The choice of anchoring protein influences interaction of recombinant Bacillus spores with the immune system

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently there are no technical possibilities to predict, whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study we compared four variants of B. subtilis spores presenting a fragment of FliD protein of Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their phagocytosis and activate both, J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent on its abundance and localization in the spore coat structure.

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SpoVID Guides SafA to the Spore Coat inBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.183.10.3041-3049.2001 ◽

2001 ◽

Vol 183 (10) ◽

pp. 3041-3049 ◽

Cited By ~ 56

Author(s):

Amanda J. Ozin ◽

Craig S. Samford ◽

Adriano O. Henriques ◽

Charles P. Moran

Keyword(s):

Direct Interaction ◽

Spore Coat ◽

Coat Proteins ◽

Yeast Two Hybrid System ◽

Spore Coat Proteins ◽

Basement Layer ◽

Two Hybrid ◽

Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

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Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

The Journal of Cell Biology ◽

10.1083/jcb.116.5.1291 ◽

1992 ◽

Vol 116 (5) ◽

pp. 1291-1300 ◽

Cited By ~ 8

Author(s):

C M West ◽

G W Erdos

Keyword(s):

Flow Cytometry ◽

Coat Protein ◽

Viscous Fluid ◽

Fluorescent Antibody ◽

Fluid Phase ◽

Spore Coat ◽

Coat Proteins ◽

Assembly Process ◽

Spore Coat Proteins ◽

Spore Coat Protein

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

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Analyse ultrastructurale et biochimique de mutants pléiotropes de Bacillus subtilis affectés dans le contrôle de la sporulation

Canadian Journal of Microbiology ◽

10.1139/m84-219 ◽

1984 ◽

Vol 30 (11) ◽

pp. 1367-1376

Author(s):

Joseph Zucca ◽

Serge Renaudin

Keyword(s):

Bacillus Subtilis ◽

Pleiotropic Effect ◽

Spore Coat ◽

Coat Proteins ◽

Extracellular Proteases ◽

Transmission Electron ◽

Mutant Strains ◽

Spore Coat Proteins ◽

Spore Ultrastructure ◽

Spore Coat Protein

Derived from a Bacillus subtilis unstable mutant strains which hyperproduce extracellular proteases were found to contain mutations at loci scoC, scoD, or scoE; these were observed by transmission electron microscopy. In scoC and scoD strains, spores formed overproduced spore coat proteins and the spore ultrastructure was deeply altered. In scoE strains, stage III of the sporulation was blocked. The pleiotropic effect of scoC mutations was either positive or negative. In unstable strains, mutations at the scoC locus stimulated α-amylase and levane sucrase hyperproduction. In stable strains, the same mutations led to an absence of α-amylase synthesis but to a normal levane sucrase production. ScoD mutations only induced α-amylase hyperproduction, while scoE mutations allowed neither levane sucrase production nor spore coat protein synthesis.

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

Journal of Medical Microbiology ◽

10.1099/jmm.0.057372-0 ◽

2013 ◽

Vol 62 (9) ◽

pp. 1379-1385 ◽

Cited By ~ 25

Author(s):

Alessandro Negri ◽

Wojciech Potocki ◽

Adam Iwanicki ◽

Michał Obuchowski ◽

Krzysztof Hinc

Keyword(s):

Bacillus Subtilis ◽

Clostridium Difficile ◽

Spore Coat ◽

Coat Proteins ◽

Infectious Agents ◽

Heterologous Proteins ◽

Oral Vaccines ◽

Dot Blot ◽

Protective Layers ◽

Spore Coat Proteins

The endospores of Bacillus subtilis can serve as a tool for surface presentation of heterologous proteins. The unique properties of the spore protective layers make them perfect vehicles for orally administered vaccines. In this study, we successfully displayed a fragment of Clostridium difficile FliD protein on the surface of B. subtilis spores using the CotB, CotC, CotG and CotZ spore coat proteins. The presence of the fusion proteins in the spore coat was verified by Western blotting and immunofluorescence microscopy. The amount of recombinant proteins was assessed by a dot-blot technique. C. difficile is one of the most common infectious agents in nosocomial infections and is especially associated with antibiotic therapies. FliD is a flagellar cap protein of C. difficile and is known to be one of the immunogenic surface antigens of this bacterium. Therefore, its use in vaccine formulations gives a good perspective for successful immunization with a FliD-based vaccine. The recombinant spores presented here may be good candidates for C. difficile oral vaccines.

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Phosphorylation of spore coat proteins during development of Dictyostelium discoideum

Biochemical Journal ◽

10.1042/bj2120699 ◽

1983 ◽

Vol 212 (3) ◽

pp. 699-703 ◽

Cited By ~ 3

Author(s):

S J Delaney ◽

D G Wilkinson ◽

B D Hames

Keyword(s):

Coat Protein ◽

Dictyostelium Discoideum ◽

Spore Coat ◽

Coat Proteins ◽

Cellular Slime Mould ◽

Slime Mould ◽

Cellular Slime ◽

Spore Coat Proteins ◽

Spore Coat Protein

Immunological evidence is presented which confirms that pp95, one of the major phosphoproteins accumulated in development of the cellular slime mould Dictyostelium discoideum, is identical with spore coat protein SP13. The site of phosphorylation is identified as phosphoserine. The second major phosphorylated component, pp74, corresponds to two co-migrating spore coat proteins known collectively as SP74.

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Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

Molecular and Cellular Biology ◽

10.1128/mcb.4.11.2273-2278.1984 ◽

1984 ◽

Vol 4 (11) ◽

pp. 2273-2278

Author(s):

B C Dowds ◽

W F Loomis

Keyword(s):

Dictyostelium Discoideum ◽

Cdna Clone ◽

Single Gene ◽

Polyclonal Antiserum ◽

Cloning And Expression ◽

Spore Coat ◽

Coat Proteins ◽

Developmentally Regulated ◽

Gene Coding ◽

Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

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