This work was supported by &#34;Finanziamento di Ricerca di Ateneo&#34; to L. Baccigalupi, project title &#34;SP-LAY: Bacterial spores as live platform for proteins display&#34;.

1. Adsorption reaction
<ol>
	<li>Incubate 2, 5, and 10 &#181;g of mRFP with 2 x 109 of B. subtilis wild-type purified spores in 200 &#181;L of binding buffer, 50 mM sodium citrate, pH 4.0 (16.7 mM sodium citrate dihydrate; 33.3 mM citric acid) for 1 h at 25 &#176;C on a rocking shaker (Figure 1).</li>
	<li>Centrifuge the binding mixtures (13,000 x g for 10 min) to fractionate pellets (P2, P5, and P10) and supernatants (S2, S5, and S10). Store the supernatants f...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+probiotics.">Bacillus probiotics.</a> Food Microbiology. 28, 214-220 (2011).</li><li>Baccigalupi, L., Ricca, E., Ghelardi, E., Venema, K., Do Carmo, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-LAB+Probiotics:+Spore+Formers.">Non-LAB Probiotics: Spore Formers.</a> Probiotics and Prebiotics: Current Research and Future Trends. , 93-103 (2014).</li><li>Cutting, S. M., Hong, H. A., Baccigalupi, L., Ricca, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Vaccine+Delivery+by+Recombinant+Spore+Probiotics.">Oral Vaccine Delivery by Recombinant Spore Probiotics.</a> International Reviews of Immunology. 28, 487-505 (2009).</li><li>Lee, S. 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This spore adsorption protocol is very simple and straightforward. The reaction is strictly dependent on the pH of the reaction buffer and the efficiency of adsorption is optimal at acidic pH values (pH 5.0 or lower). At neutral pH conditions, the efficiency of adsorption is low, and at alkaline pH values, adsorption may not occur. Optimal adsorption is obtained using a volume of 200 &#181;L in 1.5 mL tubes (or keeping a similar ratio) on a rocking shaker.
Adsorption is very tight, and washes ...

Display systems are aimed at presenting biologically active molecules on the surface of microorganisms and finding applications in a variety of fields, from industrial to medical and environmental biotechnologies. In addition to phages1,2 and cells of various Gram-negative and -positive species3,4,5,6,7, bacterial spores have also been proposed as display systems by two approaches8,9.
Because of its peculiar structure, namely a dehydrated cytoplasm surrounded by a series of protective layers10, the spore provides several advantages over phage- and cell-based display systems8,9. A first advantage comes from the extreme robustness and stability of spores at conditions that would be deleterious to all other cells10,11. Spore-displayed antigens and enzymes are stable after prolonged storage at room temperature12 and protected from degradation at low pH and high temperatures13. A second advantage of spores is the safety of many spore-forming species. B. subtilis, B. clausii, B. coagulans, and several other species are used worldwide as probiotics and have been on the market for human or animal use for decades14,15. This exceptional safety record is an obvious general requirement for a surface display system and is of particular relevance when the system is intended for human or animal use16. A third, important advantage of a spore-based display system is that it does not have limitations for the size of the molecule that has to be exposed. In phage-based systems, a large heterologous protein may affect the structure of the capsid, while in cell-based systems, it may affect the structure of the membrane or may limit/impair the membrane translocation step17. The protective layers surrounding the spore are composed of more than 70 different proteins10 and are flexible enough to accept large foreign proteins without any evident structural defect or functional impairment8. In addition, with both spore-based display systems, the membrane translocation of the heterologous protein is not required8,9. Indeed, heterologous proteins are either produced in the mother cell cytoplasm and assembled on the spore that is forming in the same cytoplasm or adsorbed on the mature spore8,9.
Spore display was initially obtained by developing a genetic system to engineer the spore surface18. This genetic system was based on i) the construction of a gene fusion between the gene coding for a spore coat protein (used as a carrier) and the gene coding for the protein to be displayed - the presence of the transcriptional and translational signals of the endogenous gene will control the expression of the fusion, and ii) integration of the chimeric gene on the B. subtilis chromosome to grant genetic stability. A variety of antigens and enzymes have been displayed by this recombinant approach, using various spore surface proteins as carriers and aiming at various potential applications, ranging from mucosal vaccine to biocatalyst, biosensor, bioremediation, or bioanalytical tool8,13.
More recently, a different approach of spore display has been developed19. This second system is nonrecombinant and relies on the spontaneous and extremely tight adsorption of molecules on the spore surface9. Antigens19,20 and enzymes13,21 have been efficiently displayed and have revealed that this method is significantly more efficient than the recombinant one. This nonrecombinant approach allows the display of proteins in the native form20 and can also be used with autoclaved, death spores19. The molecular mechanism of adsorption has not been fully clarified yet. The negative charge and the hydrophobicity of the spore have been proposed as properties relevant for the adsorption13,19,22. Recently, it has been shown that a model protein, the red autofluorescent protein (mRFP) of the coral Discosoma, when adsorbed to the spore, was able to infiltrate through the surface layers localizing in the inner coat23. If proved true for other proteins, the internal localization of the heterologous proteins could explain their increased stability when adsorbed to spores23.
In a recent study, two enzymes catalyzing two successive steps of the xylan degradation pathway were independently displayed on spores of B. subtilis and, when incubated together, were able to perform both degradation steps21. Spores collected after the reaction were still active and able to continue the xylan degradation upon the addition of fresh substrate21. Even if a loss of about 15% of the final product was observed in the second reaction21, the reusability of adsorbed enzymes for single, as well as multi-step, reactions is an additional important advantage of the spore display system.
Pan et al.24 reported an additional approach to display heterologous proteins on the spore surface: heterologous proteins (an endoglucanase protein and a beta-galactosidase one) produced in the mother cell during sporulation were spontaneously encased in the forming spore coat, without the need of a carrier. This additional spore display system is a combination of the two approaches described so far. Indeed, it is recombinant since the heterologous proteins were engineered to be expressed in the mother cell during sporulation, while their assembly within the coat was spontaneous and, therefore, nonrecombinant24. However, the efficiency of display of this additional approach remains to be tested and compared with the other two approaches by using the same heterologous proteins.
The present protocol excludes the processes of spore production and purification, which have been described extensively elsewhere24. It includes the adsorption reaction, the evaluation of the efficiency of adsorption by dot-blotting and fluorescence microscopy, and the recycling of adsorbed enzymes for additional reaction rounds.

