In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Summary

This protocol describes a method to ubiquitinate Streptococcus pneumoniae using mammalian cell lysate or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 protein complex to assess its bacteriolytic activity, enabling the study of ubiquitin-dependent immune effectors.

Abstract

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, pathogenic bacteria are initially enclosed within an endosome, from which they break free and escape into the cytosol to avoid lysosomal degradation. Once in the cytosol, bacteria are rapidly tagged with ubiquitin chains by host E3 ligases, marking them for clearance via multiple effector pathways, including autophagy and the proteasome. To delineate the contribution of individual pathways in bacterial elimination, in-vitro reconstitution offers a controlled and unambiguous approach. Here, using Streptococcus pneumoniae (SPN) as a model bacterial pathogen, we present a detailed protocol for its ubiquitination using mammalian cell lysates or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 complex to assess its bacteriolytic activity independent of other cellular factors. Overall, this procedure holds the potential to illuminate the function of immunity-linked ubiquitin ligases and effectors in a cell-free context, where dissecting such mechanisms can be challenging in intact cells.

Introduction

Ubiquitin (Ub) is a small 8.6 kDa protein that modifies target substrates by attaching covalently to them at its C-terminal glycine, creating an isopeptide bond with the ε-amino group of lysine residues in a process called ubiquitination¹. This phenomenon is governed by a series of orchestrated steps involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase^1,2,3. Ub can be attached as a monomer or as chains of different lengths through its seven lysine residues (Linear/M1, K6, K11, K27, K29, K33, K48, and K63)⁴. These unique Ub chains act as molecular markers for various cellular functions, such as proteasomal degradation, cellular trafficking, DNA repair, and selective autophagy³.

While the canonical role of ubiquitination has long been associated with maintaining cellular homeostasis, such as degrading misfolded proteins via proteasomes or directing specific cargoes towards selective autophagy^5,6, it is now evident that ubiquitin also functions as a crucial sensing mechanism of the cytosolic immune system against bacterial infections. Intracellular bacteria frequently infiltrate host cells to avoid detection by the extracellular immune response and reproduce within a safeguarded environment^7,8. Quite a few pathogens employ secretory systems and toxins to irreparably damage the vacuoles carrying them, enabling escape into the cytosol to obtain nutrients^9,10. Once in the cytosol, these bacteria are decorated with polyubiquitin chains by specific E3 ligases, acting as a molecular "death tag", which directs them towards selective elimination. For example, the E3 ligase SMURF1 engages Mycobacterium tuberculosis by initiating K48-linked polyubiquitination directly on its surface, which leads to its degradation via the proteasome¹¹. Parkin accumulates in vacuoles containing bacteria and forms K63-linked ubiquitin chains¹², while ARIH1 and LRSAM1 modify the surfaces of bacteria with K48 and K6 chains, respectively, thereby inhibiting bacterial growth^13,14. While the majority of E3 ligases primarily target host protein substrates, some ligases display notable specificity for microbial components. RNF213 identifies and ubiquitinates lipopolysaccharides (LPS) found on Salmonella enterica serovar Typhimurium¹⁵, and the SCF^FBXO2 complex recognizes bacterial glycans present in Group A Streptococcus as a substrate for ubiquitination¹⁶, facilitating xenophagy. SCF^FBXW7 complex, on the other hand, recognizes degron motifs within surface proteins of Streptococcus pneumoniae (SPN). These include β-galactosidase A (BgaA) and Pneumococcal surface protein A (PspA), which, following tagging with K48-linked polyubiquitin chains, were confirmed using K48-linked Ub-specific antibody, presumably attract the proteasomal system, allowing for effective bacterial elimination¹⁷.

Although the process of labelling these cytosolic invaders with ubiquitin is well understood, the subsequent mechanism by which the proteasomal system removes such large microbial entities remains doubtful. Recent research has revealed that the host AAA+ ATPase, VCP/p97 (Valosin-containing protein), plays a crucial role in this process. In collaboration with cofactors like UFD1 and NPLOC4, VCP can identify bacteria marked with polyubiquitin and physically extracts ubiquitinated proteins from the bacterial membrane, thus rupturing them and aiding in their elimination from the cytosolic milieu without depending on the proteasomal machinery¹⁸.

