Name: spore adsorption as a nonrecombinant display system for enzymes and antigens
Uploaded: 2019-03-19T14:30:00
Description: Watch this Scientific Journal Video about Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens at JoVE.com

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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Y., Choi, J. H., Xu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+cell-surface+display.">Microbial cell-surface display.</a> Trends in Biotechnology. 21, 45-52 (2003).</li><li>Isticato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+display+of+recombinant+proteins+on+Bacillus+subtilis+spores.">Surface display of recombinant proteins on Bacillus subtilis spores.</a> Journal of Bacteriology. 183, 6294-6301 (2001).</li><li>Huang, J. 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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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			<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>
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			<td height="26" style="height:27px;width:247px;">0.45 &#181;m Nitrocellulose Blotting Membrane</td>
			<td>Sartorius</td>
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			<td height="26" style="height:27px;width:247px;">100&#215; objective UPlanF1&#160;</td>
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			<td height="26" style="height:27px;width:247px;">Bacillus subtilis&#160;strain NCIB3610</td>
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			<td height="26" style="height:27px;width:247px;">BD ACCURI C6 PLUS</td>
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			<td height="26" style="height:27px;width:247px;">BX51</td>
			<td style="width:151px;">Olympus</td>
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			<td height="26" style="height:27px;width:247px;">Clarity</td>
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			<td height="26" style="height:27px;width:247px;">DP70 digital camera a</td>
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			<td height="42" style="height:42px;width:247px;">Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC</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>
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			<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>
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			<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>
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			<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>
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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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...