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

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.

Quick and wide-range taxonomical repertoire establishment of the cystic fibrosis lung microbiota by tandem mass spectrometry on sputum samples

“…Although a protocol for metaproteomic analysis of CF sputum samples has been previously proposed ( Graf et al, 2021 ), the need to create dedicated databases obtained after 16S RNA gene amplicon sequencing on the sputum sample and the multiple steps for enrichment in microorganisms make the approach incompatible with routine microbiological diagnosis. The protocol described in the present study does not require time-consuming steps of mechanical and enzymatic homogenization, differential centrifugations, and filtrations.…”

“…Thus, sputum has the advantage of possible iterative sampling for fine-tuned monitoring ( Françoise and Héry-Arnaud, 2020 ). CF sputum samples are among the most challenging samples because of their heterogeneity and viscosity and the low ratio of microbial protein load ( Graf et al, 2021 ). The latest published protocol for proteomics comprises a significant number of steps for the preprocessing of sputum, microbial enrichment, protein extraction, and proteolysis ( Graf et al, 2021 ).…”

“…CF sputum samples are among the most challenging samples because of their heterogeneity and viscosity and the low ratio of microbial protein load ( Graf et al, 2021 ). The latest published protocol for proteomics comprises a significant number of steps for the preprocessing of sputum, microbial enrichment, protein extraction, and proteolysis ( Graf et al, 2021 ). Taxonomical identification of pathogens by mass spectrometry proteotyping, i.e., Matrix Assisted Laser Desorption Ionisation - Time of Flight (MALDI-ToF)-based identification, is rapidly obtainable following isolate cultivation and, over the last decade, has become a standard diagnostic tool in the microbiological laboratory ( Suarez et al, 2013 ).…”

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

“…To obtain insight into the pathophysiology of the microbial community in lungs of CF patients, Graf et al [ 46 ] developed a reliable and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, and protein extraction. A detailed metaproteome analysis was also performed on a restricted number of CF sputum samples.…”

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels

“…A more recent version of such protocol is based on mechanical homogenization combined with extracellular DNA aggregates digestion by DNAase I and differential centrifugation steps for the enrichment of microbial cells. The metaproteomics analysis identified enriched proteins from various bacterial genera and highlighted that arginine deiminase pathway and multiple proteases could be contributors to the pathophysiology of cystic fibrosis ( Graf et al, 2021 ).…”