Bacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. Increasing knowledge about spore coat structure enables selection of new anchor proteins such as CotZ and CgeA. Here we describe a system of vectors for display of proteins on the surface of B. subtilis spores.

Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of interest as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliD-adsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immunized mice. Our results indicate that such dual display technology may be useful in creation of spores simultaneously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile.

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.

Mucosal immunizations are convenient ways of vaccination, which do not require any trained personnel for administration. One of the major challenges for developing an effective mucosal vaccine is finding appropriate adjuvant. Bacillus subtilis endospores have been shown to help solving these obstacles while serving as a platform for presentation of both, antigens and adjuvants. In this study, we have successfully designed and constructed recombinant spores displaying an antigen/adjuvant chimeric protein. We have used a fragment of Clostridium difficile flagellar cap FliD protein as antigen and VQGEESNDK peptide, a fragment of human IL-1β, as adjuvant. Recombinant spores presenting FliD were able to elicit immune response in orally immunized mice which could be evaluated by detection of FliD-specific IgA antibodies in feces of immunized animals. Moreover, the presence of IL-1β fragment significantly changed characteristics of elicited immune response. Obtained results show that recombinant spores presenting an antigen/adjuvant chimeric protein exhibit both properties in mucosal immunization of mice. Moreover, IL-1β fragment could serve as valuable adjuvant in B. subtilis spore-based mucosal vaccines.

Microorganisms in their natural ecological niches are constantly challenged by other inhabitants. Antagonisms exhibited by interacting microbial species are directed towards survival and increasing of their fitness. The Soft Rot Pectobacteriaceae (SRP) is a good model to study these complex microbial interactions. Along with being present in various environments, SRPs are often transferred between environments, allowing the bacteria to encounter members of other species. In this study, we investigated interactions between Dickeya solani, a representative of SRPs and a causative agent of potato soft rot, and Bacillus subtilis, which is known to be a potent producer of secondary metabolites mediating antibiosis. We have found that the soil isolate B. subtilis MB73/2 not only suppresses in vitro soft-rotting of infected potato tubers but is also able to cause directional, coordinated escape of natural isolates D. solani IFB0102 and IPO2222. While this coordinated movement of D. solani depends on surfactin produced by B. subtilis MB73/2, we show that both Dickeya strains exhibit different antagonistic interaction phenotypes toward the competing Bacillus. We prove that this antagonism depends on a single nucleotide polymorphism in one of transcriptional regulators of D. solani belonging to the LysR family.