In a proof-of-concept work, the spore coat protein CotB of B. The rigidity and compactness of the spore suggested the possibility of using this unusual cell as a platform to display heterologous proteins. megaterium, the outermost layer of the coat is the exosporium, a protective shell mainly made of glycoproteins. subtilis, spores are formed by a partially dehydrated cytoplasm (core) surrounded by several protective layers: the thick peptidoglycan-like cortex, the multilayered, proteinaceous coat and the crust, the outermost layer formed of proteins and glycans. Spore germination and resistance are in part due to the peculiar structure of the spore, that has been studied in detail in Bacillus subtilis, the model system for spore formers. Although metabolically quiescent, the spore is able to sense the environment and respond to conditions that allow cell growth by germinating and generating a new vegetative cell. ![]() In the spore form, these bacterial species can survive conditions, such as the prolonged absence of water and nutrients, the exposure to extremes of temperature and pH, to UV irradiations and to toxic chemicals, that would be lethal for other cell forms. Our results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.Įndospores (spores) are quiescent cell forms produced by over 1000 bacterial species when the environmental conditions do not allow cell growth to continue. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 ☌ slightly more efficient than 25 ☌ spores in adsorbing at least some of the model proteins tested. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. ![]() ![]() Immune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 ☌. subtilis spores prepared at 25, 37 or 42 ☌ were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach. subtilis builds structurally and functionally different spores when grown at different temperatures based on this finding B. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Bacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts.
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