Pseudomonas aeruginosa Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung

Abstract

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb-1 and cbb-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

Environment‐driven changes of mRNA and protein levels in Pseudomonas aeruginosa

“…Among them 4190 could also be quantified (71.1% of all proteins, 93.6% of the identified). This is comparable to a recent P. aeruginosa proteome study with state of the art liquid chromatography–mass spectrometry (LC–MS) techniques where 3967 proteins of the type strain PAO1 could be quantified (71% of the predicted PAO1 proteins) (Kamath et al ., ). Details on differentially expressed proteins between various environmental conditions are provided in Supporting Information Table S1.…”

SWATH mass spectrometry as a tool for quantitative profiling of the matrisome

“…SWATH MS combines the advantages of high reproducibility and sensitivity of targeted methods like selected reaction monitoring/multiple reaction monitoring (SRM/MRM) with the increased proteome depth typically seen with DDA MS [ 18 , 19 ]. SWATH MS is versatile and has been used in diverse applications including the quantification of proteins in a number of model organisms, diseases states and bacteria [ [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ]. SWATH MS has also been useful in characterising of low abundance sub-proteomes including post-translational modifications such as acetylation, succinylation and glycosylation [ [29] , [30] , [31] , [32] ].…”

Adaptation and diversification in virulence factors among urinary catheter-associated Pseudomonas aeruginosa isolates

“…The EPS of the biofilm matrix is negatively charged and hydrophobic, thereby enabling it to concentrate ions and dissolved organic compounds from the host system to survive the stressful conditions without releasing the planktonic cells (Kamath et al . ). Twitching motility was also found higher in the strong biofilm formers.…”