Assembling the Bacillus subtilis Spore Coat Basement Layer on Spherical Supported Lipid Bilayers

Abstract

Micro- and nano- particles are often designed by mimicking naturally occurring structures. Bacterial spores are dormant cells elaborated by some Gram-positive bacteria during poor growth conditions to protect their genetic material from harsh environmental stresses. In Bacillus subtilis, this protection is, in part, conferred by a proteinaceous shell, the “coat”, which is composed of ~80 different proteins. The basement layer of the coat contains two unusual proteins, which we have recently reconstituted around silica beads to generate synthetic spore-like particles termed “SSHELs”. Here, we describe the protocol for generating SSHEL particles, and describe the procedure to covalently link molecules of interest (in this case an anti-HER2 affibody) to SSHEL surfaces. SSHELs therefore represent a versatile platform for the display of ligands or antigens for the site-specific delivery of cargo or vaccines.

1. Introduction

Certain bacteria initiate a stress response to starvation conditions by metamorphosing into a dormant cell, called an “endospore” (or, simply, a “spore”) [1]. Spores are incredibly resistant to harsh environmental conditions [2], but nonetheless resume normal growth when nutrients are again available [3]. This developmental program is called “sporulation” and is limited to a small group of Gram-positive bacteria in the phylum Firmicutes [4]. Bacillus subtilis is a genetically tractable, non-pathogenic bacterium that has been a leading model organism to study sporulation, a process that is also conserved in human pathogens such as Clostridium difficile and Bacillus anthracis [5,6].

A hallmark of sporulation is the elaboration of an internal spherical daughter cell, the “forespore”, by the rod-shaped bacterium, which contains a copy of the bacterium’s chromosome. The forespore then gradually shuts down metabolically as it matures into a spore, whereupon it is released into the environment by the programmed lysis of the outer “mother cell”. Part of the maturation of the forespore is the deposition of ~80 different mother cell-produced proteins on the forespore surface that forms a stable shell surrounding the mature spore called the “coat” [7]. The first coat protein to localize to the forespore surface is SpoVM, a 26 amino acid-long amphipathic helical protein that preferentially embeds in positively curved (convex) membranes [8,9]. SpoVM then recruits the morphogenic protein SpoIVA which uses ATP hydrolysis to drive structural changes that promote the irreversible polymerization of SpoIVA around the forespore [10,11]. Polymerized SpoIVA provides a sturdy platform for the subsequent deposition of the ~80 proteins that comprise the coat of the mature spore [7].

We previously reconstituted the basement layer of the spore coat using purified SpoVM and SpoIVA that assemble in an ATP-dependent fashion around 1 μm-diameter silica beads coated with a lipid bilayer [12]. We refer to these resulting spore-like particles as synthetic spore husk-encased lipid bilayers, or “SSHELs” (Fig. 1). To modify SSHEL surfaces for use as a delivery platform, we exploited engineered cysteine residues, or primary amine groups, on SpoIVA for chemical modification using ‘click’ chemicals such as trans-cyclooctene (TCO) [13]. By utilizing a cognate click chemistry molecule, such as tetrazine, ligated onto various molecules of interest, such as peptides, proteins, or small molecules, the molecule of interest may be covalently displayed on the SSHEL surface via the fast inverse-electron demand Diels-Alder cyclo-addition reaction. SSHELs with ligated molecules can be used for antigen presentation of vaccines or targeted cell therapies against cancer [14,15]. Here we detail the protocol for construction of SSHELs with the functional TCO group conjugated to an engineered cysteine residue on the SpoIVA protein that can react to proteins and molecules with the tetrazine functional group. As an example, we present the protocol to display an anti-HER2 affibody (αHER2) on the SSHEL surface.

Figure 1. Schematic of SSHEL assembly.

1 μm diameter silica beads (gray) are covered in a lipid bilayer composed of DOPC (yellow). Next, SpoVM peptide (blue) and SpoIVA (green) are added. In the presence of ATP, SpoIVA polymerizes around the membrane-encased bead, anchored to the bead surface by SpoVM. Inset: SpoIVA can be engineered with a single Cys conjugated with TCO, which can covalently attach to a tetrazine-conjugated molecule of interest (blue star).

2. Materials

Prepare all solutions using analytical grade reagents and deionized water that has attained a sensitivity of 18 MΩ-cm at 25 °C. Prepare and store all reagents at room temperature (unless indicated otherwise).

