Potocki et al. Microb Cell Fact (2017) 16:151 DOI 10.1186/s12934-017-0765-y
Microbial Cell Factories Open Access
RESEARCH
The combination of recombinant and non‑recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore Wojciech Potocki1, Alessandro Negri1,4, Grażyna Peszyńska‑Sularz2, Krzysztof Hinc3, Michał Obuchowski3 and Adam Iwanicki3*
Abstract Background: Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of inter‑ est as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. Results: We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliDadsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immu‑ nized mice. Conclusions: Our results indicate that such dual display technology may be useful in creation of spores simultane‑ ously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile. Keywords: Bacillus subtilis, Recombinant spores, Adsorption, Mucosal immunization, FliD, Clostridium difficile Background Bacillus subtilis spores are dormant forms of this microorganism, well known for their resistance to harsh environmental conditions. Their properties, combined with the easiness of genetic modification of this bacterium, make them a very convenient platform for presentation of heterologous proteins (reviewed in [1]). An interesting application of this technology is preparation of sporebased mucosal vaccines [2]. Use of such system enabled elicitation of protective immunity against infections with
*Correspondence: [email protected] 3 Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, Gdańsk, Poland Full list of author information is available at the end of the article
such pathogens as C. perfringens [3], C. tetani [4], C. difficile [5], or rotavirus [6]. There are two main approaches to display antigens on the surface of spores. Recombinant approach, developed as the first, is based on modification of bacterial genome in a way to express a protein of interest in fusion with one of spore coat proteins. As a result, a fusion protein is expressed in the cell and incorporated into the forming spore coat. Such method enables relatively simple construction of spores presenting heterologous proteins using basic methods of molecular biology. Non-recombinant approach is based on use of unmodified spores and adsorption of a purified protein of interest [7]. This method enables presentation of larger amounts of protein as compared to recombinant spores. Moreover, it
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Potocki et al. Microb Cell Fact (2017) 16:151
does not lead to creation and use of GMO and thus can be much easier applied for animal or human use. Spore-based vaccines can stimulate both systemic and localized immune responses with balanced Th1/Th2 polarization [8]. Although unmodified B. subtilis spores can be used as mucosal adjuvants in some applications [9] they can also be engineered to display immunomodulatory molecules and be administered as adjuvants in formulations with antigen-presenting spores [10]. It is worth noting, that no successful display of more than one recombinant protein on the surface of a single spore has been described in the literature. Such construct displaying molecules of both, an antigen and adjuvant, would be of special interest. Following this line of reasoning, in this study we decided to use previously described recombinant spores presenting human IL-2 [10] and apply non-recombinant adsorption technique to display on their surface FliD flagellar cap protein of Clostridium difficile. The choice of antigen was not random, since Clostridium difficile is a well-known pathogen responsible for antibioticassociated diarrheas and pseudomembranous colitis. Moreover, FliD protein possesses strong immunogenic properties [11, 12]. The results of performed immunization experiments suggest that such combined approach is promising and could be used for preparation of efficient spore-based formulations able to elicit antigen-specific immune responses with polarization driven by adjuvant presented on the surface of the same spore.
