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

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 chron...

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Autores principales: Graf, Alexander C., Striesow, Johanna, Pané-Farré, Jan, Sura, Thomas, Wurster, Martina, Lalk, Michael, Pieper, Dietmar H., Becher, Dörte, Kahl, Barbara C., Riedel, Katharina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432295/
https://www.ncbi.nlm.nih.gov/pubmed/34513734
http://dx.doi.org/10.3389/fcimb.2021.724569
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author Graf, Alexander C.
Striesow, Johanna
Pané-Farré, Jan
Sura, Thomas
Wurster, Martina
Lalk, Michael
Pieper, Dietmar H.
Becher, Dörte
Kahl, Barbara C.
Riedel, Katharina
author_facet Graf, Alexander C.
Striesow, Johanna
Pané-Farré, Jan
Sura, Thomas
Wurster, Martina
Lalk, Michael
Pieper, Dietmar H.
Becher, Dörte
Kahl, Barbara C.
Riedel, Katharina
author_sort Graf, Alexander C.
collection PubMed
description 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 pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be 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 to combat chronic recurrent airway infection in CF.
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spelling pubmed-84322952021-09-11 An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum Graf, Alexander C. Striesow, Johanna Pané-Farré, Jan Sura, Thomas Wurster, Martina Lalk, Michael Pieper, Dietmar H. Becher, Dörte Kahl, Barbara C. Riedel, Katharina Front Cell Infect Microbiol Cellular and Infection Microbiology 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 pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be 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 to combat chronic recurrent airway infection in CF. Frontiers Media S.A. 2021-08-27 /pmc/articles/PMC8432295/ /pubmed/34513734 http://dx.doi.org/10.3389/fcimb.2021.724569 Text en Copyright © 2021 Graf, Striesow, Pané-Farré, Sura, Wurster, Lalk, Pieper, Becher, Kahl and Riedel https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular and Infection Microbiology
Graf, Alexander C.
Striesow, Johanna
Pané-Farré, Jan
Sura, Thomas
Wurster, Martina
Lalk, Michael
Pieper, Dietmar H.
Becher, Dörte
Kahl, Barbara C.
Riedel, Katharina
An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title_full An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title_fullStr An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title_full_unstemmed An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title_short An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
title_sort innovative protocol for metaproteomic analyses of microbial pathogens in cystic fibrosis sputum
topic Cellular and Infection Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432295/
https://www.ncbi.nlm.nih.gov/pubmed/34513734
http://dx.doi.org/10.3389/fcimb.2021.724569
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