title: Secretome of transmissible Pseudomonas aeruginosa AES-1R grown in a cystic fibrosis lung-like environment.
abstract: Pseudomonas aeruginosa is the predominant cause of mortality in patients with cystic fibrosis (CF). We examined the secretome of an acute, transmissible CF P. aeruginosa (Australian epidemic strain 1-R; AES-1R) compared with laboratory-adapted PAO1. Culture supernatant proteins from rich (LB) and minimal (M9) media were compared using 2-DE and 2DLC-MS/MS, which revealed elevated abundance of PasP protease and absence of AprA protease in AES-1R. CF lung-like artificial sputum medium (ASMDM) contains serum and mucin that generally preclude proteomics of secreted proteins. ASMDM culture supernatants were subjected to 2DLC-MS/MS, which allowed the identification of 57 P. aeruginosa proteins, and qualitative spectral counting was used to estimate relative abundance. AES-1R-specific AES_7139 and PasP were more abundant in AES-1R ASMDM culture supernatants, while AprA could only be identified in PAO1. Relative quantitation was performed using selected reaction monitoring. Significantly elevated levels of PasP, LasB, chitin-binding protein (CbpD), and PA4495 were identified in AES-1R ASMDM supernatants. Quantitative PCR showed elevated pasP in AES-1R during early (18 h) ASMDM growth, while no evidence of aprA expression could be observed. Genomic screening of CF isolates revealed aes_7139 was present in all AES-1 and one pair of sequential nonepidemic isolates. Secreted proteins may be crucial in aiding CF-associated P. aeruginosa to establish infection and for adaptation to the CF lung.
authors.affiliation: School of Molecular Bioscience, The University of Sydney , Building GO8, Maze Crescent, Sydney, New South Wales 2006, Australia.
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Hare
authors.forename: Melanie Y
authors.initials: MY
authors.lastname: White
authors.forename: Jim
authors.initials: J
authors.lastname: Manos
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
mesh_terms: D000595:Amino Acid Sequence; D001315:Australia; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D040761:Biomimetic Materials / Q000737:chemistry; D003470:Culture Media; D003550:Cystic Fibrosis / Q000382:microbiology; D015180:Electrophoresis, Gel, Two-Dimensional; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011552:Pseudomonas Infections / Q000382:microbiology / Q000635:transmission; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013183:Sputum / Q000737:chemistry / Q000382:microbiology; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D010447:Peptide Hydrolases