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Results Non‑recombinant display of FliD protein
Our idea was to present on spore surface both, an adjuvant and antigen, therefore we decided to use recombinant spores produced by BKH121 strain, which display human IL-2 as fusion with CotB protein joined by a peptide linker. Single spore of this strain presents an average number of 9.5 × 104 IL-2 molecules [10]. Since robust display of proteins on spore surface can be achieved using adsorption method [13–15] we also decided to apply this approach to present FliD protein of C. difficile. The entire FliD was overproduced in E. coli, purified and used for adsorption on surface of spores produced by the wild-type strain 168 and the recombinant strain BKH121. We estimated amounts of spore-adsorbed FliD by measuring unbound protein in adsorption mixture and all subsequent washes (Additional file 1: Tables S1, S2). In the case of spores of both strains the results were comparable and reached levels of 4.13 × 104 FliD molecules/ spore and 3.66 × 104 FliD molecules/spore for 168 and BKH121, respectively. Western blotting analysis of spore coat extracts showed presence of 56 kDa band which reacted with anti-FliD antibodies (Fig. 1a) indicating presence of this protein adsorbed on the spores. To visualize surface exposition of adsorbed protein we made use of immunofluorescence microscopy. Upon incubation with anti-FliD primary antibodies and anti-mouse IgG–Cy3 we observed in microscope fluorescent signal around spores of both strains which were subjected to FliD adsorption
Fig. 1 a Western blotting analysis of spore adsorption with purified FliD protein. Upon adsorption with purified FliD spore surface proteins were extracted by SDS-DTT treatment, fractionated on SDS-PAGE and analyzed by Western blotting. Spore coat extracts were prepared with spores of wild-type strain 168 and recombinant BKH121 alone or with adsorption with FliD (indicated with − and +). Purified FliD (1 μg) was used as positive control. b The same volumes of spore coat extracts were simultaneously loaded onto the second SDS-PAGE gel and analyzed by Western blotting using anti-CotZ antibodies to verify the equality of protein loading. The calculated molecular mass of CotZ is 16.4 kDa
Potocki et al. Microb Cell Fact (2017) 16:151
procedure (Fig. 2). In both experiments signal observed for 168/FliD spores was stronger than in the case of BKH121/FliD spores. These observations are in agreement with increased amount of FliD adsorbed on spores of the wild-type strain 168 in comparison to spores produced by BKH121. This suggests that the surface of recombinant spores used in the study possess diminished adsorption capability. Intranasal immunization of animal leads to development of FliD‑specific immune response
Having verified the display of FliD protein on surface of spores, we used them for mucosal immunizations of mice applying two different administration routes: oral and intranasal. First immunization scheme (oral) consisted of a total of nine doses of 1 × 1010 spores delivering 39.2 μg of protein for FliD-adsorbed spores produced by 168 strain and 34.8 μg of protein for spores produced by BKH121 strain. In the case of this administration route we failed to observe eliciting FliD-specific immune response as assessed by production of antigen-specific antibodies in immunized animals (data not shown). Intranasal immunizations were performed by administration a total of eight doses of 5 × 109 spores delivering 19.6 μg (168/FliD) and 17.4 μg (BKH121/FliD) of protein per dose. In the case of this administration route we detected FliD-specific IgG antibodies in sera the immunized
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animals. Increased levels of FliD-specific IgG antibodies were detected in sera of animals immunized with purified FliD protein and BKH121/FliD spores. The highest titers were observed for animals immunized with IL-2 presenting spores adsorbed with FliD protein (BKH121/ FliD) (Fig. 3a). We also collected entire gastrointestinal tracts (GITs) and lungs of mice used in this experiment, and performed saponin extraction to assess levels of FliD-specific IgA antibodies in the analyzed material. We detected these antibodies in extracts prepared with both, lungs and GITs, with the highest levels observed in the case of mice immunized with FliD-adsorbed spores produced by the BKH121 (GIT extracts, Fig. 3b) and comparable levels in the case of both, 168/FliD and BKH121/ FliD spores (lungs extracts, Fig. 3c). Immunization of mice with purified FliD protein did not significantly increase levels of IgA in both, GITs and lungs. Characterization of elicited immune response