The host cytosol contains a complex network of immune pathways that function in parallel and in a fail-safe manner to guarantee the removal of pathogens. This redundancy is crucial for a strong defense, but it complicates efforts to identify and examine the role of individual immune factors¹⁹. In this regard, in-vitro reconstitution systems offer a useful platform to analyze specific pathways under controlled conditions. Here, we introduce a cell-free system that recapitulates the process of ubiquitination and loss of viability of SPN using mammalian cell lysates or E1-E2-E3-Ub enzymes along with purified host effector complexes. In this two-step protocol, we first facilitate the conjugation of polyubiquitin chains on the bacteria, followed by assessing their elimination by VCP/p97 in association with UFD1 and NPLOC4. By reconstituting this pathway in-vitro, we bypass cellular complexities and specifically investigate VCP's ability to neutralize the pathogen. This precise setup allows for a detailed exploration of substrate recognition, chain specificity, effector's mode of action, and the essential components needed to initiate an effective response.

Protocol

1. Cell culture and lysate preparation

1. Thaw the A549 cells and grow them in a T25 flask using complete DMEM with 10% fetal bovine serum (FBS). Incubate at 37 °C in a humidified environment with 5% CO₂. Pass the cells through three passages for maintenance, then transfer 2 × 10⁶ cells into a T75 flask. Continue the culture until the cell density reaches 8-10 × 10⁶ cells.
2. Scrape the cells from the T75 flask at 4 °C using a reaction buffer that contains 200 mM HEPES, 100 mM MgCl₂, 1 M KCl, and 5% glycerol at pH 7.4. Centrifuge the scraped cells at 120 × g for 10 min to pellet them, then resuspend in the reaction buffer to achieve a final volume of approximately 0.6 mL in a 1.5 mL centrifuge tube.
   NOTE: The entire sample must be processed in ice.
3. Sonicate the cell suspension using a narrow probe (diameter tip, 2 mm) at an amplitude of 40 μm with 2 s on/off pulses for a duration of 2-2.5 min, keeping the mixture on ice. Centrifuge the sonicated samples at 21,000 × g for 45 min, collect the resulting supernatant, and add 2-3x of 50x protease inhibitor cocktail.
   NOTE: Either use the lysate immediately for the ubiquitination reaction on the same day or flash freeze it using liquid nitrogen and store it at -80 °C; however, make sure to use it within 3 days at most for effective ubiquitination. Lysate concentration ranging from 2 to 4 mg/mL is suitable for ubiquitination.

2. Bacterial culture

1. Grow the bacteria, Streptococcus pneumoniae (SPN) R6 strain (Serotype II), overnight at 37 °C with 5% CO₂ in a THY medium made up of Todd Hewitt broth powder (3.7 g/100 mL) and yeast extract powder (1.5 g/100 mL).
   NOTE: Avoid inoculating the bacteria if the medium has changed to a dark brown color. Streptococcus pneumoniae is classified as a Biosafety Level 2 (BSL-2) pathogen; take precautions and follow the correct BSL-2 containment protocols, including the use of a certified biosafety cabinet.
2. Next day, inoculate from the overnight culture into fresh THY media (1:10) at 37 °C with 5% CO₂ and grow the SPN till the Optical Density at 600 nm (OD_{600 nm}) reaches 0.4. Prepare a glycerol stock of SPN (900 µL of SPN culture and 600 µL of Glycerol (50%) and store it in -80 °C.
3. On the day of the ubiquitination experiment, thaw a new stock of SPN from -80 °C and completely inoculate it into a 5 mL of THY medium and allow it to grow until OD_{600 nm} reaches 0.4.
   NOTE: The bacterial culture was grown to an OD_{600 nm} of 0.4, which corresponds to approximately 10⁸ SPN cells per mL. This correlation was established by enumerating colony-forming units (CFUs), determined by serially diluting the culture in 1x PBS to decrease the bacterial cell concentration so that countable colonies can be obtained and spotting on BHI agar plates, a more enriched medium than THY.

