The protein inventory of Clostridium difficile grown in complex and minimal medium

Abstract

The intestinal pathogen Clostridium difficile is causing an increasing number of infections often characterized by severity and high relapse rates. Profound knowledge of the physiology of the pathogen could help to develop new treatment strategies. Proteomics, a valuable tool to study bacterial physiology, was used in this work to establish a benchmark proteome of reference strain C. difficile 630Δerm with MS-based details on all identified proteins. Our elaborate annotation and visualization of C. difficile 630Δerm 3764 ORFs will serve as a valuable base for researchers having to evaluate global expression studies. To exemplify expression variability, protein expression of late exponentially growing cells in complex brain-heart infusion medium and C. difficile minimal medium was compared. Noteworthy results of this comparison are as follows: (i) the higher expression of enzymes for the biosynthesis of some vitamins and purine and (ii) downregulation of proteins involved in butanoate fermentation in C. difficile minimal medium. However, the abundance of proteins involved in DNA metabolism, protein synthesis, and the cell envelope showed no variation between the two growth media.

Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach

“…The Perseus software platform [34] was used to carry out ANOVA testing and to generate heatmaps on the basis of DESeq2 rlog transformed count data. Voronoi treemaps of DESeq2 data were constructed using the Paver software (DECODON GmbH, Germany) based on an assignment of corresponding gene products to TIGRFAM roles [35] as described recently [36].…”

“…Cells were lyzed, protein cysteine residues were differentially labeled according to the diaCys approach and the proteins were then separated via SDS-PAGE according to Sievers et al [37]. Gel lanes were cut into 10 slices, proteins trypsinized in-gel, peptides eluted and desalted as described previously [36].…”

“…Liquid chromatographic separation of peptides was achieved using the EASY-nLC 1000 system (Thermo Scientific) and subsequent peptide detection by an Orbitrap Elite Hybrid mass spectrometer (Thermo Scientific) was performed as described elsewhere [36]. Database searches and quantification were performed within the framework of the MaxQuant software suite (Vers.…”

Work horse strainClostridioides difficile630Δermis oblivious to its anaerobic lifestyle

“…In previous studies, we observed a high abundance of oxidative stress-related proteins in C. difficile 630erm already at conditions devoid of any oxidizing agents (6) and no significant induction when the bacterium was shifted to micro-aerobic conditions (7). Several of the corresponding genes are encoded at one genetic locus comprising a rubrerythrin (rbr1), the transcriptional repressor PerR (perR), a desulfoferrodoxin (rbo) and a glutamate dehydrogenase with an N-terminal rubredoxin fold (CD630_08280).…”

“…Several of the corresponding genes are encoded at one genetic locus comprising a rubrerythrin (rbr1), the transcriptional repressor PerR (perR), a desulfoferrodoxin (rbo) and a glutamate dehydrogenase with an N-terminal rubredoxin fold (CD630_08280). Rbr1 even represents the second most abundant protein after the S-layer protein SlpA (6). We enquired, why these genes are highly expressed in the absence of any oxidative stress and focused on the repressor protein PerR, which regulates its own transcription and the one of genes involved in oxidative stress and metal homeostasis as described in Bacillus subtilis and in other Gram-positive bacteria (8,9).…”