ARTICLE pubs.acs.org/jpr
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 Nathan J. Hare,† Cho Zin Soe,‡ Barbara Rose,§ Colin Harbour,§ Rachel Codd,‡ Jim Manos,§ and Stuart J. Cordwell*,†,|| ‡
School of Molecular Bioscience, The University of Sydney, 2006 Australia Discipline of Pharmacology, §Discipline of Infectious Diseases and Immunology, and Discipline of Pathology, School of Medical Sciences, The University of Sydney, 2006 Australia )
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bS Supporting Information 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-toperson 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. KEYWORDS: artificial sputum medium, cystic fibrosis, Pseudomonas aeruginosa, pyochelin, pyocyanin, transmissibility
’ INTRODUCTION Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is able to survive in a wide variety of environments and cause an array of diseases, including postoperative, burn or wound infections, urinary tract infections, microbial keratitis and meningitis. P. aeruginosa is the predominant determinant of morbidity and mortality in patients suffering from the autosomal recessive disorder, cystic fibrosis1 (CF). CF results from defects in the CF transmembrane conductance regulator (CFTR), a chloride ion channel located in mucosal cells. CF patients thus display impaired electrolyte transport, which in the lungs is characterized by a thickened and dehydrated mucus layer. Mucociliary clearance is also dramatically impaired, resulting in the CF lung being an ideal environment for bacterial infection. r 2011 American Chemical Society
The CF lung is colonized by a variety of pathogens, typically including Staphylococcus aureus and Haemophilus influenzae during childhood. Antibiotics may clear these organisms, along with primary P. aeruginosa infections; however, P. aeruginosa eventually dominates the microbial population and persists into biofilm formation associated with enhanced antibiotic resistance and chronic infection.2 P. aeruginosa infections correlate with poor pulmonary status and prognosis in CF patients.3,4 Infection typically occurs by mid-childhood (
