Project description:To investigate the impact transcription that the Spore Forming (SF) Community of bacteria have on the small intestine, B6 mice on a autoclaved diet that were germfree (GF) or colonized with the SF community were investigated. Using laser capture microdissection the intestinal epithelial cells on the villi and the crypts were dissected out and bulk RNA seq was performed.
Project description:Bacterial endospores, the transmissible forms of pathogenic bacilli and clostridia, are heterogeneous multilayered structures composed of proteins. These proteins protect the spores against variety of stresses, thus helping spore survival, and assist in germination, by interacting with the environment to form vegetative cells. Owing to the complexity, insolubility, and dynamic nature of spore proteins, it has been difficult to obtain their comprehensive protein profiles. The intact spores of Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile and their vegetative counterparts were disrupted by bead-beating in 6M urea under reductive conditions. The heterogeneous mixture was then double-digested with LysC and trypsin. Next, the peptide mixture was pre-fractionated with Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) followed by reverse phase LC-FT-MS analysis of the fractions. ‘One-pot’ method is a simple, robust method that yields identification of >1000 proteins with high confidence, across all spore layers from Bacillus subtilis, Bacillus cereus, and Peptoclostridium difficile. This method can be employed for proteome-wide analysis of non-spore-forming as well as spore-forming pathogens. Analysis of spore protein profile will help to understand the sporulation and germination processes and to distinguish immunogenic protein markers.
Project description:Bacillus weihenstephanensis is a subspecies of the Bacillus cereus sensu lato group of spore forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7°C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. In order to gain insight into to the mechanistic basis of its psychrotolerant phenotype, as well as elucidate relevant aspects of its toxigenic profile, the proteome profiles of cells grown at either 6°C or 30°C were compared.