3. In-vitro ubiquitination assay from mammalian lysate

1. Take approximately 10⁷ SPN cells and wash thoroughly at least three times with the reaction buffer (200 mM HEPES, 100 mM MgCl₂, 1 M KCl, and 5% glycerol at pH 7.4) by resuspending the bacteria in the following buffer, followed by centrifugation at 21,000 × g for 5 min, finally pelleting them down via centrifugation at 21,000 × g for 5 min at room temperature.
2. Resuspend the SPN pellet with A549 mammalian cell lysate along with purified ubiquitin (350 µM) and 1 mM ATP and incubate for 1-2 h at 27 °C.
   NOTE: Avoid freezing and thawing the mammalian cell lysate. Best if the lysate is prepared on the same day of the ubiquitination assay.
3. Following incubation, wash the bacteria with reaction buffer containing 1 M Urea once, followed by two washes with only reaction buffer, followed by fixation with 1% paraformaldehyde (PFA). After fixation, incubate cells with PBS containing glycine (1 mg/mL) for 15 min, followed by two 1x PBS washes.
   NOTE: A 1 M urea wash is used to mildly disrupt noncovalent interactions, ensuring that only covalently ubiquitinated bacteria remain. Afterward, it is important to wash thoroughly with reaction buffer to remove residual urea, as its presence can interfere with the in-vitro killing assay.
   As a negative control, prepare the entire reaction mixture and incubate it with bacteria on ice to inhibit enzymatic activity. Right after the incubation, rinse the sample and then fix it using 1% PFA to maintain the reaction's condition.

4. In-vitro ubiquitination assay from the E1-E2-E3 enzyme complex

1. Prepare a reaction mixture first consisting of ubiquitin (350 µM), UBE1 (400-800 nM), UbE2C (3-5 µM), Rbx1-Skp1-Cul1-Fbxw7 complex (500 nM) and 2 mM ATP in reaction buffer (200 mM HEPES, 100 mM MgCl₂, 1 M KCl, and 5% glycerol at pH 7.4), to make the total reaction volume of 100-150 µL.
   NOTE: Store all enzymes at -80 °C. To avoid losing activity from multiple freeze-thaw cycles, create small aliquots and utilize new ones for each ubiquitination reaction.
2. Resuspend the bacteria (10⁷ CFU) in this ubiquitin enzyme-containing reaction mixture and incubate for 2 h at 27 °C. Subsequently, wash the bacteria with reaction buffer containing 1 M urea for at least 3x, followed by fixation with 1% paraformaldehyde (PFA). After fixation, incubate cells with PBS containing glycine (1 mg/mL) for 15 min, followed by two 1x PBS washes.
   NOTE: If there is excessive cell loss, one can reduce the number of washes.

5. Visualization of ubiquitinated bacteria

1. After fixing the sample with 1% PFA, incubate it in 3% BSA for 1 h at 27 °C, then perform two washes with 1x PBS.
2. Resuspend the ubiquitinated bacteria using a primary antibody at concentrations higher than those typically utilized in immunofluorescence assays. Use a bacteria-specific antibody along with an anti-ubiquitin antibody (1:100), diluted in PBS containing 1% BSA, for staining at 27 °C for 1 h.
   NOTE: For optimal antibody staining, use a low protein-binding centrifuge tube to minimize the antibody loss or use a higher antibody concentration to get the best results.
3. Subsequently, wash the bacteria twice with 1x PBS and then resuspend them with the corresponding secondary antibody conjugated with Alexa Fluor 488 and 555, respectively, for 1 h at 27 °C.
4. Apply around 10-20 µL of the labeled bacterial sample by drop casting and mix in 5-10 µL of mounting medium with or without DAPI, then place a coverslip over it and use tissue paper to absorb any excess liquid. Finally, observe the sample under a confocal microscope on the same day.