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		<tr height="26">
			<td height="26" style="height:27px;width:247px;">0.1% Poly-L-lysine solution</td>
			<td style="width:151px;">Sigma</td>
			<td style="width:173px;">P8920</td>
			<td style="width:337px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:247px;">0.45 &#181;m Nitrocellulose Blotting Membrane</td>
			<td>Sartorius</td>
			<td style="width:173px;">M_Blotting_Membranes</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:247px;">100&#215; objective UPlanF1&#160;</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:173px;">microscope equipment</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:247px;">Bacillus subtilis&#160;strain NCIB3610</td>
			<td style="width:151px;">Bacillus Genetic Stock Center</td>
			<td style="width:173px;">3A1</td>
			<td></td>
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			<td height="26" style="height:27px;width:247px;">BD ACCURI C6 PLUS</td>
			<td style="width:151px;">BD</td>
			<td style="width:173px;">flow cytometer</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:247px;">BX51</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:173px;">Fluorescent microscope</td>
			<td></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:247px;">Clarity</td>
			<td style="width:151px;">Biorad</td>
			<td style="width:173px;">1705060</td>
			<td></td>
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			<td height="26" style="height:27px;width:247px;">DP70 digital camera a</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:173px;">microscope equipment</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC</td>
			<td style="width:151px;">Thermo fisher</td>
			<td style="width:173px;">F-2754</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Goat Anti-Rabbit IgG H&#38;L (HRP)&#160;</td>
			<td style="width:151px;">Abcam</td>
			<td style="width:173px;">ab6721</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Monoclonal Anti-polyHistidine&#8722;Peroxidase antibody</td>
			<td style="width:151px;">Sigma</td>
			<td style="width:173px;">A7058-1VL</td>
			<td style="width:337px;">used to detect adsorbed proteins presenting a 6xhistidine-tag at C- or N- terminal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">SecureSlip&#160;glass coverslip</td>
			<td style="width:151px;">Sigma</td>
			<td style="width:173px;">S1815-1PAK</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Superwhite Uncharged Microscope Slides</td>
			<td style="width:151px;">VWR</td>
			<td>75836-190</td>
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			<td height="21" style="height:21px;width:247px;">U-CA Magnification Changer&#160;</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:173px;">microscope equipment</td>
			<td></td>
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spore adsorption as a nonrecombinant display system for enzymes and antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

Successful adsorption can be assessed by western blotting. Upon the reaction, the mixture is fractionated by centrifugation and washed, and the pellet fraction (Figure 1) is used to extract the surface proteins. The extract is fractionated by SDS polyacrylamide gel electrophoresis (PAGE), electrotransferred to a polyvinylidene fluoride (PVDF) membrane, and reacted against primary and secondary antibodies. The presence of proteins of the expected size, only in...

Watch this Scientific Journal Video about Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens at JoVE.com

