The adsorption of antigens and enzymes to bacterial spores has been reported previously and the involvement of a combination of physicochemical forces has been suggested [18–20]. In this frame, our work was aimed at gaining a better understanding of the spore-enzyme interaction. We used spores of a laboratory strain of B. subtilis, for which a collection of isogenic mutants altered in spore surface proteins was available, and as a model enzyme the well characterized β-Gal of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius.
Our results indicate that spores bind β-Gal without affecting its properties but, instead, stabilizing it at acidic pH and high temperatures. Spore adsorption is a very efficient process with each wild type spore able to adsorb about 7.75×103 molecules of β-Gal. Gene fusion-based display of heterologous antigens was slightly less efficient than adsorption, with 1.5×103 molecules of TTFC (5) and 4.8×103 molecules of LTB (15) exposed on each spore. Spores lacking the outermost structures, the crust and the outer coat layer, have an adsorption efficiency even higher than wild type spores, with 1.55×104 molecules of β-Gal adsorbed on each spore. This indicates that those structures, mainly formed by proteins and glycoproteins , have an inhibitory effect on the adhesion of the enzyme. While the efficiency of adhesion is improved, the stabilization at low pH is reduced in the mutants after 24 hours of incubation at pH 4.0. In the case of β-Gal, the loss of activity (30%) at low pH is compensated by the high amount of enzyme adsorbed. Although other enzymes have not been tested yet, our data suggest that the use of wild type or mutants spores can be planned according to the specific application and the heterologous enzyme to be displayed.
The carboxyl groups were identified as the major ionizable groups in the spore and proton diffusion was found much lower in the spore core than within the coats and cortex, suggesting the inner membrane, separating core from the external layers of the spore, as a major permeability barrier for protons . Then, the carboxyl groups in the coat and in the cortex have been suggested as responsible of the negative charge of spores . The different effects of wild type and mutant spores on the pH of an aqueous solution (Table2), indicate that wild type spores attract more protons than cotE or cotH mutant spores and therefore, suggest that thay have a net negative charge higher than that of the mutant spores. Since spores of those mutants lack either the entire outer coat (cotE) or a large part of it (cotH), they have a reduced number of proteins, and as a consequence of carboxyl groups, in their cortex and coat. Based on this we hypothesize that the reduced number of carboxyl groups present in the regions of the spore more permeable to proton diffusion  is responsible of the reduced number of protons attracted by mutant spores. However, the different efficiency of β-Gal adsorption observed between wild type and mutant spores is not due to a different effect on the pH, since the effect of spores on the pH is buffered at the adsorption conditions (citrate buffer pH 4.0).
Our results, indicating that spores with altered surface structures have altered adsorption efficiency, point to the physicochemical properties of the spore surface as responsible of the interaction with the model enzyme. β-Gal, having a deduced isoelectric point of 5.77, at the adsorption conditions (pH 4.0) is expected to have a net positive charge and to be attracted by negatively charged spores. In spite of this, β-Gal binds more efficiently to mutant (lower negative charge) than wild type (higher negative charge) spores. We conclude that, at least in the case of β-Gal, the electrostatic force does not seem to be the predominant force involved in the interaction with the spore.
The hypothesis that the different negative charge of wild type and mutant spores is somehow responsible of the different stabilization effect observed at pH 4.0 is an intriguing possibility that needs to be addressed. Answering the questions of what is the basis of spore adsorption and of enzyme stabilization are challenging future goals that will necessarily require further experiments and the use of sophisticated physicochemical tools.
Although the molecular details of adsorption and enzyme stabilization have not been totally elucidated, spores have shown clear potentials as a novel, surface display system that, being non-recombinant, able to protect the heterologous enzyme from acidic pH and based on a host with a remarkable safety record [10, 11], appear particularly well suited for the delivery of biotherapeutic molecules to animal and human mucosal surfaces.