6. Protein expression and purification

1. For the expression of wild-type VCP/p97, transform the VCP-containing pET28a plasmid into E. coli BL21-DE3 strain via a standard E. coli transformation protocol²⁰. Incubate colonies formed after the transformation overnight in Luria broth (LB) at 37 °C. The following day, inoculate 1:50 culture into a well-aerated flask containing LB and allow it to grow until OD_{600 nm} reaches 0.7-0.8. Add 200 µM of IPTG to the culture and incubate for 5 h at 37 °C with shaking at 180 rpm; confirm the protein expression using an SDS-PAGE.
   NOTE: For the entire bacterial growth and expression step, Kanamycin (50 µg/mL) was used as selection pressure.
2. For the expression of wild-type UFD1, transform the UFD1-containing pET41b plasmid into E. coli BL21-Rosetta strain via standard E. coli transformation protocol²⁰. Incubate colonies formed after the transformation overnight in Luria broth (LB) at 37 °C. The following day, inoculate 1:50 culture into a well-aerated flask containing LB and allow it to grow until OD_{600 nm} reaches 0.7-0.8. Add 1 mM of IPTG to the culture and incubate for 5 h at 37 °C with shaking at 180 rpm; confirm the protein expression using an SDS-PAGE.
3. To express wild-type NPLOC4, introduce the NPLOC4-containing pET41b plasmid into E. coli BL21-Rosetta strain using the standard E. coli transformation method²⁰. Incubate the colonies that arise from the transformation in Luria broth (LB) overnight at 37 °C. The next day, transfer a 1:50 dilution of the culture into a well-aerated flask filled with LB, and let it grow until the optical density at OD_{600 nm} reaches 0.7-0.8. After that, add 1 mM of IPTG to the culture and continue incubation for 16 h at 18 °C while shaking at 180 rpm; verify the protein expression by performing an SDS-PAGE analysis.
   NOTE: For the entire bacterial growth and expression step done in E. coli BL21-Rosetta strain, Kanamycin (50 µg/mL) and Chloramphenicol (20 µg/mL) were used as selection pressure. Sequences of pET28a-p97 and pET41b-NPLOC4 is provided in Supplemental File 1.
4. After protein expression, centrifuge the bacterial cells at 21,000 × g for 10 min and resuspend the pellet in lysis buffer containing 50 mM Tris-Cl, 300 mM NaCl, 5 mM β-mercaptoethanol, 1 mM PMSF, 5% glycerol, pH 8.
5. Sonicate the cells for 1 h using a narrow probe (tip diameter, 6 mm) at an amplitude of 50 μm, and with a pulse of 20 sec on/off at 4 °C. Pellet down the debris using 15,000 × g for 1 h and collect the supernatant.
   NOTE: Total sonication was carried out for 1 h (30 min ON and 30 min OFF as 20 s pulses), and the process was continued until the turbid cell suspension turned translucent to transparent. High-speed centrifugation is required to ensure that cell debris remains pelleted and does not dislodge during loading of the lysate onto the protein purification column.
6. Pass the supernatant through a 0.4 µm filter and load it onto the column packed with Ni-NTA beads for affinity purification, pre-equilibrated with purification buffer (50 mM Tris-Cl, 300 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol, pH 8).
7. Rinse the protein-rich column using wash buffer that contains 50 mM Tris-Cl, 300 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol, pH 8, and 30-60 mM imidazole of approximately 400 mL, and then elute the protein with an elution buffer that includes 50 mM Tris-Cl, 300 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol, pH 8, and 250 mM imidazole.
   NOTE: The concentration of imidazole and the volume of wash may differ based on the specific protein being analyzed. To evaluate purity, SDS-PAGE should be performed. It is essential to wash the column thoroughly until the eluent shows a negative reaction with Bradford reagent. The eluted fraction displays a prominent, concentrated band of the target protein, which should predominantly represent the sample, with only minor traces, if any, of other proteins.
8. Gather various fractions, analyze them with SDS PAGE to identify which fractions contain the pure proteins, and then perform a buffer exchange into the reaction buffer (200 mM HEPES, 100 mM MgCl₂, 1 M KCl, and 5% glycerol at pH 7.4) utilizing a PD-10 column.
   NOTE: Measure the protein concentration using Bradford Reagent (Coomassie Brilliant Blue G-250). All the proteins should be flash frozen in liquid nitrogen and stored at -80 °C.

