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.
