Dataset Information

A metabolic labeling strategy for relative protein quantification in Clostridioides difficile

ABSTRACT: Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, spore-forming pathogen which cases drug-induced Clostridioides difficile-associated diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapy strategies to combat this pathogen. So far, quantitative proteome analyses could only be carried out by label-free or chemical labeling methods. However, the application of metabolic labeling would allow for accurate quantification of significant differences, even in the case of very small changes. Additionally, it would be possible to perform bias free studies of the membrane or surface proteome which require elaborated preparations and are therefore prone to higher standard deviations during the quantification. Up to now, the implementation of metabolic labeling strategies of C. difficile was hampered by the very specific metabolic requirements of this anaerobic pathogen. To solve this problem, media were evaluated and the cultivation procedure with 15N labeled media for the C. difficile 630Δerm strain was optimized to gain a high incorporation rate. In the following proof-of-principle experiment, the cytosolic sub-proteomes of C. difficile cells of three different cultivation media and two growth phases were analyzed resulting in reproducible data which are shown in detail.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Peptoclostridium Difficile (strain 630) (clostridium Difficile)

SUBMITTER: Anke Trautwein-Schult

LAB HEAD: Prof. Dr. Dörte Becher

PROVIDER: PXD010279 | Pride | 2018-10-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	171013_OV3_P5_ATS_BHI_log02_N14_N15_01.raw	Raw
	171013_OV3_P5_ATS_BHI_log02_N14_N15_02.raw	Raw
	171013_OV3_P5_ATS_BHI_log02_N14_N15_03.raw	Raw
	171013_OV3_P5_ATS_BHI_log02_N14_N15_04.raw	Raw
	171013_OV3_P5_ATS_BHI_log02_N14_N15_05.raw	Raw

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Publications

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 obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism as well as the nutritional status in the environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A biosynthesis. In contrast, several fermentation pathways, different chaperones and the cysteine, serine and pantothenate biosynthesis were upregulated. Focused on the Stickland amino acid fermentation and the central carbon metabolism, we demonstrated the ability of C. difficile to replenish its favored amino acid cysteine by a pathway from the glycolysis substrate 3-phosphoglycerate via L-serine. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first using butanoate fermentation and then to lactate. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular integrity and ensure survival under starvation conditions.

Dataset Information

A metabolic labeling strategy for relative protein quantification in Clostridioides difficile

Dataset's files

Publications

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

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