To characterize the polarization of immune response elicited by FliD-adsorbed spores we isolated spleens of immunized animals and used these organs to obtain splenocytes. Cells were stimulated with purified FliD protein and cell supernatants containing secreted cytokines were analyzed by flow cytometer using Cytometric Bead Array (CBA). With this method, we were able to measure levels of IL-2, IL-4, IL-6, IL-10, IL-17A,
Fig. 2 Spore surface display of adsorbed FliD protein as assessed by immunofluorescence microscopy. Purified, free spores of wild-type strain 168 and recombinant BKH121 alone and with adsorption of FliD protein (168/FliD, BKH121/FliD) were visualized by phase contrast and immunofluores‑ cence microscopy. The spores were incubated with mouse anti-FliD antibodies, followed by anti-mouse IgG–Cy3 conjugates. The same exposure time was used for all immunofluorescence images. Scale bar—10 μm
Potocki et al. Microb Cell Fact (2017) 16:151
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as with purified FliD (Fig. 4a). The highest level of IL-2 was observed in samples corresponding to group immunized with BKH121/FliD spores. IL-10 level, interestingly, was high in the case of cells isolated from animals immunized with BKH121/FliD spores and comparable with cells isolated from naïve mice. In the case of other experimental groups we observed much lower level of this cytokine (Fig. 4b). IL-17A level showed statistically significant (P = 0.0004) increase in supernatants of cells isolates from animals immunized with 168/FliD spores. Some increase was observed in the case of samples corresponding to experimental groups immunized with either purified FliD protein or BKH121/FliD spores, nevertheless the increase was not statistically significant (Fig. 4c). We also noticed statistically significant (P = 0.0002) increase in the level of IFN-γ in supernatant of cells isolated from animals immunized with 168/ FliD spores (Fig. 4d). We were not able to detect IL-4 and IL-6 in any of analyzed samples. Serum IgG response to native FliD in Clostridium difficile
Fig. 3 Antibody production in mice immunized with spore-adsorbed FliD. Groups (n = 6) of BALB/c mice were intranasally immunized with purified FliD, spores of alone (168, BKH121), or FliD-adsorbed spores (168/FliD, BKH121/FliD). a Anti-FliD IgG detected in mice serum at the end of treatment, anti-FliD IgA detected in saponin extracts of b gastrointestinal tracts, and c lungs of immunized animals. Antibody levels are expressed as endpoint titers. Error bars represent standard deviations. Statistical analysis performed as described in the “Meth‑ ods” section. *P
Microbial Cell Factories Open Access
RESEARCH
The combination of recombinant and non‑recombinant Bacillus subtilis spore display technology for presentation of antigen and adjuvant on single spore Wojciech Potocki1, Alessandro Negri1,4, Grażyna Peszyńska‑Sularz2, Krzysztof Hinc3, Michał Obuchowski3 and Adam Iwanicki3*
Abstract Background: Bacillus subtilis spores can be used for presentation of heterologous proteins. Two main approaches have been developed, the recombinant one, requiring modification of bacterial genome to express a protein of inter‑ est as a fusion with spore-coat protein, and non-recombinant, based on the adsorption of a heterologous protein onto the spore. So far only single proteins have been displayed on the spore surface. Results: We have used a combined approach to adsorb and display FliD protein of Clostridium difficile on the surface of recombinant IL-2-presenting spores. Such spores presented FliD protein with efficiency comparable to FliDadsorbed spores produced by wild-type 168 strain and elicited FliD-specific immune response in intranasally immu‑ nized mice. Conclusions: Our results indicate that such dual display technology may be useful in creation of spores simultane‑ ously presenting adjuvant and antigen molecules. Regarding the characteristics of elicited immune response it seems plausible that such recombinant IL-2-presenting spores with adsorbed FliD protein might be an interesting candidate for vaccine against infections with Clostridium difficile. Keywords: Bacillus subtilis, Recombinant spores, Adsorption, Mucosal immunization, FliD, Clostridium difficile Background Bacillus subtilis spores are dormant forms of this microorganism, well known for their resistance to harsh environmental conditions. Their properties, combined with the easiness of genetic modification of this bacterium, make them a very convenient platform for presentation of heterologous proteins (reviewed in [1]). An interesting application of this technology is preparation of sporebased mucosal vaccines [2]. Use of such system enabled elicitation of protective immunity against infections with
*Correspondence: [email protected] 3 Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdańsk, Gdańsk, Poland Full list of author information is available at the end of the article