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

Live bacterial spores displaying antigen or enzymes are functionally active nanoparticles that can be used as mucosal vaccines or biocatalysts. In principle, any protein can be efficiently adsorbed to spores. This technique is simple and does not require any genetic manipulation.Therefore, no release of recombinant organisms into the environment is involved. In addition to mucosal vaccinology for humans and animals, spores displaying enzymes can be developed as reusable biocatalyzers. Begin by labeling two times 10 to the ninth B.subtilis wild-type purified spores with two-five-or 10-microgram concentrations of model RFP in 200 microliters of binding buffer supplemented with 50-millimolar sodium citrate for one hour at 25 degrees Celsius on a rocking shaker.At the end of the incubation, pellet the binding mixtures by centrifugation, and transfer the supernatants into new conical tubes for subsequent adsorption efficiency analysis. Then, wash the spore pellets two times with 200 microliters of binding buffer per wash, resuspending the pellets in 100 microliters of fresh binding buffer after the second wash. For surface protein extraction, add 50 microliters of each adsorbed spore resuspension to 50 microliters of 2X sodium dodecyl sulfate-dithiothreitol to solubilize the surface spore proteins.After 45 minutes at 65 degrees Celsius, collect the mixtures by centrifugation, and run 10 microliters of each volume of extracted proteins supernatant on a western blot, using a monoclonal anti-His antibody recognizing the His tag present at the N-terminus of mRFP to identify the labeled surface proteins. To localize and quantify the adsorbed molecules by fluorescence microscopy, add five microliters of the adsorbed spore resuspension to 95 microliters of PBS, and add five microliters of each resulting suspension onto individual microscope slides. Place a poly-L-lysine-treated coverslip onto each slide, and place one slide onto a fluorescence microscope stage.Then, for each field, save the phase-contrast and fluorescence microscopy images. For image analysis, open the fluorescence microscopy images in ImageJ, and select Image and Type to confirm that all of the images are in eight-bit format. From the Analyze menu, select Set Measurements, and confirm that Area, Integrated density, and Mean gray value are selected.Use a drawing or selection tool to draw a line around the spore of interest, and select Measure from the Analyze menu. A popup box with a stack of values for the selected spore will appear. After at least 50 spores have been selected, select several regions without any spores, and repeat the measurement to obtain a reading for the background fluorescence.After obtaining several background measurements, copy all of the data in the Results window into a spreadsheet, and calculate the mean of the integrated density in the area of the selected spores and the background fluorescence values to obtain the corrected total-per-cell fluorescence. For indirect adsorption efficiency evaluation, perform six two-fold serial dilutions of purified mRFP at a 0.5-nanogram-per-microliter concentration to a final volume of 250 microliters per dilution in binding buffer and an appropriate experimental number of two-fold serial dilutions of 100 microliters of each reserved supernatant sample containing the unbound mRFP fraction of the adsorption reaction with 100 microliters of binding buffer per dilution. Next, cut a nitrocellulose membrane with a 0.45-micrometer cutoff to a nine-by-10-centimeter size, to cover a five-sample-by-six-dots-of-dilution area without extending beyond the edge of the gasket of the dot blot apparatus.Place the prewet membrane in the dot blot apparatus. Check the correct size of the membrane, and remove any air bubbles trapped between the membrane and the gasket. Assemble the dot blot apparatus as described by the manufacturer, and cover the unused portion of the apparatus to prevent air from moving through those wells.When the apparatus is ready, load the standard in the two most external lanes and the samples in the middle lanes with 100 microliters of each appropriate dilution per well. When all the samples have been loaded, turn on the vacuum pump for two minutes, before stopping the vacuum and allowing the samples to filter through the membrane by gravity. After 10 minutes, wash each well with 100 microliters of PBS.Run the vacuum pump for another five minutes. When the washing buffer has completely drained from the apparatus, while the vacuum is on, loosen the screws and carefully open the dot blot apparatus. Then, process the membrane according to standard western blot protocols.For densitometric analysis of the filter, open ImageJ and outline each dot to measure the integrated density using the Analyze, Measure command as demonstrated. To make a background correction of the image, draw a circle in an empty area, and measure its integrated density as demonstrated. Correlate the integrated density of the standard dots with the amount of loaded protein to obtain a calibration line, and use the calibration curve to extrapolate the concentration of mRFP of each sample dot.The concentration of the mRFP remaining in the unbound fractions can then be calculated. The presence of proteins of the expected size only in the lanes loaded with an extract of the adsorbed spores is indicative of a successful adsorption reaction. Direct evaluation of the adsorption efficiency depends on the heterologous protein that has been used and can be performed by fluorescence microscopy and cytofluorimetry of the pellet fraction after the fractionation of the adsorption reaction.Quantification of the fluorescent signals present on spores can then be performed using ImageJ as demonstrated. An indirect analysis of the adsorption efficiency can be performed by a dot blotting analysis of the supernatant fraction containing the unbound protein, and subsequent densitometric analysis of the unbound protein allows the indirect calculation of the amount of protein adsorbed onto the spores. Although, in principle, any protein can be adsorbed for use as mucosal vaccines or biocatalysts, in practice the efficiency of the adsorption depends on the protein sites and chemical properties.

spore-adsorption-as-nonrecombinant-display-system-for-enzymes

Research

JoVE Journal

Immunology and Infection

6.5K vues.  Federico II University. Les spores bactériennes vivantes présentant des antigènes ou des enzymes sont des nanoparticules fonctionnellement actives qui peuvent être utilisées comme vaccins muqueux ou biocatalyseurs. En principe, n’importe quelle protéine peut être efficacement adsorbée aux spores. Cette technique est simple et ne nécessite aucune manipulation génétique.Par conséquent, aucun rejet d’organismes recombinants dans l’environnement n’est en cause. En plus de la vaccinologie muque...