2.1. Small Unilamellar Vesicle (SUV) Production

1. 25 mg/ml DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) in chloroform (see Note 1)

2. Borosilicate glass tubes 16 × 100 mm

3. Vacuum centrifuge desiccator

4. Branson Ultrasonic Bath 1800

2.2. Spherical Supported Lipid Bilayer (SSLB) Production

1. Mesoporous silica beads (1 μm diameter) (Glantreo)

2.3. SpoIVA Purification

1. Luria-Bertani (LB) Broth: 10g of Yeast Extract, 10 g of NaCl, and 10g of Peptone from casein.

2. Terrific Broth Modified (Fisher) (see Note 2)

3. Kanamycin disulfate salt

4. Isopropyl β-D-1-thiogalactopyranoside (IPTG)

5. Dry incubator shaker

6. E. coli strain BL21 (DE3) Competent Cells

7. E. coli strain BL21 (DE3) containing plasmid pJP120 that expresses His₆-tagged cysteine-less spoIVA harboring an engineered N-terminal Cys [12]

8. Lysis buffer: 150mM NaCl, 50mM Tris-HCl, pH 7.5

9. French Pressure Cell Press

10. Ultracentrifuge and appropriate rotor to accommodate 30 ml sample tubes

11. Ni²⁺-NTA Agarose (Qiagen)

12. Wash buffer 1: 20mM imidazole, 50mM Tris-HCl, pH 7.5, 150mM NaCl.

13. Wash buffer 2: 80mM imidazole, 50mM Tris-HCl, pH 7.5, 150mM NaCl.

14. Elution buffer: 250mM imidazole, 50mM Tris-HCl, pH 7.5, 150mM NaCl.

15. PD-10 desalting columns (GE)

16. Fast Protein Liquid Chromatography (FPLC)

17. FPLC MonoQ column

18. FPLC Buffer A: 50mM Tris-HCl, pH 7.5

19. FPLC Buffer B: 50mM Tris-HCl, pH 7.5, 1M NaCl

2.4. SSHELs preparation

1. SSHEL assembly solution: 50mM Tris, pH 7.5, 10μM SpoVM, 4mM ATP, 10mM MgCl₂, 400mM NaCl and 1.5μM SpoIVA-αHER2

2. Trans-Cyclooctene-PEG3-Maleimide (TCO) (Click Chemistry Tools)

3. Dimethyl Sulfoxide (DMSO)

4. Zeba Spin Desalting Columns, 7K MWCO, 5ml (Thermo Scientific)

5. Anti-ErbB2 affibody molecule (Abcam)

6. Methyltetrazine-PEG4-Malemeide (Click Chemistry Tools)

7. Tris-(2-Carboxyethyl)phosphine, Hydrochloride (TCEP)

8. SpoVM peptide (MKFYTIKLPKFLGGIVRAMLGSFRKD) (see Note 3)

9. Tris Buffer: 50mM Tris-HCl, pH 7.5, 400mM NaCl

10. Phosphate buffered saline (PBS) at pH 7.4

11. Adenosine 5’-triphosphate disodium (ATP)

12. 1M MgCl₂

13. Quantum Alexa Fluor 488 MESF kit (Bangs Laboratories)

3. Methods

Carry out all procedures at room temperature unless otherwise specified.

SSHELs production

3.1. SpoIVA Purification

1. Make 4 × 5 ml overnight cultures of E. coli BL21 (DE3) pJP120 in LB supplemented with kanamycin (50μg/ml) for plasmid maintenance, and use each 5 ml culture to inoculate 4 × 500ml of Terrific Broth media supplemented with kanamycin (50 μg/ml).

2. Grow cultures for 2h in dry shaker set at 37 °C, shaking at 250 rpm.

3. Add IPTG for a final 1mM concentration and continue growing for 4.5h.

4. Centrifuge each culture at 4,000 × g at 4 °C. Remove and discard supernatant (see Note 4).

5. Resuspend each of the four pellets on ice with 30ml ice-cold lysis buffer and disrupt cells by French Press twice using a pressure setting of 1200psi (see Note 5).

6. Centrifuge each 30ml lysate at 30,000 × g for 1h at 4 °C. Collect supernatant in 50ml conical tubes and place on ice.

7. During centrifugation, equilibrate 4ml of Ni²⁺-NTA agarose in a gravity column with 15ml ice cold lysis buffer using gravity flow. Cap the column with resin suspended in ~1ml lysis buffer and place on ice for future use.

8. Add entire cell supernatant (~120ml) to Ni²⁺-NTA agarose column equilibrated with lysis buffer (see Note 6).

9. Wash column with 50ml cold wash buffer 1, followed by 4ml cold wash buffer 2 (see Note 6).

10. Add 10ml elution buffer, cap the column, and incubate on ice for 30 min (see Note 7).

11. While column is incubating, equilibrate 3 x PD-10 desalting columns with ice cold 15ml lysis buffer using manufacturer’s instructions.

