One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

Summary

Proteinases are vital for bacterial pathogenesis, enabling invasion and immune evasion. Single-step extraction of cytoplasmic and membrane proteins of bacteria in their active form is harrowing. This video article demonstrates the extraction of whole cellular proteins of Leptospira for zymography analysis to detect and characterize gelatinases.

Abstract

Proteinases of pathogenic bacteria contribute to invasion and pathogenesis by actively degrading the host's extracellular matrix (ECM) components and cellular barriers, modifying the host immune response and degrading the host defense system. This makes the proteinases one of the most virulent molecules and their identification and characterization are essential for preventing and controlling disease. Many bacteria also possess outer membrane-bound proteinases. Isolation and characterization of outer membrane proteins in their active forms present significant challenges due to their localization within the outer membrane. In this study, we demonstrate a single-step method for extracting both cytoplasmic and outer membrane-associated gelatinases from Leptospira and demonstrate their enzymatic activity using zymography. In this method, we use sodium deoxycholate to disrupt the cell membrane and release the bound proteins and 6-aminocaproic acid to solubilize the released membrane protein in its native form. Further, we show a zymographic analysis of the leptospiral gelatinases. This approach provides a versatile framework for isolating, purifying, and characterizing metalloproteases, collagenases, and gelatinases from various bacterial pathogens.

Introduction

Most bacterial pathogens must encounter the host's extracellular matrix (ECM) during invasion and dissemination to target organs during pathogenesis. Collagen forms the major component in the ECM of animal tissues¹. Gelatinases are a group of proteases that act on collagen and its hydrolyzed form, gelatin. The ECM degradation is primarily executed by a group of proteases, which includes collagenases and gelatinases^2,3. Bacterial pathogens use collagenases and gelatinases to remodel or break down the host ECM for their convenience in invasion and pathogenesis^4,5. Bacterial ECM-degrading enzymes are potential candidates for vaccine and drug targeting⁵.

Bacterial proteases are found in secretory and membrane-bound forms^6,7,8,9. Many methods for cellular enzyme extraction are ineffective for isolating membrane-bound bacterial gelatinases and collagenases, as these procedures may not efficiently preserve the native form of membrane-bound proteins. Similarly, there are membrane-specific methods that extract only membrane-bound proteases and gelatinases. Conventional extraction methods for total proteins, including membrane proteins, often involve detergents and chaotropic ions, which can impact enzymatic activity. An extraction procedure that maintains enzyme activity and solubility is essential for electrophoretic separation and enables zymography development to detect protease activity.

Sodium deoxycholate, an ionic detergent that can lyse cells by disrupting the cell membrane, enables the release of the cell contents and membrane protein. The 6-aminocaproic acid can solubilize the released membrane protein in its native form. Solubilization by 6-aminocaproic acid has an advantage that it does not affect electrophoresis. Here, we demonstrate the extraction of cellular and membrane-bound bacterial proteinases using sodium deoxycholate and 6-aminocaproic acid, stabilizing them in their native soluble form^10,11,12. This method effectively solubilizes proteins from the cell and the membrane that can be used directly without interfering electrophoresis^13,14. Various enzymatic characterizations can be performed by changing the protease inhibitors and metal ions used in the activation buffer, depending on the application.

Protocol

1. Extraction of bacterial gelatinases

NOTE: See Table 1 for preparation of reagents and materials.

