Dataset Information

Identification of Clostdridium difficile Immunoreactive Spore Proteins of the Epidemic strain R20291

ABSTRACT: Clostridium difficile infections are the leading cause of diarrhea associated to the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. Nowadays, the development of vaccine and passive immunization therapies against C. difficile have focused on toxins A and B. In the present study, we used an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C. difficile spores as potential candidates for the development of immunotherapy and/or diagnostic methods against this devastating infection. Experimental design. To identify potential immunogenic proteins on the surface of C. difficile R20291, spore coat/exosporium extracts were separated by two-dimensional electrophoresis (2-DE) and analyzed for reactivity against C. difficile spore-specific goat sera. Finally, the proteins present at the spot of the interest were in-gel digested with chymotrypsin, and the peptides generated were separated by nanoUPLC followed by MS/MS using a Quad-TOF MS, and corroborated by Ultimate 3000RS nano UHPLC coupled to QExactive Plus Orbitrap MS.

INSTRUMENT(S): Synapt MS, Q Exactive

ORGANISM(S): Peptoclostridium Difficile (strain R20291) (clostridium Difficile)

TISSUE(S): Spore

DISEASE(S): Severe Diarrhea

SUBMITTER: Eduardo Callegari

LAB HEAD: Daniel Paredes-Sabja

PROVIDER: PXD008592 | Pride | 2018-07-05

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	DDAS_Experimental_2.pdf	Pdf
	DP_Processingparameters_PRIDE.docx	Other
	DP_Processingparameters_PRIDE.pdf	Pdf
	DP_Processingparameters_PRIDE.txt	Txt
	DP_Project_description_PRIDE.pdf	Pdf

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Identification of Clostridium difficile Immunoreactive Spore Proteins of the Epidemic Strain R20291.

Pizarro-Guajardo Marjorie M Ravanal María Cristina MC Paez Maria Daniela MD Callegari Eduardo E Paredes-Sabja Daniel D

Proteomics. Clinical applications 20180418 5

<h4>Purpose</h4>Clostridium difficile infections are the leading cause of diarrhea associated with the use of antibiotics. During infection, C. difficile initiates a sporulation cycle leading to the persistence of C. difficile spores in the host and disease dissemination. The development of vaccine and passive immunization therapies against C. difficile has focused on toxins A and B. In this study, an immunoproteome-based approach to identify immunogenic proteins located on the outer layers of C ...[more]

PMID: 29573213

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Project description:The virulence factors of Clostridium difficile have been studied for many years, but the main in vivo pathogenesis processes of this bacterium has to be investigated. Especially, data on the early mechanisms of C. difficile adaptation to the host is not yet available. The objective of our study was to improve the understanding of the pathogenesis of C. difficile by the analysis of the genome-wide temporal expression of C. difficile genes during the first hours of infection. Three groups of 4 axenic mice each were challenged by vegetative cells of the 630 C. difficile strain, and sacrified at 8, 14, and 38 hours post-infection. Pure prokaryotic RNA was obtained from caecal bacteria. Comparative hybridizations on strain 630 microarrays were done using cDNA issued from an 8-hours in vitro culture as control, with a dye-swap protocol for each sample. Normalisation and statistical analysis of all data were done with different functions of the limma package. The pathogenesis of C. difficile could be seen as a result of the successful metabolic adaptation of the bacteria to its host, contributing to its persistence and multiplication, and the coordinate expression of virulence factors. Analysis of our data enlightened some of these aspects. The results support strongly a two-step infection model, since there is, during the course of infection, a significant increase in the toxins expression contrasting with a decreased expression of most of the putative colonization factors. Several paralogs of the HMW S-layer protein are also down-regulated, some of them could be strong candidates as colonisation factors. Bacterial adaptation to the microenvironment of the host is assessed by the regulation of numerous metabolic pathways, i.e., the upregulation of the ethanolamine catabolic operon or the modulation of several PTS systems. Inactivation of some putative virulence factors identified by this methodology will complete this analysis. Transcriptional profiling of the C. difficile 630E strain after 8h, 14h or 38h in TY. Two-condition experiments: 630E strain 14h or 38h in TY vs. 630E strain 8h in TY. 2 biological replicates for each condition in dye swap.