12. Collect column elution in 3.3ml-aliquots (see Note 6). Add one 3.3ml elution to each PD-10 desalting column, previously equilibrated with lysis buffer (step 11), and elute with 4 ml cold lysis buffer, collecting 1 ml aliquots in Eppendorf tubes for a total of 12 × 1ml aliquots (see Note 6).

13. Using an FPLC, equilibrate the MonoQ column with 10ml Buffer A (see Note 8).

14. Apply ~10ml of desalted Ni²⁺-NTA agarose-purified SpoIVA to the MonoQ column (see Note 9) and run the following stepwise program: first 10ml of 0% Buffer B; second 10ml of 15% Buffer B; third 10ml of 30% buffer B; fourth 10ml of 40% Buffer B; and fifth 10ml of 100% buffer B, all at 1 ml/min flow rate with 1 ml fraction collection.

15. Collect fractions 33 through 35, corresponding to the third peak on the A₂₈₀ chromatogram and quantitate molarity using UV-spectrophotometer, measuring A₂₈₀ and using the Beer–Lambert law (SpoIVA MW = 57.71 kDa and ε = 44.35) or Bradford assay using bovine serum albumin (BSA) standards (see Note 6).

16. Store 100μl aliquots at −80 °C for future use (see Note 10).

3.2. Labeling SpoIVA with TCO

1. Slowly thaw frozen aliquots of purified SpoIVA on ice. Centrifuge at 14,000 × g at 4 °C to remove any precipitated protein (see Note 11).

2. Resuspend stock TCO powder in DMSO to 20mM and add 10-fold molar excess of TCO reagent directly to SpoIVA. Wrap tube in foil to block light (see Note 12).

3. Incubate 1–4 h at room temperature or overnight at 4 °C on a shaker.

4. Remove unreacted TCO with Zeba-desalting column equilibrated with 50mM Tris-HCl, pH 7.5, 400mM NaCl buffer (see Note 13).

5. Store SpoIVA-TCO at 4 °C (covered from light) for up to 1 month (see Note 14).

3.3. Labeling anti-HER2 affibody (αHER2) with Methyl-Tetrazine

1. Reduce anti-ERB2 (HER2) affibody with TCEP according to manufacturer’s instructions.

2. Measure the concentration of reduced affibody using UV-spectrophotometer, measuring A₂₈₀ and using the Beer–Lambert law (MW=6.8 kDa, ε= 8480; see Note 15).

3. Resuspend stock Methyltretazine-PEG4-malemeide powder in DMSO (see Note 12) to 20mM and add 10-fold molar excess directly to the HER2 affibody.

4. Incubate 1– 4 h at room temperature on a shaker. (see Note 16).

5. Desalt the methyl-tetrazine-labeled affibody using PD MiniTrap G-25 columns equilibrated with PBS using manufacturer’s instructions.

3.4. SUV Production

1. Transfer 200 μL of (25 mg/ml) DOPC in chloroform into a 16 × 100 mm borosilicate test tube and desiccate overnight at room temperature using speed-vacuum centrifuge (see Note 17).

2. Hydrate each tube with 1 ml of 0.5X PBS and incubate for 1 h in a water bath set at 42 °C, vortexing vigorously every 15 min.

3. Freeze the sample in an ethanol/dry ice mixture (see Note 18), then thaw the sample in a water bath set at 42 °C, then vortex vigorously. Repeat five times.