such pathogens as C. perfringens [3], C. tetani [4], C. difficile [5], or rotavirus [6]. There are two main approaches to display antigens on the surface of spores. Recombinant approach, developed as the first, is based on modification of bacterial genome in a way to express a protein of interest in fusion with one of spore coat proteins. As a result, a fusion protein is expressed in the cell and incorporated into the forming spore coat. Such method enables relatively simple construction of spores presenting heterologous proteins using basic methods of molecular biology. Non-recombinant approach is based on use of unmodified spores and adsorption of a purified protein of interest [7]. This method enables presentation of larger amounts of protein as compared to recombinant spores. Moreover, it
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Potocki et al. Microb Cell Fact (2017) 16:151
does not lead to creation and use of GMO and thus can be much easier applied for animal or human use. Spore-based vaccines can stimulate both systemic and localized immune responses with balanced Th1/Th2 polarization [8]. Although unmodified B. subtilis spores can be used as mucosal adjuvants in some applications [9] they can also be engineered to display immunomodulatory molecules and be administered as adjuvants in formulations with antigen-presenting spores [10]. It is worth noting, that no successful display of more than one recombinant protein on the surface of a single spore has been described in the literature. Such construct displaying molecules of both, an antigen and adjuvant, would be of special interest. Following this line of reasoning, in this study we decided to use previously described recombinant spores presenting human IL-2 [10] and apply non-recombinant adsorption technique to display on their surface FliD flagellar cap protein of Clostridium difficile. The choice of antigen was not random, since Clostridium difficile is a well-known pathogen responsible for antibioticassociated diarrheas and pseudomembranous colitis. Moreover, FliD protein possesses strong immunogenic properties [11, 12]. The results of performed immunization experiments suggest that such combined approach is promising and could be used for preparation of efficient spore-based formulations able to elicit antigen-specific immune responses with polarization driven by adjuvant presented on the surface of the same spore.
Page 2 of 11
Results Non‑recombinant display of FliD protein
Our idea was to present on spore surface both, an adjuvant and antigen, therefore we decided to use recombinant spores produced by BKH121 strain, which display human IL-2 as fusion with CotB protein joined by a peptide linker. Single spore of this strain presents an average number of 9.5 × 104 IL-2 molecules [10]. Since robust display of proteins on spore surface can be achieved using adsorption method [13–15] we also decided to apply this approach to present FliD protein of C. difficile. The entire FliD was overproduced in E. coli, purified and used for adsorption on surface of spores produced by the wild-type strain 168 and the recombinant strain BKH121. We estimated amounts of spore-adsorbed FliD by measuring unbound protein in adsorption mixture and all subsequent washes (Additional file 1: Tables S1, S2). In the case of spores of both strains the results were comparable and reached levels of 4.13 × 104 FliD molecules/ spore and 3.66 × 104 FliD molecules/spore for 168 and BKH121, respectively. Western blotting analysis of spore coat extracts showed presence of 56 kDa band which reacted with anti-FliD antibodies (Fig. 1a) indicating presence of this protein adsorbed on the spores. To visualize surface exposition of adsorbed protein we made use of immunofluorescence microscopy. Upon incubation with anti-FliD primary antibodies and anti-mouse IgG–Cy3 we observed in microscope fluorescent signal around spores of both strains which were subjected to FliD adsorption
Fig. 1 a Western blotting analysis of spore adsorption with purified FliD protein. Upon adsorption with purified FliD spore surface proteins were extracted by SDS-DTT treatment, fractionated on SDS-PAGE and analyzed by Western blotting. Spore coat extracts were prepared with spores of wild-type strain 168 and recombinant BKH121 alone or with adsorption with FliD (indicated with − and +). Purified FliD (1 μg) was used as positive control. b The same volumes of spore coat extracts were simultaneously loaded onto the second SDS-PAGE gel and analyzed by Western blotting using anti-CotZ antibodies to verify the equality of protein loading. The calculated molecular mass of CotZ is 16.4 kDa
Potocki et al. Microb Cell Fact (2017) 16:151
