Proteomics

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A combined metaproteomics and metabolomics approach for the in vivo analysis of cystic fibrosis sputum focusing on microbial pathogens


ABSTRACT: Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This paves the way for the colonization by microbial pathogens and the concomitant establishment of chronic infections leading to lung tissue damage, reduced lung function, and to decreased life expectancy. Although microbial infections play a key role during disease progression, only a few studies investigated the pathophysiology of the microbial community in vivo so far. Moreover, no CF study so far applied metaproteomics, a powerful approach to unravel molecular mechanisms of microbial infection, mainly reasoned due to (I) the challenging processability of inhomogeneous, viscous, slimy sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. Consequently, we developed a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, and subsequent metaproteomics analyses with a focus on microbial pathogens overcoming the aforementioned challenges. Metaproteomics data were complemented and validated by 16S sequencing, metabolomic as well as microscopic analyses. In total, we processed 21 CF sputum samples and selected three for detailed metaproteome analysis. The number of bacterial proteins/protein groups increased from 199-425 to 392-868. Moreover, our data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera are so far underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies against mucoviscidosis.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Sputum

DISEASE(S): Cystic Fibrosis

SUBMITTER: Daniela Zuehlke  

LAB HEAD: Katharina Riedel

PROVIDER: PXD025134 | Pride | 2021-11-03

REPOSITORIES: Pride

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Publications

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum.

Graf Alexander C AC   Striesow Johanna J   Pané-Farré Jan J   Sura Thomas T   Wurster Martina M   Lalk Michael M   Pieper Dietmar H DH   Becher Dörte D   Kahl Barbara C BC   Riedel Katharina K  

Frontiers in cellular and infection microbiology 20210827


Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathoph  ...[more]

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