1. Grow the organism in Ellinghausen McCullough Johnson Harris (EMJH) medium containing 10 mg/mL bovine serum albumin at 30 °C. Take 10 mL of Leptospira culture in the mid-log phase of growth containing 3.5 × 10⁸ cells/mL for the extraction proteinases^13,14. Use the direct microscopic count with a Petroff-Hausser counting chamber under a dark-field microscope to observe the mid-log phase of growth.
2. Harvest the cells by centrifugation at 6,000 × g for 10 min, and discard the supernatant in a container for decontamination by autoclaving at 121 °C for 60 min.
3. Transfer the pellet by suspending it in 1 mL of wash buffer to a 1.5 mL microcentrifuge tube and centrifuge at 6,000 × g for 5 min. Repeat this wash.
4. Extract the protein from the pellet by adding 100 µL of extraction buffer for a Leptospira pellet from 10 mL of culture and vortex for 5 min. Continue the extraction by incubating at 4 °C for 1 h. Collect the extracted protein by centrifugation at 15,000 × g at 4 °C for 10 min, and discard the pellet in a container for decontamination by autoclaving at 121 °C for 60 min.
5. Use the protein extract directly for zymography by adding an equal volume of 2x sample buffer.
6. Use the Bradford estimation assay to determine the protein concentration.
7. If the protein concentration is low, precipitate it with acetone by mixing the protein with a 4x volume of acetone for precipitation and incubate it overnight at -20 °C. The next day, centrifuge the sample at 10,000 × g for 10 min, air dry the resulting pellet, and resuspend it in PBS or dissolve in the desired buffer and store it at -20 °C for up to 1 month. Use lyophilization for long-term storage or transport.
   NOTE: Due to active proteases in the extract and the absence of protease inhibitors, proteins may degrade in the sample.

2. Zymography of gelatinases

1. Preparation of the gelatin co-polymerized SDS PAGE gel
   1. Resolving gel
      1. Make the resolving gel mix by adding the components in the required amounts to cast the gel. To 3.86 mL of water in a 50 mL beaker, add 2.5 mL of Tris-HCl (pH 8.8), 0.1 mL from the 10% gelatin stock solution, 3.34 mL of acrylamide-bis acrylamide solution (ABA), 0.1 mL of 10% SDS, 0.006 mL of TEMED, and 0.1 mL of 10% ammonium persulfate and mix well without foaming and bubbles. The system mentioned (Regular Glass Plates; 18 x 8 cm) requires 10 mL of resolving gel mix.
         NOTE: If the gelatin is from a frozen aliquot, thaw it till it melts to clear the solution without any clumps. Add ammonium persulfate just before casting because the polymerization will start immediately after adding ammonium persulfate.
      2. Pour the resolving gel mix into the casting chamber up to ~3-5 mm below the marking for the bottom of the wells. Avoid air bubbles.
      3. Slowly pour some water to obtain an even 1 mm layer on the top of the gel and remove bubbles. Allow the gel to be set for 1 h.
         NOTE: Gel formation can be confirmed by looking for a phase separation between the layer of water and the gel; the gel cannot be poured out even if the gel cassette is tilted.
   2. Stacking gel
      1. Prepare the stacking gel by mixing the following components. To 1.84 mL of water in a 10 mL beaker, add 2.5 mL of Tris-Cl (pH 6.6), 0.35 mL of ABA, 0.03 mL of 10% SDS, and 0.006 mL of TEMED. The 8 x 10 cm system of 1 mm thickness requires 3 mL of resolving gel mix.
      2. From the gel cassette, decant the water formed at the top of the gel by tilting the gel setup to one side and blotting the collected water from the corner using a narrow strip of filter paper.
      3. Add 0.03 mL of 10% APS to the mixture mentioned in the step 2.1.2.1 and mix without forming bubbles.
      4. Pour the stacking gel onto the resolving gel and immediately place the comb in position without forming air bubbles under the comb teeth, leaving a thickness of 3-5 mm of stacking gel between the resolving gel and the bottom of the comb teeth. Wait for at least 3 h for complete polymerization and crosslinking of the gelatin.
      5. Take out the lower spacer and fix the gel on the electrophoresis apparatus.
      6. Slowly place the gel in the tank containing 1x electrode buffer, ensuring there is no trapped air in the bottom of the gel.
2. Preparation of sample
   1. Add an equal volume of sample buffer to the extract and incubate at 37 °C for 30 min to allow SDS to bind to the protein. Use a sample containing 2 µL of human serum with an equal volume of sample buffer as a positive control.
3. Loading the sample
   1. Carefully load the sample into the wells with a narrow-tipped pipette or a Hamilton syringe (0.5-1 mL). Gradually add electrode buffer to fill the remaining portions of the wells, the top of the gel, and the upper tank of the electrophoresis unit without disturbing the loaded samples.
      NOTE: The Hamilton syringe should be thoroughly washed between the samples to avoid cross-contamination.
4. Running electrophoresis
   1. Start the electrophoresis at 6 mA per gel and run until the dye front enters the resolving gel. Then, increase the current to 12 mA per gel and continue until the dye front reaches 1 cm above the bottom of the gel.
      NOTE: The running time may change depending on the concentration of ABA used and the size of the target protein.
5. Marking the orientation of the bands of the standard
   1. After completing the electrophoresis, carefully dismantle the glass plates using a plate separation tool or plastic spatula and mark the orientation of sample loading by cutting the right lower corner of the gel and mark the prestained marker bands by punching holes nearby or scanning the gel.
6. Renaturation of resolved enzymes
   1. Submerge the gel in renaturing solution containing Triton X-100 in a gel tray and incubate for 30 min at room temperature without shaking.
      NOTE: Triton X-100 renatures the proteins, as the zymogram gel containing resolved proteins may be denatured and lose activity in the presence of SDS.
7. Incubation for protease activity
   1. Replace the renaturing solution with an activation buffer that completely submerges the gel and incubate at 37 °C for 18 h in an incubator.
      NOTE: Modify the activation buffer by adding inhibitors or activators of proteinases of interest to characterize proteinase activity as needed.
8. Staining of the zymogram
   1. After incubation, remove the activation buffer and rinse the gel briefly with water for 1 min to remove any remaining Triton X-100 and to prevent foaming during shaking.
   2. Add the staining solution to the tray, ensuring the gel is completely submerged. Shake the gel gently at a low speed to prevent damage. Continue staining until the gel turns completely blue.
   3. Remove excess stain from the gel surface by rinsing it with water for 1 min. Add the destaining solution to the tray and shake gently. Replace the destaining solution with fresh solution several times until the desired band contrast is achieved against the blue background.
      NOTE: While performing the destaining, continuously monitor the gel to ensure it does not become completely destained, allowing observation and recording of the proteolytic bands.
   4. Scan the gel in a gel documentation system or document scanner, and determine the molecular mass of the protein bands by comparing them to the protein standards.

