Manual curation and reannotation of the genomes of Clostridium difficile 630Δerm and C. difficile 630

Dannheim, Henning; Riedel, Thomas; Neumann‐Schaal, Meina; Bunk, Boyke; Schober, Isabel; Spröer, Cathrin; Chibani, Cynthia Maria; Gronow, Sabine; Liesegang, Heiko; Overmann, Jörg; Schomburg, Dietmar

doi:10.1099/jmm.0.000427

Cited by 49 publications

(66 citation statements)

References 54 publications

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“…However, both cultures were apparently stressed by the increased agitation due to the gas flow. The influence of agitation on C. difficile has been observed before and may be attributed to a disruption of the fragile exopolysaccharide matrix [39]. Oxygen stressed cultures turned darker compared to reference cultures which may be attributed to increased iron sulfide precipitation.…”

Section: General Observationsmentioning

confidence: 68%

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

et al. 2018

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Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype.

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Section: General Observationsmentioning

confidence: 68%

“…For analysis of the metabolome, cell lysis and metabolite extraction were performed for five independent experiments as described previously [39]. Extracellular samples were prepared and volatile and non-volatile compounds in the culture supernatants and cell extracts were analyzed as described by Neumann-Schaal et al [24].…”

Section: Metabolomics Analysismentioning

confidence: 99%

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

et al. 2018

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“…Despite the considerable effect of CD2068 on drug resistance and transport in E. coli , the susceptibility profiles of the cd2068 mutant and the complemented strains revealed that CD2068 could significantly confer the resistance to certain antibiotics with a relatively low level in C. difficile . It might be explained by the facts that (i) CD2068 may be poorly expressed in its native host, C. difficile , under our experimental settings, however, it can still effectively pump drugs when overexpressed from a plasmid in E. coli ; (ii) there are a total of 243 genes annotated to encode for putative ABC transporters present in the genome of C. difficile 630 34 and they may exhibit an overlapping activity to compensate the loss of CD2086 in the mutant strain; and (iii) the resistance to certain antibiotics may also be mediated by other mechanisms in C. difficile .…”

Section: Discussionmentioning

confidence: 99%

CD2068 potentially mediates multidrug efflux in Clostridium difficile

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Sreesai

Harnvoravongchai

et al. 2017

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Clostridium difficile is a major cause of antibiotic-associated diarrhea and the treatment thereof becomes more difficult owing to a rise of multidrug resistant strains. ATP-binding cassette (ABC) transporters are known to play a crucial role in the resistance to multiple antibiotics. In this study, the potential contribution of an ABC transporter in C. difficile multidrug resistance was investigated. The expression level of the cd2068 gene in C. difficile encoding an ABC transporter was up-regulated following the exposure to certain antibiotics compared to the control cells. Heterologous expression of CD2068 in Escherichia coli revealed that it mediated the efflux of fluorescent substrates and conferred resistance to multiple drugs. The CD2068-associated ATPase activity in membrane vesicles was also stimulated by various antibiotics. Furthermore, the insertional inactivation of the cd2068 gene in C. difficile led to a significant increase in susceptibility to antibiotics, which could be genetically complemented, supporting that CD2068 was directly associated to the drug resistance. These results demonstrate the potential role for the ABC transporter CD2068 in the resistance mechanism against multiple drugs in C. difficile.

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“…Briefly, ∗ .raw files were searched with Sorcerer SEQUEST v. 27, rev. 11 (Thermo Fisher Scientific, United States) against a C. difficile 630Δ erm database ( Dannheim et al, 2017 ) containing 7646 protein entries (common laboratory contaminations and all reversed sequences added). The search was performed in two iterations using full digest with trypsin (after KR/); 10 ppm peptide mass tolerance; variable modification methionine oxidation (15.99 amu); and for the second iteration, mass shift of all amino acids completely labeled with 15 N-nitrogen.…”

Section: Methodsmentioning

confidence: 99%

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile

et al. 2018

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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 630Δerm 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.

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Manual curation and reannotation of the genomes of Clostridium difficile 630Δerm and C. difficile 630

Abstract: Based on the genomic analysis, several new metabolic features of C. difficile are proposed and could be supported by literature and subsequent experiments.

Cited by 49 publications

References 54 publications

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

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

CD2068 potentially mediates multidrug efflux in Clostridium difficile

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile

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