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Surface display systems have been developed to express target molecules on almost all types of biological entities from viruses to mammalian cells and on a variety of synthetic particles. Various approaches have been developed to achieve the display of many different target molecules, aiming at several technological and biomedical applications. Screening of libraries, delivery of drugs or antigens, bio-catalysis, sensing of pollutants and bioremediation are commonly considered as fields of potential application for surface display systems. In this review, the non-recombinant approach to display antigens and enzymes on the surface of bacterial spores is discussed. Examples of molecules displayed on the spore surface and their potential applications are summarized and a mechanism of display is proposed. Unlabelled Image • Bacterial spores can be used to display heterologous proteins. • Spore-display can be recombinant and non-recombinant. • The non-recombinant spore display system (Spore-adsorption) is safe for human and field use. • Adsorbed proteins are tightly bound to and protected by the spore. • Heterologous proteins infiltrate through semi-permeable pores present on the spore surface. [ABSTRACT FROM AUTHOR]

Halotolerant (HT) bacteria are a group of microorganisms able to thrive in environments with relatively high salt concentrations. HT-microorganisms with plant growth-promoting (PGP) characteristics have been proposed to increase plant tolerance in salty soil. Here, we evaluated the PGP properties at increasing NaCl concentrations of HT-Bacillus strains, previously shown to have beneficial effects under physiological conditions. Most of the isolated showed indole acetic acid and ammonia production and were able to solubilize phosphate and suppress the proliferation of the phytopathogenic fungus Macrophomina phaseolina 2013–1 at high salt concentrations. One of the selected strains, Bacillus amyloliquefaciens RHF6, which retained its beneficial properties up to 400 mM NaCl in vitro , was tested on the legume model plant Lotus japonicus cv Gifu under salt stress. The inoculation with RHF6 significantly improved the survival of plants under high salinity conditions, as reflected in seedling root and shoot growth and total fresh weight (increased by 40%) when compared with non-inoculated plants. The ability of RHF6 to induce a plant antioxidant response, secrete the osmoprotectant proline and reduce ethylene level via the enzymatic ACC deaminase activity indicated this strain as a potentially helpful PGPB for the treatment of degraded soils. [ABSTRACT FROM AUTHOR]

• Shouchella clausii SF174 has a beneficial effect in the context of high fructose diet. • Shouchella clausii SF174 vegetative cells and spores are able to digest part of the fructose at the ileum level. • Both Shouchella clausii SF174 vegetative cells and spores are the major players of the observed probiotic beneficial effects. • Future possibilities to use Shouchella clausii SF174 in most commercial products. The oral administration of probiotics is nowadays recognized as a strategy to treat or prevent the consequences of unhealthy dietary habits. Here we analyze and compare the effects of the oral administration of vegetative cells or spores of Shouchella clausii SF174 in counteracting gut dysfunctions induced by 6 weeks of high fructose intake in a rat model. Gut microbiota composition, tight junction proteins, markers of inflammation and redox homeostasis were evaluated in ileum and colon in rats fed fructose rich diet and supplemented with cells or spores of Shouchella clausii SF174. Our results show that both spores and cells of SF174 were effective in preventing the fructose-induced metabolic damage to the gut, namely establishment of "leaky gut", inflammation and oxidative damage, thus preserving gut function. Our results also suggest that vegetative cells and germination-derived cells metabolize part of the ingested fructose at the ileum level. [Display omitted] [ABSTRACT FROM AUTHOR]

Bacteria classified as Bacillus cereus sensu stricto cause two different type of gastrointestinal diseases associated with food poisoning. Outbreaks of this opportunistic pathogen are generally due to the resistance of its spores to heat, pH and desiccation that makes hard their complete inactivation from food products. B. cereus is commonly isolated from a variety of environments, including intestinal samples of infected and healthy people. We report the genomic and physiological characterization of MV19, a human intestinal strain closely related (ANI value of 98.81%) to the reference strain B. cereus ATCC 14579. MV19 cells were able to grow in a range of temperatures between 20 and 44 °C. At the optimal temperature the sporulation process was rapidly induced and mature spores efficiently released, however these appeared structurally and morphologically defective. At the sub-optimal growth temperature of 25 °C sporulation was slow and less efficient but a high total number of fully functional spores was produced. These results indicate that the reduced rapidity and efficiency of sporulation at 25 °C are compensated by a high quality and quantity of released spores, suggesting the relevance of different performances at different growth conditions for the adaptation of this bacterium to diverse environmental niches. [ABSTRACT FROM AUTHOR]

