Dataset Information

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile.

ABSTRACT: Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, anaerobe, spore-forming pathogen, which causes drug-induced diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapeutic strategies to combat this pathogen. The application of metabolic labeling (ML) would allow for accurate quantification of significant differences in protein abundance, even in the case of very small changes. Additionally, it would be possible to perform more accurate studies of the membrane or surface proteomes, which usually require elaborated sample preparation. Such studies are therefore prone to higher standard deviations during the quantification. The implementation of ML strategies for C. difficile is complicated due to the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism of this anaerobic pathogen. Hence, quantitative proteome analyses could only be carried out by label free or chemical labeling methods so far. In this paper, a ML approach for C. difficile is described. A cultivation procedure with 15N-labeled media for strain 630?erm was established achieving an incorporation rate higher than 97%. In a proof-of-principle experiment, the performance of the ML approach in C. difficile was tested. The proteome data of the cytosolic subproteome of C. difficile cells grown in complex medium as well as two minimal media in the late exponential and early stationary growth phase obtained via ML were compared with two label free relative quantification approaches (NSAF and LFQ). The numbers of identified proteins were comparable within the three approaches, whereas the number of quantified proteins were between 1,110 (ML) and 1,861 (LFQ) proteins. A hierarchical clustering showed clearly separated clusters for the different conditions and a small tree height with ML approach. Furthermore, it was shown that the quantification based on ML revealed significant altered proteins with small fold changes compared to the label free approaches. The quantification based on ML was accurate, reproducible, and even more sensitive compared to label free quantification strategies.

SUBMITTER: Trautwein-Schult A

PROVIDER: S-EPMC6198727 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile.

Trautwein-Schult Anke A Maaß Sandra S Plate Kristina K Otto Andreas A Becher Dörte D

Frontiers in microbiology 20181016

Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, anaerobe, spore-forming pathogen, which causes drug-induced diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapeutic strategies to combat this pathogen. The application of metabolic labeling (ML) would allow for accurate quantification of significant differences in protein abundance, even in the case of very small changes. Additionall ...[more]

PMID: 30386308

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Project description:The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB,) of the F0F1-type was found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming ATPase (atpDBAFCEKI, was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

Dataset Information

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile.

Publications

A Metabolic Labeling Strategy for Relative Protein Quantification in <i>Clostridioides difficile</i>.

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