Consistently, in Patient A we detected an alkyl hydroperoxide reductase (sp
Consistently, in Patient A we detected an alkyl hydroperoxide reductase (sp.), a Glutathione S-transferase (sp.), an iron-sulfur-cluster repair protein (sp.), glutathione-disulfide reductase (sp.), and the molecular chaperones DnaK (sp.) and GroL (various species) as well as the protease ClpP (sp.), which all are involved in protein protection, repair, or degradation of proteins and inducible after various stress conditions like oxidative stress (Michta et al., 2014; Ezraty et al., 2017). CF lungs remains poorly comprehended. Particular challenges in the analysis of the microbial population present in CF sputum is usually (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 C an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway contamination in CF. and represent early colonizers, which are followed by other bacterial pathogens including e.g. complex and but also fungal pathogens like and and (Rogers et al., 2014; Filkins et al., 2015). Of note, CF Loviride airways are characterized by an inflammatory milieu, which can be attributed to the microbial colonization/contamination eliciting a host immune response characterized by the dysregulation of epithelial innate immunity and airway leukocytes. Proteolytic and oxidative products derived from an exuberant immune response in combination with microbial virulence factors are the main reasons for lung tissue damage, which ultimately lead to respiratory failure and death (Cohen and Prince, 2012; Eiserich et al., 2012; Kamath et al., 2015). Thus, deeper insights into these complex polymicrobial infections, focusing on the (patho-)physiology of the microbial CF lung community as well as host-microbe interactions are of essential importance for a better understanding of the disease progression and the development of novel treatment strategies. In the past, (meta-)proteomics approaches were used as a powerful tool to investigate the physiological alterations of lung tissues and body fluids (e.g. bronchoalveolar lavage, blood, feces, and sputum) in CF patients as well as CFTR post-translational modifications and CF biomarkers (Eiserich et al., 2012; Kamath et al., 2015; Debyser et al., 2016; Liessi et al., 2020). However, most of these studies were limited by focusing on the host perspective while overlooking the microbial side of contamination. Studies characterizing the bacterial and fungal pathogens of CF lungs were typically performed analyses of the microbial pathophysiology directly at the Loviride site of contamination are urgently needed. Here, we present the first 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). In this protocol, a combination of differential centrifugation and filtration is used as key elements for the enrichment of bacterial cells significantly increasing bacterial protein identification coverage. 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. Experimental Procedures Study Cohort, Ethics Statement, and Sputum Sampling In total, 24 sputum samples derived from 20 different patients were collected. The study was approved by the institutional ethics review board Mnster, Germany (2010-155-f-S). Of the 24 sputum samples, 21 were used as test samples to establish a reliable, reproducible and widely applicable protocol suitable for sputum processing, nucleic acid HDAC5 extraction, microbial enrichment and subsequent protein and metabolite extraction. As a proof of concept, three samples, which were derived from three individual patients (designated Patient A, Patient B, and Patient.Here, the samples were centrifuged at 500 g for 5 min (keeping cell lysis as low as possible) and the pellet made up of human cells and bigger aggregates was discarded. role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly comprehended. Particular challenges in the analysis of the microbial population present in CF sputum is usually (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 Loviride 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 C an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway contamination in CF. and represent early colonizers, which are followed by other bacterial pathogens including e.g. complex and but also fungal pathogens like and and (Rogers et al., 2014; Filkins et al., 2015). Of note, CF airways are characterized by an inflammatory milieu, which can be attributed to the microbial colonization/contamination eliciting a host immune response characterized by the dysregulation of epithelial innate immunity and airway leukocytes. Proteolytic and oxidative products derived from an exuberant immune response in combination with microbial virulence factors are the main reasons for lung tissue damage, which ultimately lead to respiratory failure and death (Cohen and Prince, 2012; Eiserich et al., 2012; Kamath et al., 2015). Thus, deeper insights into these complex polymicrobial infections, focusing on the (patho-)physiology of the microbial CF lung community as well as host-microbe interactions are of essential importance for a better understanding of the disease progression and the development of novel treatment strategies. In the past, (meta-)proteomics approaches were used as a powerful tool to investigate the physiological alterations of lung tissues and body fluids (e.g. bronchoalveolar lavage, blood, feces, and sputum) in CF patients as well as CFTR post-translational modifications and CF biomarkers (Eiserich et al., 2012; Kamath et al., 2015; Debyser et al., 2016; Liessi et al., 2020). However, most of these studies were limited by focusing on the host perspective while overlooking the microbial side of infection. Studies characterizing the bacterial and fungal pathogens of CF lungs were typically performed analyses of the microbial pathophysiology directly at the site of infection are urgently needed. Here, we present the first 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). In this protocol, a combination.