To investigate whether short term fructose-rich diet induces changes in the gut microbiota as well as in skeletal muscle and adipose tissue physiology and verify whether they persist even after fructose withdrawal, young rats of 30 d of age were fed for 3 weeks a fructose-rich or control diet. At the end of the 3-weeks period, half of the rats from each group were maintained for further 3 weeks on a control diet. Metagenomic analysis of gut microbiota and short chain fatty acids levels (faeces and plasma) were investigated. Insulin response was evaluated at the whole-body level and both in skeletal muscle and epididymal adipose tissue, together with skeletal muscle mitochondrial function, oxidative stress, and lipid composition. In parallel, morphology and physiological status of epididymal adipose tissue was also evaluated. Reshaping of gut microbiota and increased content of short chain fatty acids was elicited by the fructose diet and abolished by switching back to control diet. On the other hand, most metabolic changes elicited by fructose-rich diet in skeletal muscle and epididymal adipose tissue persisted after switching to control diet. Increased dietary fructose intake even on a short-time basis elicits persistent changes in the physiology of metabolically relevant tissues, such as adipose tissue and skeletal muscle, through mechanisms that go well beyond the reshaping of gut microbiota. This picture delineates a harmful situation, in particular for the young populations, posed at risk of metabolic modifications that may persist in their adulthood. [Display omitted] [ABSTRACT FROM AUTHOR]

The 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. [ABSTRACT FROM AUTHOR]

Abstract: We report the use of recombinant spores of Bacillus subtilis as a potential bioremediation tool for adsorption of nickel ions. The spore surface protein CotB, previously used for the display of heterologous antigens, was engineered to express eighteen histidine residues within the spore coat. Wild type and recombinant spores were then analyzed to assess their efficiency in adsorbing nickel ions, and the latter proved to be significantly more efficient than wild type spores in metal-binding. The quantities of spores used in the adsorption reaction significantly affected nickel binding, while other factors such as pH and temperature did not show relevant effects. In addition, simple washing procedures were used to partially release spore-bound nickel ions by wild type and recombinant spores. The efficiency of nickel binding, together with the simple purification procedure, the high robustness and safety of B. subtilis spores and the possibility of recovering bound nickel, makes the recombinant spore a new and potentially powerful tool for the treatment of contaminated ecosystems. [ABSTRACT FROM AUTHOR]

Abstract: In order to perform selective isolation of bacteria tightly bound to the human gut, ileal biopsies of healthy volunteers were treated to wash out the mucus layer and loosely bound bacterial cells. Rod-shaped anaerobic bacteria that had remained attached to the epithelial cells were isolated and identified at the species level. One isolate was identified as belonging to the Bifidobacterium breve species, while all the others were lactobacilli of only two species, Lactobacillus mucosae and Lactobacillus gasseri. Members of these species were found previously in intestinal samples, but their predominance among bacteria strictly associated with the epithelium was not suspected before and suggests that these species may represent a specific subpopulation of tissue-bound bacteria. Physiological analysis indicated that all isolates were able to produce antimicrobials, grow and form biofilm in simulated intestinal fluid after exposure to gastric conditions. Some isolates were able to degrade mucin while none showed cytotoxicity in vitro on HT29 cells. The tight association of the strains isolated with ileal epithelial cells is presumably indicative of a direct interaction with the host cells. For this reason and for the absence of cytotoxicity in vitro, those isolates can be proposed as potential probiotic strains for human use. [Copyright &y& Elsevier]

Abstract: Our understanding of the genetics and physiology of the spore-forming genus Bacillus is remarkable. On the other hand, though, where these Gram-positive bacteria live and grow is far from clear. The soil, once considered their habitat, may simply serve as a reservoir. A growing number of studies show that Bacillus spores can be found in the intestinal tracts of animals, raising the question of whether this could be where they live and grow. In this study, we have conducted the first evaluation of Bacillus spore formers in soil and in human faeces. Our aim is simply to determine the abundance of aerobic spore-formers. Our results show that soil carries approximately ∼106 spores/g while human faeces an average of up to 104 spores/g. The numbers of spores found in faeces, we reason, is too high to be accounted for principally by ingestion of food contaminated with spores from soil. This provides further evidence that Bacillus spore formers may have adapted to survival within the intestinal tract of insects and other animals that ingest them; if so they may well be hitherto undiscovered gut commensals. [Copyright &y& Elsevier]

Abstract: Probiotic strains have been reported to exert immunomodulatory activities in the gut-associated lymphoid tissue. In this study we explored the effect of Lactobacillus casei in transgenic mice expressing the human DQ8 heterodimer, a HLA molecule linked to Celiac Disease (CD). DQ8 mice, mucosally immunized with the gluten component gliadin, mounted an intestinal Th1-like response as observed in CD, without developing enteropathy. Co-administration of L. casei in sensitized mice specifically enhanced the gliadin-specific response mediated by CD4+ T cells. Notably, both a strong increase of the gliadin-specific IFNγ expression and a pro-inflammatory polarization of the cytokine milieu in the small intestinal mucosa were associated to the presence of the probiotic strain. However, this condition did not bring on any mucosal alteration. These findings suggest that the gliadin-specific enteropathy is not merely related to the HLA DQ8-restricted massive production of IFNγ, but additional parameters are involved. Moreover, our data imply that the intrinsic adjuvanticity of L. casei can be exploited to further enhance both mucosal and systemic T cell-mediated responses. [Copyright &y& Elsevier]

