Dataset Information

Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites

ABSTRACT: Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites such as burned wounds, lung and urinary tract. To shed light on the expression rates of cytoplasmic P. aeruginosa proteins, commonly expressed by eleven different clinical isolates, absolute protein quantities were determined using P. aeruginosa PAO1 as a reference strain and employing a highly precise gel-free and data-independent LC-IMSE approach. Moreover, the metabolic diversity of these isolates has been investigated by 13C-metabolic flux analyses. 903 proteins were reproducibly identified and absolutely quantified for P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all of the tested clinical isolates. The vast majority of these proteins is expressed in constant amounts in all strains and exhibits a relatively low relative standard deviation. In contrast, the expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in isolates from burned wounds when compared to isolates from lung or urinary tract. Moreover, proteins involved in the uptake of iron and amino acids (i.e. HitA, BfrB, PA5217, BraC, PA5153) were found to be more abundant in urinary tract isolates compared to lung isolates. The fluxome data revealed a conserved glycolysis, and a niche-specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated analysis of proteome and fluxome did not indicate straightforward correlation between the amount of proteins and their flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Pseudomonas Aeruginosa

SUBMITTER: Christian Lassek

LAB HEAD: Katharina Riedel

PROVIDER: PXD002373 | Pride | 2016-03-29

REPOSITORIES: Pride

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Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

Lassek Christian C Berger Antje A Zühlke Daniela D Wittmann Christoph C Riedel Katharina K

Proteomics 20160413 9

Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel-free and data-independent LC-IMS(E) approach. Moreover, the metabolic diversity of these isolates was investigated by (13) C-metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified ...[more]

PMID: 26959854

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Project description:Pseudomonas aeruginosa is one of the most frequent pathogen dominant in complicated urinary tract infections (UTI). To unravel the adaptation strategies of P. aeruginosa to the conditions in the urinary tract and to define the underlying regulatory network an artificial growth system mimicking the conditions in the urinary tract was established. Transcriptome analyses were used to investigate the physiological status of P. aeruginosa under this conditions. We performed comparisons to identify genes induced under artificial urinary tract conditions to unravel the adaptive strategies and the underlying regulatory network used by Pseudomonas aeruginosa during urinary tract infections using Affimetrix GeneChips. Pseudomonas aeruginosa wild type strain PAO1 was grown in an artificial in vitro growth system mimicking the conditions in the urinary tract. Therefore, biofilms were grown on the surface of membrane filters placed on agar plates at 37 Â°C up to the late logarithmic state under aerobic and anaerobic conditions (incubated in an anaerobic beanch). An artificial urine medium (AUM) simulating the averaged urine of an human adult was used as nutrient souce. 10-fold diluted Luria Bertani (LB)-medium was used as reference medium. For growth under oxygen depletion the media were supplemented with 50 mM KNO3 to sustain anaerobic respiration. The biofilms were harveted at this time points and resuspsended in 0.9% (w/v) NaCl. The OD578 of biofilm suspension was 0.8 for all tested conditions. First comparison: Identification of genes induced or repressed under aerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown aerobically for 18 h to the late logarithmic phase in biofilms on AUM with the transcriptome profile of the PAO1 strain, which was grown aerobically for 18 h to the late logarithmic phase in biofilms on 10-fold diluted LB. Second comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown anaerobically for 2 days up to the late logarithmic phase in biofilms on AUM supplemented with 50 mM nitrate with the transcriptome profile of the PAO1 strain, which was grown anaerobically for 2 days up to the late logarithmic phase in biofilms on 10-fold diluted LB supplemented with 50 mM nitrate.

Project description:Pseudomonas aeruginosa is a Gram-negative, opportunistic bacterium and a major etiological agent in monogenic disease cystic fibrosis (CF). High density colonies of P. aeruginosa are often isolated from hypoxic mucus plugs in respiratory tract of CF patients, indicating high adaptive capacities of the bacterium. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to this hypoxic environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT labelled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2<1%) and aerobic conditions in media that mimics the nutrient components of the CF lung. 3,967 P. aeruginosa proteins were quantitated (FDR <1%) across all three strains, 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 two-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 (cbb3-1 and cbb3-2 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.

Dataset Information

Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites

Publications

Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites.

Similar Datasets

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