pr4007365_si_001.pdf (4.04 MB)
Secretome of Transmissible Pseudomonas aeruginosa AES-1R Grown in a Cystic Fibrosis Lung-Like Environment
journal contribution
posted on 2013-12-06, 00:00 authored by Nichollas
E. Scott, Nathan
J. Hare, Melanie Y. White, Jim Manos, Stuart J. CordwellPseudomonas 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.