4. Sonicate the sample in a bath sonicator for ~30 min or until the sample becomes transparent (see Note 19).

5. Transfer lipid solution to microcentrifuge tube and spin down at 13,000 × g for 10 min.

6. Collect supernatant (containing the SUVs) and discard any residual pellet.

7. Repeat steps 5 and 6 for additional purity, as needed (see Note 20).

8. Store SUVs in glass tube with Teflon-coated cap at 4 °C for up to one week (see Note 21).

3.5. SSLB Production

1. Centrifuge silica beads (100 μl of 10% w/v stock = 10 mg of beads) 2 min at 13000 × g and remove supernatant.

2. Wash pellet with 1ml H₂O three times (see Note 22).

3. Wash pellet with 1ml Methanol (100%) three times (see Note 22).

4. Wash pellet with 1ml NaOH (1M) three times (see Note 22).

5. Wash pellet with 1ml H₂O three times (see Note 22).

6. Resuspend pellet in 200μl H₂O (see Note 23).

7. Add 200μl of SUVs (5mg) to 200 μl washed silica beads (10mg).

8. Incubate for 1h at room temperature with gentle inversion.

9. Centrifuge sample for 2 min at 13,000 × g and wash three times with 1ml PBS (see Note 22).

10. Resuspend SSLB pellet in 1 ml PBS. Final concentration will be 10mg/ml (~1 × 10⁷ beads/μl).

11. Store SSLBs at 4 °C for up to 1 week (see Note 24).

3.6. Click Chemistry Reaction

1. Incubate desalted SpoIVA-TCO and Methyltetrazine-αHER2 affibody in a 1:1 ratio.

2. Incubate the reaction for 1 to 4 h at room temperature protected from light (see Note 25).

3.7. SSHEL assembly

1. Resuspend SSLB (final concentration 2.5 mg/ml) in SSHEL assembly solution.

2. Wrap samples in foil, incubate overnight at room temperature, using a shaker or inverter.

3. The next day, centrifuge the sample and check the concentration of SpoIVA-αHER2 using MESF method or plate reader with a calibration curve if the affibody or the molecule of interest was previously labeled.

4. If using an αHER2 affibody that has been previously labeled with a fluorophore, the distribution of the affibody around SSHEL particles may be optionally observed using fluorescence microscopy (Fig. 2).

5. Labeled SSHELs can be stored indefinitely at −80 °C covered from light.

Figure 2. SSHELs can covalently display anti-HER2 affibodies.

SpoIVA on the SSHEL surface were covalently attached to αHER2 affibodies that were labeled with AlexaFluor 488 (AF488) and examined using fluorescence microscopy, visualized using (A) fluorescence from affibody, or (B) differential interference contrast (DIC).

4. Notes

1. Optional: purchase lyophilized DOPC from Avanti and resuspend in chloroform.

2. Terrific Broth Modified produces a greater protein yield but one may use LB media with 10 g yeast extract, 10 g NaCl, and 10 g Tryptone per liter.

3. One may also purify His₆-tagged SpoVM using Ni²⁺ -NTA agarose as described in reference (8).

4. Pellets can be stored at −80 °C indefinitely.

5. To prevent protein degradation, pre-chill the steel French Press cell and piston at 4 °C prior to use. Collect sample from French Press at ~ 2 ml/min.

6. Optional: collect 20 μl column flowthrough for analysis by SDS PAGE.

7. For better yield combine supernatant and Ni⁺²-NTA agarose in 50 ml tube and rotate on a nutator mixer for 1 h at 4 °C prior to adding to the column.

8. The bed volume of the MonoQ column will dictate the volume of buffer A needed to equilibrate. See manufacturer’s instructions.

9. One may use a 10 ml superloop with 1/16” fitting (130 × 30 mm) (avantor™, Cat# 10497–994).

10. SpoIVA elutes from the MonoQ column at 40% Buffer B for a final buffer composition of 50 mM Tris at pH 7.5, 400 mM NaCl.

11. If noticeable precipitated protein is observed and removed, re-quantify remaining soluble protein using UV-spectrophotometer or Bradford assay.

12. Unused TCO (or Methyltetrazine) in DMSO can be stored for several months at −20 °C protected from light.

13. Optional: quantify SpoIVA-TCO concentration using UV-spectrophotometer or Bradford assay. Expect about a 10% loss of sample.

14. SpoIVA-TCO can be aliquoted and stored indefinity at −80 °C for future use.

15. Expect ≥ 90% recovery.

16. Optional: shake or rotate reaction overnight at 4 °C.

17. Optional: one may desiccate DOPC in a vacuum centrifuge for or 1 h at 45°C.

18. Optional: one may freeze sample in methanol and dry ice mixture.

19. When using a bath sonicator, fix the 1 ml lipid solution in place just below the surface of the water bath where the top of the water bath is aligned in the middle of the sample. Optional: use a tip sonicator (4 mm titanium tip) for 30 seconds in water – ice bath.

20. Each additional centrifugation should result in reduced observed pellet.

21. When storing SUVs, cap the sample under nitrogen gas and wrap parafilm around vial cap to prevent oxidation. SUVs can be stored for up to a week according to Avanti indications. Do not freeze SUVs.

22. When washing the pellet, use a 200 μl pipet tip to gently pipet up and down to ensure the beads are adequately resuspended and to remove beads adhering to the side of the centrifuge tube. Note: the beads will naturally sediment to the bottom of the centrifuge tube over time.

23. Optional: washed beads can be stored at 4 °C for up to several months in Eppendorf tubes wrapped in parafilm. Pay attention if bacterial contamination occurs.

24. When storing DOPC-coated beads at 4 °C, cap the sample under nitrogen gas and wrap parafilm around vial cap to prevent oxidation.

25. Optional: one may incubate the SpoIVA-TCO and methyl-tetrazine-αHER2 affibody solution overnight at 4 °C with gentle inverting or mixing protected from light.