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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.


(a) Dot blot of released mRFP after three washes (W1, W2 and W3) with PBS buffer at pH 3 or pH 7 or after extraction with 1 M NaCl, 0.1 % Triton X-100 (Ext) (“Methods” section). Purified recombinant mRFP (mRFP) and unbound mRFP (u-mRFP) after the adsorption reaction were used as markers. b Kinetic of mRFP release based on the densitometric analysis (Additional file 4: Table S3) of mRFP released upon incubation either in PBS buffer pH 7.0 (triangles) or 1 M NaCl, 0.1 % Triton X-100 (squares). c Western blot of mRFP extracted by a SDS–DTT or after consecutive SDS–DTT and urea treatments of wild type and cotH mutant spores. Immuno reactions were performed with anti-His primary antibody anti-His primary antibody conjugated with the horseradish peroxidase (“Methods” section)
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Fig3: (a) Dot blot of released mRFP after three washes (W1, W2 and W3) with PBS buffer at pH 3 or pH 7 or after extraction with 1 M NaCl, 0.1 % Triton X-100 (Ext) (“Methods” section). Purified recombinant mRFP (mRFP) and unbound mRFP (u-mRFP) after the adsorption reaction were used as markers. b Kinetic of mRFP release based on the densitometric analysis (Additional file 4: Table S3) of mRFP released upon incubation either in PBS buffer pH 7.0 (triangles) or 1 M NaCl, 0.1 % Triton X-100 (squares). c Western blot of mRFP extracted by a SDS–DTT or after consecutive SDS–DTT and urea treatments of wild type and cotH mutant spores. Immuno reactions were performed with anti-His primary antibody anti-His primary antibody conjugated with the horseradish peroxidase (“Methods” section)

Mentions: Upon adsorption to spores, mRFP is not easily released even after multiple washes. Spores adsorbed with mRFP were washed three times with 100 μl PBS buffer at pH 3.0 or pH 7.0 or extracted with 1 M NaCl, 0.1 % Triton X-100 [3]. With wild type spores no mRFP was released by any of these treatments, while with cotH mutant spores some mRFP was released by the washes at pH 7.0 and by the NaCl−Triton extraction (Fig. 3a). To evaluate the kinetic of mRFP-release adsorbed mutant spores were washed two times, resuspended in PBS pH 7.0 or 1 M NaCl, 0.1 % Triton X-100 for 5, 10, 15 or 30 min and the supernatant fractions analyzed by dot blot (not shown). A densitometric analysis of the dot blot (Additional file 4: Table S3) was performed and Fig. 3b reports the percentage of mRFP released by the two treatments at the different time points. The pH 7.0 buffer extracted about 5 % of the adsorbed mRFP within the first 5 min of incubation and did not extract more protein over time. The NaCl–Triton solution extracted about 3 % of mRFP in the first 5 min and the amount of extracted mRFP increased over time in an almost linear way reaching over 9 % of mRFP released after 30 min of treatment (Fig. 3b).Fig. 3


Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis
(a) Dot blot of released mRFP after three washes (W1, W2 and W3) with PBS buffer at pH 3 or pH 7 or after extraction with 1 M NaCl, 0.1 % Triton X-100 (Ext) (“Methods” section). Purified recombinant mRFP (mRFP) and unbound mRFP (u-mRFP) after the adsorption reaction were used as markers. b Kinetic of mRFP release based on the densitometric analysis (Additional file 4: Table S3) of mRFP released upon incubation either in PBS buffer pH 7.0 (triangles) or 1 M NaCl, 0.1 % Triton X-100 (squares). c Western blot of mRFP extracted by a SDS–DTT or after consecutive SDS–DTT and urea treatments of wild type and cotH mutant spores. Immuno reactions were performed with anti-His primary antibody anti-His primary antibody conjugated with the horseradish peroxidase (“Methods” section)
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Related In: Results  -  Collection

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Fig3: (a) Dot blot of released mRFP after three washes (W1, W2 and W3) with PBS buffer at pH 3 or pH 7 or after extraction with 1 M NaCl, 0.1 % Triton X-100 (Ext) (“Methods” section). Purified recombinant mRFP (mRFP) and unbound mRFP (u-mRFP) after the adsorption reaction were used as markers. b Kinetic of mRFP release based on the densitometric analysis (Additional file 4: Table S3) of mRFP released upon incubation either in PBS buffer pH 7.0 (triangles) or 1 M NaCl, 0.1 % Triton X-100 (squares). c Western blot of mRFP extracted by a SDS–DTT or after consecutive SDS–DTT and urea treatments of wild type and cotH mutant spores. Immuno reactions were performed with anti-His primary antibody anti-His primary antibody conjugated with the horseradish peroxidase (“Methods” section)
Mentions: Upon adsorption to spores, mRFP is not easily released even after multiple washes. Spores adsorbed with mRFP were washed three times with 100 μl PBS buffer at pH 3.0 or pH 7.0 or extracted with 1 M NaCl, 0.1 % Triton X-100 [3]. With wild type spores no mRFP was released by any of these treatments, while with cotH mutant spores some mRFP was released by the washes at pH 7.0 and by the NaCl−Triton extraction (Fig. 3a). To evaluate the kinetic of mRFP-release adsorbed mutant spores were washed two times, resuspended in PBS pH 7.0 or 1 M NaCl, 0.1 % Triton X-100 for 5, 10, 15 or 30 min and the supernatant fractions analyzed by dot blot (not shown). A densitometric analysis of the dot blot (Additional file 4: Table S3) was performed and Fig. 3b reports the percentage of mRFP released by the two treatments at the different time points. The pH 7.0 buffer extracted about 5 % of the adsorbed mRFP within the first 5 min of incubation and did not extract more protein over time. The NaCl–Triton solution extracted about 3 % of mRFP in the first 5 min and the amount of extracted mRFP increased over time in an almost linear way reaching over 9 % of mRFP released after 30 min of treatment (Fig. 3b).Fig. 3

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.