procedure (Fig. 2). In both experiments signal observed for 168/FliD spores was stronger than in the case of BKH121/FliD spores. These observations are in agreement with increased amount of FliD adsorbed on spores of the wild-type strain 168 in comparison to spores produced by BKH121. This suggests that the surface of recombinant spores used in the study possess diminished adsorption capability. Intranasal immunization of animal leads to development of FliD‑specific immune response
Having verified the display of FliD protein on surface of spores, we used them for mucosal immunizations of mice applying two different administration routes: oral and intranasal. First immunization scheme (oral) consisted of a total of nine doses of 1 × 1010 spores delivering 39.2 μg of protein for FliD-adsorbed spores produced by 168 strain and 34.8 μg of protein for spores produced by BKH121 strain. In the case of this administration route we failed to observe eliciting FliD-specific immune response as assessed by production of antigen-specific antibodies in immunized animals (data not shown). Intranasal immunizations were performed by administration a total of eight doses of 5 × 109 spores delivering 19.6 μg (168/FliD) and 17.4 μg (BKH121/FliD) of protein per dose. In the case of this administration route we detected FliD-specific IgG antibodies in sera the immunized
Page 3 of 11
animals. Increased levels of FliD-specific IgG antibodies were detected in sera of animals immunized with purified FliD protein and BKH121/FliD spores. The highest titers were observed for animals immunized with IL-2 presenting spores adsorbed with FliD protein (BKH121/ FliD) (Fig. 3a). We also collected entire gastrointestinal tracts (GITs) and lungs of mice used in this experiment, and performed saponin extraction to assess levels of FliD-specific IgA antibodies in the analyzed material. We detected these antibodies in extracts prepared with both, lungs and GITs, with the highest levels observed in the case of mice immunized with FliD-adsorbed spores produced by the BKH121 (GIT extracts, Fig. 3b) and comparable levels in the case of both, 168/FliD and BKH121/ FliD spores (lungs extracts, Fig. 3c). Immunization of mice with purified FliD protein did not significantly increase levels of IgA in both, GITs and lungs. Characterization of elicited immune response
To characterize the polarization of immune response elicited by FliD-adsorbed spores we isolated spleens of immunized animals and used these organs to obtain splenocytes. Cells were stimulated with purified FliD protein and cell supernatants containing secreted cytokines were analyzed by flow cytometer using Cytometric Bead Array (CBA). With this method, we were able to measure levels of IL-2, IL-4, IL-6, IL-10, IL-17A,
Fig. 2 Spore surface display of adsorbed FliD protein as assessed by immunofluorescence microscopy. Purified, free spores of wild-type strain 168 and recombinant BKH121 alone and with adsorption of FliD protein (168/FliD, BKH121/FliD) were visualized by phase contrast and immunofluores‑ cence microscopy. The spores were incubated with mouse anti-FliD antibodies, followed by anti-mouse IgG–Cy3 conjugates. The same exposure time was used for all immunofluorescence images. Scale bar—10 μm
Potocki et al. Microb Cell Fact (2017) 16:151
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as with purified FliD (Fig. 4a). The highest level of IL-2 was observed in samples corresponding to group immunized with BKH121/FliD spores. IL-10 level, interestingly, was high in the case of cells isolated from animals immunized with BKH121/FliD spores and comparable with cells isolated from naïve mice. In the case of other experimental groups we observed much lower level of this cytokine (Fig. 4b). IL-17A level showed statistically significant (P = 0.0004) increase in supernatants of cells isolates from animals immunized with 168/FliD spores. Some increase was observed in the case of samples corresponding to experimental groups immunized with either purified FliD protein or BKH121/FliD spores, nevertheless the increase was not statistically significant (Fig. 4c). We also noticed statistically significant (P = 0.0002) increase in the level of IFN-γ in supernatant of cells isolated from animals immunized with 168/ FliD spores (Fig. 4d). We were not able to detect IL-4 and IL-6 in any of analyzed samples. Serum IgG response to native FliD in Clostridium difficile
Fig. 3 Antibody production in mice immunized with spore-adsorbed FliD. Groups (n = 6) of BALB/c mice were intranasally immunized with purified FliD, spores of alone (168, BKH121), or FliD-adsorbed spores (168/FliD, BKH121/FliD). a Anti-FliD IgG detected in mice serum at the end of treatment, anti-FliD IgA detected in saponin extracts of b gastrointestinal tracts, and c lungs of immunized animals. Antibody levels are expressed as endpoint titers. Error bars represent standard deviations. Statistical analysis performed as described in the “Meth‑ ods” section. *P
