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 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.
Highlights
Cystic fibrosis is the most common inherited monogenic disorder in Caucasian populations with an incidence of approx. one in 3,000 births (O’Sullivan and Freedman, 2009)
Our study represents a fundamental basis for follow-up studies investigating the microbial metaproteome and bacterial pathophysiology in cystic fibrosis (CF) sputum, which is an essential prerequisite for the development of innovative antimicrobial treatment approaches
In order to address these critical open questions, we developed an in vivo approach with a specific focus on the metaproteomic analyses of the CF microbiome, driven by 16S sequencing community composition analyses
Summary
Cystic fibrosis is the most common inherited monogenic disorder in Caucasian populations with an incidence of approx. one in 3,000 births (O’Sullivan and Freedman, 2009). We present the first in vivo microbial metaproteome analysis, complemented by 16S sequencing, metabolomics, and microscopic analyses to study microbial communities and facultative microbial pathogens within CF sputum To this end, we established an innovative sputum processing protocol, which overcomes major technical and analytical challenges of CF sputum including (I) limited sample volume, (II) challenging processability of CF sputum due to its viscous and slimy character, (III) extraction of nucleic acids, proteins and metabolites out of a single sputum sample, (IV) enormous dominance of human proteins (e.g. mucins, albumins, immunoglobulins) over microbial proteins of interest, and (V) high abundance of (neutrophil-derived) proteases unspecifically digesting microbial proteins of interest (Kamath et al, 2015). Our study represents a fundamental basis for follow-up studies investigating the microbial metaproteome and bacterial pathophysiology in CF sputum, which is an essential prerequisite for the development of innovative antimicrobial treatment approaches
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