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Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis

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ABSTRACT

Background: Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet.

Results: We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface.

Conclusions: Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-016-0551-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Cartoon model of mRFP localization in wild type (a) and cotH mutant (b) spores. Wild type and mutant spores are over imposed with red dots representing mRFP molecules
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Fig9: Cartoon model of mRFP localization in wild type (a) and cotH mutant (b) spores. Wild type and mutant spores are over imposed with red dots representing mRFP molecules

Mentions: Wild type spores of B. subtilis adsorb and tightly bind mRFP that is not displayed on the spore surface. We propose that it infiltrates through crust and outer coat layers, localizes in the inner coat and appears more abundantly concentrated at the spore poles (Fig. 9a). The observation that mRFP crosses crust and outer coat indicates these structures as permeable to a 27 kDa protein. Permeability of the spore surface is not totally surprising since germinants (small molecules with molecular masses typically <200 Da) present in the environment have to cross the external layers of the spore to reach their receptors. In addition, studies conducted on B. megaterium suggested that the spore surface is permeable to the passage of molecules with masses somewhere between 2 and 40 kDa [34, 35].Fig. 9


Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis
Cartoon model of mRFP localization in wild type (a) and cotH mutant (b) spores. Wild type and mutant spores are over imposed with red dots representing mRFP molecules
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016992&req=5

Fig9: Cartoon model of mRFP localization in wild type (a) and cotH mutant (b) spores. Wild type and mutant spores are over imposed with red dots representing mRFP molecules
Mentions: Wild type spores of B. subtilis adsorb and tightly bind mRFP that is not displayed on the spore surface. We propose that it infiltrates through crust and outer coat layers, localizes in the inner coat and appears more abundantly concentrated at the spore poles (Fig. 9a). The observation that mRFP crosses crust and outer coat indicates these structures as permeable to a 27 kDa protein. Permeability of the spore surface is not totally surprising since germinants (small molecules with molecular masses typically <200 Da) present in the environment have to cross the external layers of the spore to reach their receptors. In addition, studies conducted on B. megaterium suggested that the spore surface is permeable to the passage of molecules with masses somewhere between 2 and 40 kDa [34, 35].Fig. 9

View Article: PubMed Central - PubMed

ABSTRACT

Background: Bacterial spores have been proposed as vehicles to display heterologous proteins for the development of mucosal vaccines, biocatalysts, bioremediation and diagnostic tools. Two approaches have been developed to display proteins on the spore surface: a recombinant approach, based on the construction of gene fusions between DNA molecules coding for a spore surface protein (carrier) and for the heterologous protein to be displayed (passenger); and a non-recombinant approach based on spore adsorption, a spontaneous interaction between negatively charged, hydrophobic spores and purified proteins. The molecular details of spore adsorption have not been fully clarified yet.

Results: We used the monomeric Red Fluorescent Protein (mRFP) of the coral Discosoma sp. and Bacillus subtilis spores of a wild type and an isogenic mutant strain lacking the CotH protein to clarify the adsorption process. Mutant spores, characterized by a strongly altered coat, were more efficient than wild type spores in adsorbing mRFP but the interaction was less stable and mRFP could be in part released by raising the pH of the spore suspension. A collection of isogenic strains carrying GFP fused to proteins restricted in different compartments of the B. subtilis spore was used to localize adsorbed mRFP molecules. In wild type spores mRFP infiltrated through crust and outer coat, localized in the inner coat and was not surface exposed. In mutant spores mRFP was present in all surface layers, inner, outer coat and crust and was exposed on the spore surface.

Conclusions: Our results indicate that different spores can be selected for different applications. Wild type spores are preferable when a very tight protein-spore interaction is needed, for example to develop reusable biocatalysts or bioremediation systems for field applications. cotH mutant spores are instead preferable when the heterologous protein has to be displayed on the spore surface or has to be released, as could be the case in mucosal delivery systems for antigens and drugs, respectively.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-016-0551-2) contains supplementary material, which is available to authorized users.

No MeSH data available.