1,721,020 research outputs found
Spore Surface Display
No full textA variety of bioactive peptides and proteins have been successfully displayed on the surface of recombinant spores of Bacillus subtilis and other sporeformers. In most cases spore
display has been achieved by stably anchoring the foreign molecules to endogenous surface
proteins or part of them. Recombinant spores have been then proposed for a large number of potential applications, ranging from oral vaccine vehicles to bioremediation tools, and including biocatalysts, probiotics for animal or human use as well as the generation and screening of mutagenesis libraries. In addition, a non-recombinant approach has been
recently developed and proposed to adsorb antigens and enzymes on the spore surface. This non-recombinant approach appears particularly well suited for applications involving the delivery of active molecules to human or animal mucosal surfaces. Both the recombinant and non-recombinant spore display systems have a number of advantages over cell- or phage-based systems. The stability, safety and amenability to laboratory manipulations of
spores of several bacterial species, together with the lack of some constrains limiting the use of other systems, make the spore a highly efficient platform to display heterologous protein
CotE binds to CotC and CotU and mediates their interaction during spore coat formation in Bacillus subtilis
No full textCotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that
surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer
coat components, but such interactions were hypothesized from genetic experiment results and have never been
directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention
on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We
report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other
coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to
allow formation of the CotC-CotU heterodimer in a heterologous host
CotG-Like modular proteins are common among spore-forming bacilli
No full textCotG is an abundant protein initially identified as an outer component of the Bacillus subtilis spore coat. It has an unusual structure characterized by several repeats of positively charged amino acids that are probably the outcome of multiple rounds of gene elongation events in an ancestral minigene. CotG is not highly conserved, and its orthologues are present in only two Bacillus and two Geobacillus species. In B. subtilis, CotG is the target of extensive phosphorylation by a still unidentified enzyme and has a role in the assembly of some outer coat proteins. We report now that most spore-forming bacilli contain a protein not homologous to CotG of B. subtilis but sharing a central “modular” region defined by a pronounced positive charge and randomly coiled tandem repeats. Conservation of the structural features in most spore-forming bacilli suggests a relevant role for the CotG-like protein family in the structure and function of the bacterial endospore. To expand our knowledge on the role of CotG, we dissected the B. subtilis protein by constructing deletion mutants that express specific regions of the protein and observed that they have different roles in the assembly of other coat proteins and in spore germination.
IMPORTANCE CotG of B. subtilis is not highly conserved in the Bacillus genus; however, a CotG-like protein with a modular structure and chemical features similar to those of CotG is common in spore-forming bacilli, at least when CotH is also present. The conservation of CotG-like features when CotH is present suggests that the two proteins act together and may have a relevant role in the structure and function of the bacterial endospore. Dissection of the modular composition of CotG of B. subtilis by constructing mutants that express only some of the modules has allowed a first characterization of CotG modules and will be the basis for a more detailed functional analysis
cotH over-expression by-passes the CotE requirement for assembly of outer coat components of Bacillus subtilis
No full textThe spore surface of intestinal isolates of Bacillus subtilis
No full textBacillus subtilis has been used for over 50 years as a model organism for biochemistry, genetic, molecular biology and cell biology studies. More recently, its spore has been proposed as a platform to display heterologous proteins and as a vehicle for mucosal vaccination. We characterize here the spore surface of four human intestinal strains of B. subtilis, previously identified as able to grow anaerobically and form biofilm. These properties, lost in laboratory strains, are relevant for the colonization of human mucosal sites and likely to improve the efficiency of strains to be used for mucosal delivery. Our characterization is an essential preliminary step for the development of these intestinal strains as display systems and has indicated that spores of at least one of them are more efficient than the laboratory strain for the non-recombinant display of two model heterologous proteins
Mucosal immunity induced by gliadin-presenting spores of Bacillus subtilis in HLA-DQ8-transgenic mice