7. In-vitro bacterial killing assay

1. Combine 6 µM of VCP/p97 with 1 µM each of NPLOC4 and UFD1, then fill the volume with reaction buffer to approximately 90 µL. Allow this reaction mixture to incubate on ice for 2 h.
2. Add 1-2 mM of ATP into the reaction mixture that includes p97-UFD1-NPLOC4, and then combine it with the ubiquitinated SPN (10⁷ cells) for a duration of 2 h at 37 °C.
3. To evaluate bacterial viability, immediately following treatment, take a 1 µl aliquot of the reaction mixture, carry out a serial dilution (1:1, 1:10, 1:100, and 1:1000), and spot 10-2 µL of each dilution onto Brain Heart Infusion (BHI) agar plates. Repeat this procedure using a 1 µL aliquot from the reaction after  2 h of incubation.
   NOTE: Spot all the dilutions for better results.
4. Assess the colony-forming units (CFUs) between the  0 h and 2 h time points. Calculate bacterial viability (%) by dividing the CFU count at 2 h by the CFU count at 0 h, then multiplying the result by 100.
   NOTE: To achieve more reliable results, utilize heat-denatured VCP/p97, VCP/p97 lacking the UFD1-NPLOC4 cofactor complex, or an ATPase-deficient variant of VCP/p97 for comparison as a negative control.

Results

Our protocol details the utilization of mammalian A549 cells to produce a cell lysate that is rich in the necessary components for initiating a ubiquitination process (Figure 1A). This lysate, whether in a concentrated state or substituted with purified components such as UBE1 (the E1 ubiquitin-activating enzyme), UbE2C (an E2 ubiquitin-conjugating enzyme), and the Rbx1-Skp1-Cul1-Fbxw7 complex (the E3 ubiquitin ligase), is mixed with purified ubiquitin and AT...

Discussion

While the in-vitro reconstitution system detailed here offers a robust framework to explore the molecular intricacies of bacterial ubiquitination and elimination, several significant limitations must be considered. The reliance on supraphysiological concentrations of purified proteins, comprising the ubiquitination machinery (E1, E2, E3 ligases, ubiquitin) and the host effector complex (VCP/p97, UFD1, NPLOC4), offers key mechanistic insights into pathogen ubiquitination and clearance, but may not truly reflect t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50X Protease inhibitor cocktail	Promega	G6521
6x-His-Ubiquitin	UBPBio	E1100
A549 cell line	ATCC	ATCC CCL-185
Alexa Fluor 488	Invitrogen	A21202
Alexa Fluor 555	Invitrogen	A-31572
Anti-Ubiquitin antibody	Enzo	BML-PW8810-0100
Anti-Ubiquitin K48-Specific, clone Apu2 antibody	Sigma Aldrich	ZRB2150
ATP	Sigma Aldrich	A6419
BHI	HiMedia	M210
DMEM (high glucose)	HiMedia	AL251
FBS	Gibco (US origin)	38220090
Glycerol	PureSynth	PSR37325
HEPES	Sigma Aldrich	H4034-100G
KCl	Sigma Aldrich	P3911
MgCl2	Sigma Aldrich	TC006
Paraformaldehyde	Sigma Aldrich	E6148
pET28a-p97 plasmid	Our lab		Plasmid available upon request
pET41b-NPLOC4 plasmid	Our lab		Plasmid available upon request
pET41b-UFD1 plasmid	Addgene	Plasmid #117107
Rbx1-Skp1-Cul1-Fbxw7	Sigma Aldrich	23-030
Streptococcus pneumoniae R6 strain	ATCC	ATCC BAA-255	Risk Group -2 pathogen, to be handled in BSL-2 facility
Todd Hewitt broth powder	HiMedia	M313
UBE1	UBPBio	B1100
UbE2C	UBPBio	C1300
Urea	HiMedia	MB032
Yeast extract powder	HiMedia	RM027