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Clostridium difficile is a Gram positive pathogen of significant importance in the UK, Europe and the USA. No vaccine has been developed and current treatments are focused on hospital management and the use of antibiotics. The disease is spread in hospitals in the spore form and the role of spores in C. difficile infecton is poorly understood. In this project spores of C. difficile have been characterised. The proteins from the outermost layers of the spore were identified and the genes cloned. Three of these surface proteins have unique enzymatic properties that maybe important for symptoms of disease. The ability of C. difficile spores to adhere to intestinal cells was found to be far greater than with live cells and through this we have identified that the spore may play an important role in colonisation. The regulation of spore coat gene expression during sporulation was also examined and temporal phases of genes expression identified. A major part of this project was to develop a mucosal vaccine to C. difficile. The approach used was to clone the C-terminus of toxin A onto the surface of Bacillus subtilis spores and use these recombinant spores to immunise mice and hamsters. We found that oral delivery of these spores conferred 75% protection to C. difficile infection in a hamster model of infection. Further, parenteral immunisation of the same antigens (toxin A and B) failed to generate mucosal responses and this showed that mucosal immunisation is critical for good protection. Finally, we found that antibodies to the C-terminus of toxin A were cross reactive to the C-terminus of toxin B. This showed that mucosal delivery of just the C-terminus of toxin A is sufficient to confer protection in an animal model of infection. The outcome of this work is that we have shown the parameters for successful immunisation and vaccination against C. difficile.

La spore bactérienne est une forme microbienne multicouche extrêmement résistante aux perturbations environnementales. Cette résistance est notamment liée à sa structure unique qui est particulièrement peu perméable et compacte. Ce travail de thèse a pour but d’identifier et de caractériser les structures sporales impliquées dans ces propriétés. Des méthodes d’investigations globales comme la RMN ou l’anisotropie de fluorescence ont permis de montrer que le cortex des spores de Bacillus subtilis est modifié par la température, pour des valeurs proches de celle de l’activation de la germination. Ceci aura pour conséquence de modifier l’accès à la membrane interne. Un outil d’étude à l’échelle de la spore, l’imagerie en temps de vie de fluorescence (FLIM) couplé à l’utilisation d’un rotor moléculaire, a également été mis au point. Cet outil a permis de mettre en évidence que la membrane interne de B. subtilis possède une très forte viscosité, environ deux fois plus importante que celle de la membrane d’une cellule végétative. Cette viscosité n’est modifiée par la température qu’au-delà de 65 °C, correspondant également à l’activation de la germination. Une perturbation connue pour modifier l’intégrité de la structure de la spore a également été étudiée : l’éthanol couplé à une température importante (65 ou 70°C). Ce traitement est responsable d’une perméabilisation et d’une inactivation des spores. L’éthanol conduit notamment à l’altération de la membrane interne, dont la viscosité et la perméabilité sont modifiées. Ces résultats apportent de nouvelles données pour la compréhension des mécanismes responsables de l’inactivation des spores. Ils permettent d’envisager des applications, pour lesquelles une maitrise des modifications structurales est nécessaire, comme la microencapsulation. / The bacterial spore is a multilayer microbial form which is extremely resistant to environmental perturbations. This resistance is especially due to its unique structure which is particularly compact and weakly permeable. This work aims to identify and characterize the spore structures involved in these properties. Overall investigation methods, such as NMR and fluorescence anisotropy, have shown that the cortex of Bacillus subtilis spores is modified by temperature for level similar to that of the activation of germination. This will result in changes to the access to the inner membrane. A tool at the spore’s scale, the fluorescence lifetime imaging microscopy (FLIM) in conjunction with the use of a molecular rotor, has been set up. This tool allowed demonstrating that inner membrane of B. subtilis has a very high viscosity, about two times greater than that of the membrane of a vegetative cell. This viscosity is changed by temperature near 65 °C, which corresponds to activation of germination. A stress known to modify the structural integrity of the spore has also been studied: ethanol combined with significant temperature (65 ou 70 °C). This treatment is responsible for inactivation of spores in parallel with their permeabilization. Ethanol especially leads to alteration of the inner membrane for which the viscosity and permeability are changed. These results provide new understanding of mechanisms implicated in spores’ destruction. They allow considering some new applications, for which it is necessary to control structural changing, for example microencapsulation.