Proteomics of Pseudomonas aeruginosa Australian Epidemic Strain 1 (AES-1) Cultured under Conditions Mimicking the Cystic Fibrosis Lung Reveals Increased Iron Acquisition via the Siderophore Pyochelin

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.

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

“…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). Other modifications included the absence of bovine serum albumin and an increase to 20 mg ml À 1 porcine stomach mucin to better reflect our experience with CF sputum consistency.…”

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

“…3) (56,57), as well as for pathogenicity in a mouse model of systemic infection (29), our data suggest that specific features of the lung environment somehow foster the iron-chelating activity of pyochelin during pulmonary infection. Notably, expression of pyochelin genes was recently documented for P. aeruginosa cells infecting the lungs of CF patients (55), and pyochelin production was found to be induced in a CF epidemic strain in artificial CF sputum medium (63).…”

Epidemiology, Biology, and Impact of Clonal Pseudomonas aeruginosa Infections in Cystic Fibrosis

“…Proteomic profiles of AUST-01 show considerable differences compared to the reference strains PAO1 and PA14 (159,160). In particular, AUST-01 has been shown to overexpress many proteins involved in a range of functions, including virulence (including chitinase, hemolysin, and hydrogen cyanide synthase), siderophore biosynthe- sis and acquisition (in particular pyochelin), antibiotic resistance (MexX, a multidrug efflux pump component), and LPS biosynthesis (160).…”