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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.


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Fluorescence intensity profiles (Fl. Int.; scale in arbitrary units) of wild type and mutant spores adsorbed with mRFP. The profiles were generated from fluorescence microscopy images using the 3D Surface plotter function of Image J (http://imagej.nih.gov/ij/). a Representative fluorescence intensity profiles of a wild type (left) and cotH mutant spore (right). The fluorescence intensity is reported in arbitrary units. b Box plots displaying the total corrected cellular fluorescence (TCCF) for 80 different spores of each strain. Limits of each box represent the first and the third quartile (25 and 75 %) and the values outside the boxes represent the maximum and the minimum values. The line dividing the box indicates the median value for each strain. P value is less than 0.0001
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Fig5: Fluorescence intensity profiles (Fl. Int.; scale in arbitrary units) of wild type and mutant spores adsorbed with mRFP. The profiles were generated from fluorescence microscopy images using the 3D Surface plotter function of Image J (http://imagej.nih.gov/ij/). a Representative fluorescence intensity profiles of a wild type (left) and cotH mutant spore (right). The fluorescence intensity is reported in arbitrary units. b Box plots displaying the total corrected cellular fluorescence (TCCF) for 80 different spores of each strain. Limits of each box represent the first and the third quartile (25 and 75 %) and the values outside the boxes represent the maximum and the minimum values. The line dividing the box indicates the median value for each strain. P value is less than 0.0001

Mentions: To assess whether spore-adsorbed mRFP molecules retained their fluorescence properties and investigate their distribution around the spore we performed a fluorescence microscopy analysis. With both wild type and mutant spores red fluorescent signals were observed all around the spore (Fig. 4), and in agreement with results of Figs. 2 and 3, the fluorescence signal appeared stronger with mutant than with wild type spores (Fig. 4). In all cases the fluorescent signal was stronger at the spore poles, indicating that adsorbed mRFP molecules were not evenly distributed around the spore and accumulated at the poles (Fig. 4). We used Image J software (v1.48, NIH) to perform a quantitative fluorescence image analysis and the corrected spore fluorescence was calculated as described in the “Methods” section. The analysis of 80 spores of each strain indicated an average fluorescence intensity, in arbitrary units, of 7816 ± 2712 and of 11541 ± 2573 for wild type spores and mutant spores, respectively (Fig. 5, P  <  0.0001), confirming that cotH mutant spores adsorb more mRFP than wild type spores.Fig. 4


Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis
Fluorescence intensity profiles (Fl. Int.; scale in arbitrary units) of wild type and mutant spores adsorbed with mRFP. The profiles were generated from fluorescence microscopy images using the 3D Surface plotter function of Image J (http://imagej.nih.gov/ij/). a Representative fluorescence intensity profiles of a wild type (left) and cotH mutant spore (right). The fluorescence intensity is reported in arbitrary units. b Box plots displaying the total corrected cellular fluorescence (TCCF) for 80 different spores of each strain. Limits of each box represent the first and the third quartile (25 and 75 %) and the values outside the boxes represent the maximum and the minimum values. The line dividing the box indicates the median value for each strain. P value is less than 0.0001
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016992&req=5

Fig5: Fluorescence intensity profiles (Fl. Int.; scale in arbitrary units) of wild type and mutant spores adsorbed with mRFP. The profiles were generated from fluorescence microscopy images using the 3D Surface plotter function of Image J (http://imagej.nih.gov/ij/). a Representative fluorescence intensity profiles of a wild type (left) and cotH mutant spore (right). The fluorescence intensity is reported in arbitrary units. b Box plots displaying the total corrected cellular fluorescence (TCCF) for 80 different spores of each strain. Limits of each box represent the first and the third quartile (25 and 75 %) and the values outside the boxes represent the maximum and the minimum values. The line dividing the box indicates the median value for each strain. P value is less than 0.0001
Mentions: To assess whether spore-adsorbed mRFP molecules retained their fluorescence properties and investigate their distribution around the spore we performed a fluorescence microscopy analysis. With both wild type and mutant spores red fluorescent signals were observed all around the spore (Fig. 4), and in agreement with results of Figs. 2 and 3, the fluorescence signal appeared stronger with mutant than with wild type spores (Fig. 4). In all cases the fluorescent signal was stronger at the spore poles, indicating that adsorbed mRFP molecules were not evenly distributed around the spore and accumulated at the poles (Fig. 4). We used Image J software (v1.48, NIH) to perform a quantitative fluorescence image analysis and the corrected spore fluorescence was calculated as described in the “Methods” section. The analysis of 80 spores of each strain indicated an average fluorescence intensity, in arbitrary units, of 7816 ± 2712 and of 11541 ± 2573 for wild type spores and mutant spores, respectively (Fig. 5, P  <  0.0001), confirming that cotH mutant spores adsorb more mRFP than wild type spores.Fig. 4

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.


Related in: MedlinePlus