Results

The result shows the zymogram of leptospiral protein and gelatinases from human serum. The results, shown in Figure 1, represent proteases as white clear bands against a blue background. The bands represent areas where the copolymerized gelatin is degraded and hence, not stained, while other areas of the entire gel are stained blue due to the presence of gelatin. Further, it shows that even though gelatin is also a protein, it does not get eluted in the electrophoresis due to copolymerizatio...

Discussion

During the preparation of the acrylamide gel, gelatin co-polymerizes with the acrylamide polymer matrix. Consequently, the co-polymerized gelatin remains immobilized within the gel and does not migrate during electrophoresis, even though it is a protein. After electrophoresis, the native proteins extracted using deoxycholate and 6-aminocaproic acid buffer separate according to their mass and charge. After removing the SDS, these proteins become renatured in the presence of Triton X-100 and regain their activity. Under na...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-Aminocaproicacid	Merck	A2504
Acetic acid	Sigma	695092
Acetone	Sigma	32201
Acrylamide	Sigma	A-3553
Ammonium persulfate	Sigma	A-3678
Bis Acrylamide	Sigma	M7279
Bovine serum albumin fraction V (BSA)	Sigma	A-8022
Bromophenol Blue	Sigma	B-8026
CaCl2	Merck	C4901
Coomassie brilliant blue	Sigma	1.12553
Deoxycholate	Sigma-Aldrich	D6750
Ellinghausen–McCullough–Johnson–Harris (EMJH) medium, Difco	Difco	279410
Gelatin	Merck	G9382
Glycerol	Merck	G5516
Glycine G8790	Sigma	G-8898
Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (ATCC BAA-1198) , maintained in the biorepository ofICMR-RMRC Portblair	Maintained in the biorepository of ICMR-RMRC Port Blair		Optimum growth temperature 30 °C
Magnesium chloride M8266	Sigma	M8266
Methanol	Sigma	17995-7
Phosphate buffered saline	Sigma	P4417
Plate separation tool	Wonder Wedgle
Rotor	Thermoscientific	FIBERLite F15 8x50 cy
Sodium dodecyl sulfate (SDS)	Sigma	L-4396
TEMED	Sigma	T-9281
Triton X-100	Sigma-Aldrich	T8787
Trizma Base (TRIS)	Sigma	T6066