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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.


Western (a) and dot (b) blot of proteins extracted from wild type and cotH mutant spores after mRFP-spore adsorption. Purified recombinant mRFP is used as marker. In panel b the amount of purified recombinant mRFP (ng) and the number of spores present in each dilution are indicated. Immuno reactions were performed with anti-His primary antibody conjugated with horseradish peroxidase ("Methods'' section)
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Fig1: Western (a) and dot (b) blot of proteins extracted from wild type and cotH mutant spores after mRFP-spore adsorption. Purified recombinant mRFP is used as marker. In panel b the amount of purified recombinant mRFP (ng) and the number of spores present in each dilution are indicated. Immuno reactions were performed with anti-His primary antibody conjugated with horseradish peroxidase ("Methods'' section)

Mentions: In an initial experiment 5 μg of mRFP of the coral Discosoma sp., over-expressed in E. coli and purified by affinity chromatography with Ni–Nta columns ("Methods" section), was incubated with 2.0 × 109 purified spores of the B. subtilis strain PY79 [25] or of the isogenic strain ER209, lacking CotH [21]. The adsorption reactions were performed at pH 4.0, as previously described [15]. After the adsorption reaction spores were extensively washed, spore surface proteins were extracted as previously described [26] and analyzed by western blot with monoclonal anti-polyHistidine–peroxidase antibody (Sigma), able to recognize the his tagged N terminus of mRFP. As shown in Fig. 1a, mRFP was extracted from both wild type and cotH mutant spores, indicating that it was absorbed by both types of spores, retained during the washing steps and released by the extraction treatment. mRFP adsorption was extremely stable over time and the protein was still extracted by SDS–DTT treatment after 2 weeks of storage at room temperature of the adsorbed spores (Additional file 1: Figure S1).Fig. 1


Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis
Western (a) and dot (b) blot of proteins extracted from wild type and cotH mutant spores after mRFP-spore adsorption. Purified recombinant mRFP is used as marker. In panel b the amount of purified recombinant mRFP (ng) and the number of spores present in each dilution are indicated. Immuno reactions were performed with anti-His primary antibody conjugated with horseradish peroxidase ("Methods'' section)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016992&req=5

Fig1: Western (a) and dot (b) blot of proteins extracted from wild type and cotH mutant spores after mRFP-spore adsorption. Purified recombinant mRFP is used as marker. In panel b the amount of purified recombinant mRFP (ng) and the number of spores present in each dilution are indicated. Immuno reactions were performed with anti-His primary antibody conjugated with horseradish peroxidase ("Methods'' section)
Mentions: In an initial experiment 5 μg of mRFP of the coral Discosoma sp., over-expressed in E. coli and purified by affinity chromatography with Ni–Nta columns ("Methods" section), was incubated with 2.0 × 109 purified spores of the B. subtilis strain PY79 [25] or of the isogenic strain ER209, lacking CotH [21]. The adsorption reactions were performed at pH 4.0, as previously described [15]. After the adsorption reaction spores were extensively washed, spore surface proteins were extracted as previously described [26] and analyzed by western blot with monoclonal anti-polyHistidine–peroxidase antibody (Sigma), able to recognize the his tagged N terminus of mRFP. As shown in Fig. 1a, mRFP was extracted from both wild type and cotH mutant spores, indicating that it was absorbed by both types of spores, retained during the washing steps and released by the extraction treatment. mRFP adsorption was extremely stable over time and the protein was still extracted by SDS–DTT treatment after 2 weeks of storage at room temperature of the adsorbed spores (Additional file 1: Figure S1).Fig. 1

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.