Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores

Negri, Alessandro; Potocki, Wojciech; Iwanicki, Adam; Obuchowski, Michał; Hinc, Krzysztof

doi:10.1099/jmm.0.057372-0

Cited by 43 publications

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References 30 publications

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“…Recombinant spores expressing bacterial antigens elicited specific immune responses and provided protection following mucosal immunization of mice (Mauriello et al, 2004;Negri et al, 2013;Zhou et al, 2008a). Here we reported the first application, we believe, of spores expressing the H. pylori urease B antigen as a vaccine candidate.…”

Section: Discussionmentioning

confidence: 99%

Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores

Zhou

Gong

et al. 2015

Journal of Medical Microbiology

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Helicobacter pylori infection is a major risk factor for chronic gastritis, digestive ulcers, gastric adenocarcinoma and lymphoma. Due to the decreasing efficacy of anti-H. pylori antibiotic therapy in clinical practice, there is renewed interest in the development of anti-H. pylori vaccines. Bacillus subtilis is non-pathogenic and can produce endospores, which can survive under extreme conditions. These features make the B. subtilis spore an ideal vehicle for delivery of heterologous antigens to extreme environments such as the gastrointestinal tract. In this study, we displayed H. pylori urease B protein on the B. subtilis spore coat using the spore coat protein CotC as a fusion partner. Western blot analyses were used to verify urease B surface expression on spores. Recombinant spores displaying the urease B antigen were used for oral immunization and were shown to generate humoral response in mice. Urease B-specific secretory IgA in faeces and IgG in serum reached significant levels 2 weeks after oral dosing. In addition, oral immunization of recombinant urease B spores induced a significant reduction (84 %) in the stomach bacterial load (0.25±0.13¾10 6 c.f.u.) compared to that in the non-recombinant spores treated group (1.56±0.3¾10 6 c.f.u.; P,0.01). This report shows that urease B expressed on B. subtilis spores was immunogenic, and oral administration of urease B spores can provide protection against H. pylori infection. INTRODUCTIONHelicobacter pylori is a Gram-negative bacterium that colonizes the stomach in approximately half the world's population. Infection may progress to pathological states, such as peptic ulcer disease and gastric adenocarcinoma, resulting in significant morbidity and mortality worldwide (de Martel et al., 2012;Nakamura et al., 2012;Toller et al., 2011; Uemura et al., 2001; You et al., 2012). Current regimens for treatment of H. pylori infection consist of a proton pump inhibitor (PPI) and antibiotics including amoxicillin, clarithromycin and metronidazole. Despite an eradication rate greater than 80 %, there are some limitations to the PPI-based triple therapy, such as poor patient compliance, emerging antibiotic resistance, frequent reinfection and high cost (Lee et al., 2013;Su et al., 2013). Vaccination would therefore be a suitable alternative or complement to antibiotic treatment to eradicate the bacterium and prevent reinfection (Czinn & Blanchard, 2011).Identification of antigens that can induce effective immune responses is a crucial step in vaccine development. Many protein molecules expressed by H. pylori have been shown to possess immunogenicity, including urease, cytotoxinassociated antigen, neutrophil-activating protein A, H. pylori adhesin A, vacuolating toxin A, catalase and outermembrane protein (Czinn & Blanchard, 2011;Liu et al., 2011).Among these antigens the B subunit of the urease protein (urease B), a 61 kDa protein encoded by a 1.7 kbp gene, is the most promising one. H. pylori produces a large amount of urease, which is essential for the survival and path...

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Section: Discussionmentioning

confidence: 99%

Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores

Zhou

Gong

et al. 2015

Journal of Medical Microbiology

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“…Each pair consists of one version of vector in which recombinant protein is linked directly to Cot protein and the other with short peptide linker (-GGGGS-). The vectors with cotZ and cgeA genes are designed in a way enabling for obtaining fusions with heterologous protein attached to the C-terminus of the coat protein directly or via alpha-helical linker (-GGGEAAAKGGG-) [21]. Such set of vectors gives a flexibility in construction of strains producing recombinant spores.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting plasmid, pKH143, was verified by restriction analysis and nucleotide sequencing. The protein was purified and used for antibody production following method described previously [21]. …”

