Giuseppina Cangiano

Surface Display of Recombinant Proteins on Bacillus subtilis Spores

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 x 10(3) TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat

We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms. Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediately after their synthesis, since no accumulation of either of them was detected in the mother cell compartment, where their synthesis occurs. The other two components, 12.5 and 30 kDa, were generated 2 h later and were probably the products of posttranslational modifications of the two early forms occurring directly on the coat surface during spore maturation. None of the CotC forms was found either on the spore coat or in the mother cell compartment of a cotH mutant. This indicates that CotH serves a dual role of stabilizing the early forms of CotC and promoting the assembly of both early and late forms on the spore surface.

Direct and indirect control of late sporulation genes by GerR of Bacillus subtilis

GerR is a sporulation-specific transcriptional factor of Bacillus subtilis that has been identified as a negative regulator of genes transcribed by sigma(E)-containing RNA polymerase and as a positive effector of the expression of three late sporulation genes. Here we confirmed that gerR transcription is dependent on sigma(E)-containing RNA polymerase but also observed that it requires the transcriptional regulator SpoIIID. The study of the role of GerR in regulating the expression of several late sporulation genes allowed us to observe that its effect is strongly positive on spoVIF, cotC, and cotG, weakly positive on cotB, and negative on cotU. The results of chromatin immunoprecipitation (ChIP) experiments indicated that GerR binds to the promoter regions of some, but not all, of the GerR-controlled genes, leading us to propose that GerR controls late sporulation genes in two ways: (i) directly, by acting on the transcription of cotB, cotU and spoVIF; and (ii) indirectly, through the activation of SpoVIF, which stabilizes the transcriptional activator GerE and consequently induces the expression of the GerE-dependent genes cotC and cotG.

Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis

Development of mucosal vaccines strongly relies on an efficient delivery system and, over the years, a variety of approaches based on phages, bacteria or synthetic nanoparticles have been proposed to display and deliver antigens. The spore of Bacillus subtilis displaying heterologous antigens has also been considered as a mucosal vaccine vehicle, and shown able to conjugate some advantages of live microrganisms with some of synthetic nanoparticles. Here we review the use of non-recombinant spores of B. subtilis as a delivery system for mucosal immunizations. The non-recombinant display is based on the adsorption of heterologous molecules on the spore surface without the need of genetic manipulations, thus avoiding all concerns about the use and environmental release of genetically modified microorganisms. In addition, adsorbed molecules are stabilized and protected by the interaction with the spore, suggesting that this system could reduce the rapid degradation of the antigen, often observed with other delivery systems and identified as a major drawback of mucosal vaccines.

Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages.

Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

The sps Gene Products Affect the Germination, Hydrophobicity, and Protein Adsorption of Bacillus subtilis Spores

ABSTRACT The multilayered surface of the Bacillus subtilis spore is composed of proteins and glycans. While over 70 different proteins have been identified as surface components, carbohydrates associated with the spore surface have not been characterized in detail yet. Bioinformatic data suggest that the 11 products of the sps operon are involved in the synthesis of polysaccharides present on the spore surface, but an experimental validation is available only for the four distal genes of the operon. Here, we report a transcriptional analysis of the sps operon and a functional study performed by constructing and analyzing two null mutants lacking either all or only the promoter-proximal gene of the operon. Our results show that both sps mutant spores apparently have normal coat and crust but have a small germination defect and are more hydrophobic than wild-type spores. We also show that spores lacking all Sps proteins are highly adhesive and form extensive clumps. In addition, sps mutant spores have an increased efficiency in adsorbing a heterologous enzyme, suggesting that hydrophobic force is a major determinant of spore adsorption and indicating that a deep understanding of the surface properties of the spore is essential for its full development as a surface display platform.

Antagonistic role of CotG and CotH on spore germination and coat formation in Bacillus subtilis.

Spore formers are bacteria able to survive harsh environmental conditions by differentiating a specialized, highly resistant spore. In Bacillus subtilis, the model system for spore formers, the recently discovered crust and the proteinaceous coat are the external layers that surround the spore and contribute to its survival. The coat is formed by about seventy different proteins assembled and organized into three layers by the action of a subset of regulatory proteins, referred to as morphogenetic factors. CotH is a morphogenetic factor needed for the development of spores able to germinate efficiently and involved in the assembly of nine outer coat proteins, including CotG. Here we report that CotG has negative effects on spore germination and on the assembly of at least three outer coat proteins. Such negative action is exerted only in mutants lacking CotH, thus suggesting an antagonistic effect of the two proteins, with CotH counteracting the negative role of CotG.

The Exosporium of Bacillus megaterium QM B1551 Is Permeable to the Red Fluorescence Protein of the Coral Discosoma sp.

Bacterial spores spontaneously interact and tightly bind heterologous proteins. A variety of antigens and enzymes have been efficiently displayed on spores of Bacillus subtilis, the model system for spore formers. Adsorption on B. subtilis spores has then been proposed as a non-recombinant approach for the development of mucosal vaccine/drug delivery vehicles, biocatalysts, bioremediation, and diagnostic tools. We used spores of B. megaterium QM B1551 to evaluate their efficiency as an adsorption platform. Spores of B. megaterium are significantly larger than those of B. subtilis and of other Bacillus species and are surrounded by the exosporium, an outermost surface layer present only in some Bacillus species and lacking in B. subtilis. Strain QM B1551 of B. megaterium and a derivative strain totally lacking the exosporium were used to localize the adsorbed monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp., used as a model heterologous protein. Our results indicate that spores of B. megaterium adsorb mRFP more efficiently than B. subtilis spores, that the exosporium is essential for mRFP adsorption, and that most of the adsorbed mRFP molecules are not exposed on the spore surface but rather localized in the space between the outer coat and the exosporium.

Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes

BackgroundThe Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-d-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-d-xylans to remove successive d-xylose residues from the non-reducing termini.ResultsWe report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation.ConclusionOur results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes for single as well as multi-step reactions.