Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA 26 -39 ) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA 26 -39 (but not to toxoids), whether raised to the recombinant protein or to TcdA 26 -39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile. These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA 26 -39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

Spores of Clostridium difficile carry a chitinase enzyme whose function is to bind to mucin, leading to its degradation. We show in vivo that this is potentially an important element of colonization and virulence.

Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile.

Genetic manipulation of bacterial spores of the genus has shown potential for vaccination and for delivery of drugs or enzymes. Remarkably, proteins displayed on the spore surface retain activity and generally are not degraded. The heat stability of spores coupled with their desiccation resistance makes them suitable for delivery to humans or to animals by the oral route. Despite these attributes one regulatory obstacle has remained regarding the fate of recombinant spores shed into the environment as viable spores. We have addressed the biological containment of spore GMOs by utilizing the concept of a 'thymine-less death', a phenomenon first reported six decades ago. Using we have inserted chimeric genes in the two thymidylate synthase genes, and, using a two-step process. Insertion is made first at followed by where resistance to trimethoprim enables selection of recombinants. Importantly, this method requires introduction of no new antibiotic resistance genes. Recombinant spores have a strict dependence on thymine (or thymidine) and in their absence cells lyse and die. Insertions are stable with no evidence for suppression or reversion. Using this system we have successfully created a number of spore vaccines as well as spores displaying active enzymes. Genetic manipulation of bacterial spores offers a number of exciting possibilities for public and animal health including their use as heat stable vehicles for delivering vaccines or enzymes. Despite this, one remaining problem is the fate of recombinant spores if released to the environment where they could survive in a dormant form indefinitely. We describe a solution whereby following genetic manipulation the bacterium is rendered dependent on thymine. As a consequence spores if released would produce bacteria unable to survive and they would exhibit a thymine-less death due to rapid cessation of metabolism. The method we describe has been validated using a number of exemplars and solves a problem for containing spore GMOs in the environment.

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