Section: Methodsmentioning

confidence: 99%

A system of vectors for Bacillus subtilis spore surface display

et al. 2014

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BackgroundBacterial spores have been utilized as platforms for protein display. The best studied display systems are based on Bacillus subtilis spores in which several coat proteins have successfully been used as anchors for heterologous protein. Increasing knowledge about spore coat structure enables selection of new anchor proteins such as CotZ and CgeA. Here we describe a system of vectors for display of proteins on the surface of B. subtilis spores.ResultsWe have designed and constructed a set of 16 vectors for ectopic integration which can be used for spore surface display of heterologous proteins. There is a selection of five coat proteins: CotB, CotC, CotG, CotZ and CgeA which can be used for construction of fusions. Three of these (CotB, CotC and CotG) enable obtaining N-terminal and C-terminal fusions and other two (CotZ and CgeA) are designed to produce C-terminal fusions only. All the vectors enable introduction of an additional peptide linker between anchor and displayed protein to enhance surface display. As a selection marker trophic genes are used. Additionally we describe an example application of presented vector system to display CagA protein of Helicobacter pylori in fusion with CgeA spore coat protein.ConclusionsDescribed system of vectors is a versatile tool for display of heterologous proteins on the surface of B. subtilis spores. Such recombinant spores can be further used as for example biocatalysts or antigen-carriers in vaccine formulations. The lack of antibiotic resistance genes in the system makes such spores an interesting option for applications in which a possible release to the environment can occur.

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“…Samples were fixed directly in the medium as described by Negri A, et al [12]. Briefly, intact spores were incubated overnight at 4°C with GA2B anti-influenza A matrix protein 1 mAb (Pierce), followed by incubation with anti-mouse Cy3 (Jackson Immuno Research) overnight at 4°C.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

Oral Administration of Bacillus subtilis Endospores Displaying Influenza Virus Matrix Protein 1 Elicit Cellular Immune Response in Mice

Łęga¹,

Weiher²,

Nidzworski³

2017

SOJMID

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Open Access Short communicationimmune response targets conserved antigens and can provide cross-immunity against different subtypes [2]. Influenza virusspecific Cytotoxic T Lymphocytes (CTLs) have been shown in animal studies to limit influenza A virus replication and to protect against lethal influenza A virus challenge [3]. In last year's a new live antigen carrier system emerged [4]. Recent studies show that recombinant endospores of Bacillus subtilis can serve as antigen carriers by fusing peptide of interest to spore coat proteins [5]. In our study we constructed Bacillus subtilis strain producing spores presenting influenza M1 protein on their surface. M1 is most abundant protein in influenza virons and it is conserved among various strains. Moreover it has been shown that vaccine based on M1 protein can elicit significant cellular response in vaccinated individuals [6]. Recombinant spores were orally administrated to mice to evaluate the immunogenic properties of constructs. This work indicates that spores can serve as influenza antigen carriers and induce cell-mediated immune response. Moreover co-administration of constructed spores with spores presenting interleukin 2 significantly boosted observed cellmediated response. Materials and Methods Bacterial Strains and TransformationAll strains used in this study are listed in Table 1. All cloning experiments were done using Escherichia coli DH5α [7]. Bacteria were transformed according to the previously described methods: CaCl 2 -induced competence of E. coli cells [8] and transformation of B. subtilis [9]. Construction of Recombinant Bacillus subtilis StrainTo generate genetic fusion, gene cotZ coding for the coat protein was PCR-amplified together with its natural promoter using the B. subtilis 168 chromosomal DNA as a template and AbstractBacterium Bacillus subtilis is a gram-positive bacilli which produce endospores. Being metabolically dormant, spores are resistant to many environmental stressors such as UV radiation, desiccation, heat or freezing. There are evidence that oral or intranasal administration of spores presenting antigens induces a specific, both cellular and humoral immune response which can protect animals from infection. In our study, using a genetic approach we constructed Bacillus subtilis strains producing spores presenting influenza A virus matrix protein 1 (M1) on their surface. M1 protein was fused to spore coat CotZ protein and was stably exposed on the spore surface as demonstrated by the immunostaining of intact, recombinant spores. The immunogenicity of recombinant spores was tested by oral administration in mice. As proved by an IFN-γ ELISPOT assay, constructed spores elicited significant cell-mediated immune response.

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Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores

Cited by 43 publications

References 30 publications

Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores

Expression of Helicobacter pylori urease B on the surface of Bacillus subtilis spores

A system of vectors for Bacillus subtilis spore surface display

Oral Administration of Bacillus subtilis Endospores Displaying Influenza Virus Matrix Protein 1 Elicit Cellular Immune Response in Mice

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