a powerful tool for investigating bacterial physiology during infection in CF.

The bacterium Pseudomonas aeruginosa is commonly isolated from the general environment and also infects the lungs of patients with cystic fibrosis (CF). Iron in mammals is not freely available to infecting pathogens although significant amounts of extracellular iron are available in the sputum that occurs in the lungs of CF patients. P. aeruginosa has a large number of systems to acquire this essential nutrient and many of these systems have been characterised in the laboratory. However, which iron acquisition systems are active in CF is not well understood. Here we review recent research that sheds light on how P. aeruginosa obtains iron in the lungs of CF patients.

BackgroundSynthesis and uptake of pyoverdine, the primary siderophore of the opportunistic pathogen Pseudomonas aeruginosa, is dependent on two extra-cytoplasmic function (ECF) sigma factors, FpvI and PvdS. FpvI and PvdS are required for expression of the ferri-pyoverdine receptor gene fpvA and of pyoverdine synthesis genes respectively. In the absence of pyoverdine the anti-sigma factor FpvR that spans the cytoplasmic membrane inhibits the activities of both FpvI and PvdS, despite the two sigma factors having low sequence identity.ResultsTo investigate the interactions of FpvR with FpvI and PvdS, we first used a tandem affinity purification system to demonstrate binding of PvdS by the cytoplasmic region of FpvR in P. aeruginosa at physiological levels. The cytoplasmic region of FpvR bound to and inhibited both FpvI and PvdS when the proteins were co-expressed in Escherichia coli. Each sigma factor was then subjected to error prone PCR and site-directed mutagenesis to identify mutations that increased sigma factor activity in the presence of FpvR. In FpvI, the amino acid changes clustered around conserved region four of the protein and are likely to disrupt interactions with FpvR. Deletion of five amino acids from the C-terminal end of FpvI also disrupted interactions with FpvR. Mutations in PvdS were present in conserved regions two and four. Most of these mutations as well as deletion of thirteen amino acids from the C-terminal end of PvdS increased sigma factor activity independent of whether FpvR was present, suggesting that they increase either the stability of PvdS or its affinity for core RNA polymerase.ConclusionsThese data show that FpvR binds to PvdS in both P. aeruginosa and E. coli, inhibiting its activity. FpvR also binds to and inhibits FpvI and binding of FpvI is likely to involve conserved region four of the sigma factor protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0287-2) contains supplementary material, which is available to authorized users.