No full textThe induction of mucosal immunity requires efficient antigen delivery and adjuvant systems. Probiotic bacterial strains are considered to be very promising tools to address both of these needs. In particular, Bacillus subtilis spores are currently under investigation as a long-lived, protease-resistant adjuvant system for different antigens. Furthermore, a non-recombinant approach has been developed based on the stable adsorption of antigen on the spore surface. In the present study, we explored this strategy as a means of modulating the immune response to wheat gliadin, the triggering agent of celiac disease (CD), an enteropathy driven by inflammatory CD4+ T cells. Gliadin adsorption was tested on untreated or autoclaved wild-type (wt) and mutant (cotH or cotE) spores. We found that gliadin was stably and maximally adsorbed by autoclaved wt spores. We then tested the immune properties of the spore-adsorbed gliadin in HLA-DQ8-transgenic mice, which express one of the two HLA heterodimers associated with CD. In vitro, spore-adsorbed gliadin was efficiently taken up by mouse dendritic cells (DCs). Interestingly, gliadin-pulsed DCs efficiently stimulated splenic CD4+ T cells from mice immunised with spore-adsorbed gliadin. Nasal pre-dosing with spore-adsorbed gliadin failed to down-regulate the ongoing cellular response in gliadin-sensitised DQ8 mice. Notably, naïve mice inoculated intranasally with multiple doses of spore-adsorbed gliadin developed an intestinal antigen-specific CD4+ T cell-mediated response. In conclusion, our data highlight the ability of spore-adsorbed gliadin to elicit a T-cell response in the gut that could be exploitable for developing immune strategies in CD
The triterpenoid curcumene mediates the relative hydrophilicity of Bacillus subtilis spores
No full text: Spores of Bacillus subtilis are surrounded and protected by the coat and the crust, multi-layered structures mainly made of proteins and polysaccharides. These polysaccharides are covalently linked to some of the coat and crust proteins and influence some spore properties, such as surface adhesion and hydrophilicity. This study reports that a mutant strain lacking the spsA-L operon, encoding 11 enzymes involved in the synthesis of spore surface polysaccharides, produced spores exposing on their surface hydrophobic molecules that were responsible for the drastic reduction of hydrophilicity of the mutant spores. Biochemical and genetic data support the identification of the C35-terpenoid curcumene, a precursor of the spore-associated lipid sporulene, as the highly hydrophobic molecule present on the surface of mutant spores.IMPORTANCEBacterial spores are the most resistant cell forms on Earth. The metabolically quiescent spores withstand conditions that would be lethal for other cells, maintaining the capacity to sense the environment and respond to the presence of favorable conditions by germinating. Such remarkable resistance is also due to the complex layers that surround the spore cytoplasm and protect it against damaging factors. Altogether, the spore surface layers form a complex cell structure composed of proteins, polysaccharides, and, as highlighted by this study, also of lipids. Understanding the complexity of the spore surface and the specific molecules involved in its structure is an essential step for unraveling the mechanisms underlying the spore's resistance to environmental assaults
Structural studies on the O-specific polysaccharide of the lipopolysaccharide from Pseudomonas donghuensis strain SVBP6, with antifungal activity against the phytopathogenic fungus Macrophomina phaseolina
No full textAn O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide (LPS) of Pseudomonas donghuensis SVBP6, a bacterium with a broad-spectrum antifungal activity in vitro, particularly that against Macrophomina phaseolina. This latter is one of the most virulent and dangerous pathogens of plants, including soybean which is an economically important crop in Argentina today. The OPS was studied by sugar analysis and spectroscopy (1D and 2D 1H and 13C NMR) showing the following trisaccharide repeating unit: →6)-ɑ-D-ManpNAc-(1 → 3)-β-L-Rhap-(1 → 4)-β-D-Glcp-(1→. The crude LPS, the purified LPS and the O-chain were assayed for their antifungal activity against M. phaseolina at 25, 50, 100, and 200 μg plug−1. The results showed that the crude LPS best inhibition was at 200 μg plug−1, able to inhibit the fungus growth by about 45%, while purified LPS and the corresponding OPS, in the same condition, reduced fungus growth by 65%, and 75%, respectively. Furthermore, the purified LPS and OPS significantly reduced the growth of M. phaseolina already at 100 μg plug−1 compared to the crude LPS. The structure of the O-chain is unique among the bacterial LPS and this is the first time that both the antifungal activity of a bacterial LPS and its corresponding O-chain were described.Fil: Zdorovenko, Evelina L.. Università degli Studi di Napoli Federico II; ItaliaFil: Dmitrenok, Andrey S.. Università degli Studi di Napoli Federico II; ItaliaFil: Masi, Marco. Università degli Studi di Napoli Federico II; ItaliaFil: Castaldi, Stefany. Università degli Studi di Napoli Federico II; ItaliaFil: Muzio, Federico Matías. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Isticato, Rachele. Università degli Studi di Napoli Federico II; ItaliaFil: Valverde, Claudio Fabián. Universidad Nacional de Quilmes; ArgentinaFil: Knirel, Yuriy A.. Università degli Studi di Napoli Federico II; ItaliaFil: Evidente, Antonio. Università degli Studi di Napoli Federico II; Itali
The Bacterial Spore as a Mucosal Vaccine Delivery System
Full text linkThe development of efficient mucosal vaccines is strongly dependent on the use of appropriate vectors. Various biological systems or synthetic nanoparticles have been proposed to display and deliver antigens to mucosal surfaces. The Bacillus spore, a metabolically quiescent and extremely resistant cell, has also been proposed as a mucosal vaccine delivery system and shown able to conjugate the advantages of live and synthetic systems. Several antigens have been displayed on the spore by either recombinant or non-recombinant approaches, and antigen-specific immune responses have been observed in animals immunized by the oral or nasal route. Here we review the use of the bacterial spore as a mucosal vaccine vehicle focusing on the advantages and drawbacks of using the spore and of the recombinant vs. non-recombinant approach to display antigens on the spore surface. An overview of the immune responses induced by antigen-displaying spores so far tested in animals is presented and discussed
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