Abstract: A T helper epitope was expressed in three innovative delivery vehicles recently developed in our laboratories and based respectively, on the filamentous bacteriophage fd, the E2 protein from the PDH complex of Bacillus stearothermophilus and the protein CotC of Bacillus subtilis spores. Studies of antigenicity and immunogenicity were performed by using a specific T cell hybridoma and by priming mononuclear cells isolated from the venous blood of human donors. The results indicate that the E2 system is the best suited for inducing a specific immune response towards a CD4 T cell epitope. Importantly, TCR clonal analysis demonstrated the persistence over years of a previously described antigen specific clonotype and its presence correlates with the immunogenic strength of the antigen delivery system. [Copyright &y& Elsevier]

Abstract: Bacillus subtilis spores displaying the tetanus toxin fragment C (TTFC) on their surface have been previously shown to induce the production of specific IgG and secretory IgA in mice immunized through the oral or nasal route. Aim of this study was to analyze whether these spores were also able to induce cellular immunity, and whether such immune response was dependent on spore germination in the animal gastro-intestinal tract (GIT). We first developed a germination defective strain of B. subtilis unable to produce viable cells inside the mouse GIT. Germination-defective and congenic wild-type spores both expressing TTFC on their surface were then used to orally immunize Balb/C mice. Both types of spores induced spleen and mesenteric lymph nodes cell proliferation as well as production of IFNγ but not of IL-4 and IL-10 in both districts. Our results indicate that recombinant spores preferentially induce a strong cell-mediated immune response with a Th1 phenotype, independently from their ability to germinate in the GIT. [Copyright &y& Elsevier]

Abstract: The present work was aimed at investigating whether Bacillus subtilis spores, widely used in probiotic as well as pharmaceutical preparations for mild gastrointestinal disorders, can suppress enteric infections. To address this issue, we developed a mouse model of infection using the mouse enteropathogen Citrobacter rodentium, a member of a family of human and animal pathogens which includes the clinically significant enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli strains. This group of pathogens causes transmissible colonic hyperplasia by using attaching and effacing (A/E) lesions to colonize the host colon. Because of its similarities to human enteropathogens, C. rodentium is now widely used as an in vivo model for gastrointestinal infections. Swiss NIH mice were orally administered B. subtilis spores one day before infection with C. rodentium. Mice were sacrificed on day 15 after infection, and distal colon, liver and mesenteric lymph nodes were removed for bacteria counts, morphology, immunohistology and IFNγ mRNA analysis. We observed that spore predosing was effective in significantly decreasing infection and enteropathy in suckling mice infected with a dose of C. rodentium sufficient to cause colon colonization, crypt hyperplasia and high mortality rates. Moreover, in mice predosed with spores, the number of CD4+ cells and IFNγ transcript levels remained high. These results thus indicate that our newly established model of C. rodentium infection is a suitable system for analyzing the effects of probiotic bacteria on enteroinfections and that B. subtilis spores are efficient at reducing C. rodentium infection in mice, leaving unaltered the immune response against the pathogen. [Copyright &y& Elsevier]

Abstract: In a search for bacteria having putative probiotic activity, we screened a collection of food-isolated microorganisms for the ability to survive at low pH in the presence of bile salts and for the production of antimicrobial compounds active against a number of animal pathogens. Among these, we found a strain that we classified as a member of Lactobacillus fermentum sp., and we further investigated its features. This organism was able to adhere to human enterocyte-like (Caco-2) cells with high efficiency as compared to that of a well known indicator strain. Chromatographic analysis indicated that at least two small (less than 3 kDa) factors were involved in mediating the in vitro interaction of L. fermentum with Caco-2 cells. Adhesion activity could be abolished by mild treatment of the bacterial cells in buffer and rescued by incubating them with either the same buffer after its use in the treatment or with chromatographic fractions containing each of the two factors, which indicated that these factors were loosely associated with the cell wall and that each of them was sufficient to warrant the adhesiveness of L. fermentum to Caco-2 cells. These data are suggestive of a novel mechanism of bacterial adhesion to epithelial cells. [Copyright &y& Elsevier]

We report the use of CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of two heterologous antigens on the spore coat. Recombinant spores expressing tetanus toxin fragment C (TTFC) of Clostridium tetani or the B subunit of the heat-labile toxin of Escherichia coli (LTB) were used for oral dosing and shown to generate specific systemic and mucosal immune responses in a murine model. This report, expanding the previously described expression of TTFC on the spore surface by fusion to CotB [J Bacteriol 183 (2001) 6294] and its use for oral vaccination [Infect Immun 71 (2003) 2810] shows that different antigens can be successfully presented on the spore coat and supports the use of the spore as an efficient vehicle for mucosal immunisation. [Copyright &y& Elsevier]

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones. [Copyright &y& Elsevier]